Department of Food Hygiene and Environmental Health

Faculty of Veterinary Medicine

University of Helsinki

Finland

MUTAGENIC AND OESTROGENIC ACTIVITIES OF COMMERCIALLY

PROCESSED FOOD ITEMS AND WATER SAMPLES: A COMPARISON

BETWEEN FINLAND AND NIGERIA

Iyekhoetin Matthew Omoruyi

ACADEMIC DISSERTATION

To be presented, with permission of the Faculty of Veterinary Medicine of the University of Helsinki, for public examination in Seminar Room 11 and 12, Agnes Sjöbergin katu 2,

Helsinki, on 6th November, 2015, at 12 noon.

Helsinki, 2015

Supervisor: Professor Raimo Pohjanvirta, Ph.D., DVM.

Department of Food Hygiene and Environmental Health

Faculty of Veterinary Medicine

University of Helsinki

Finland

Director of studies: Professor Raimo Pohjanvirta, Ph.D., DVM.

Department of Food Hygiene and Environmental Health

Faculty of Veterinary Medicine

University of Helsinki

Finland

Reviewed by : Errol Zeiger, Ph.D., J.D., A.T.S.

Errol Zeiger Consulting

Chapel Hill, NC 27514

United States of America

Professor Poul Bjerregaard, Ph.D.

Department of Biology

Faculty of Science

University of Southern Denmark

Denmark

Opponent: Professor Atte Johannes Von Wright

Institute of Applied Biotechnology

University of Eastern Finland

Kuopio

Finland

ISBN 978-951-51-1423-5 (paperback)

ISBN 978-951-51-1424-2 (PDF)

Unigrafia Oy

Helsinki 2015

Abstract

Commercially processed food, drinking-water sources and effluent waters discharged

into bodies of water from wastewater treatment plants are putative but yet poorly delineated sources

of human exposure to chemical mutagens and oestrogen-like chemicals globally. To this end, this

study was aimed at determining the current situation for a possible comparison between a European

country (Finland) and an African country (Nigeria). A total of 116 commercially processed food

items and ready-to-eat snacks (three lots each) were obtained from Finland (60) and Nigeria (36) for

initial screening, as well as sachet-pure water (16 different brands) from Nigeria, bottled still and

mineral waters (10 brands each), tap water (hot and cold collected over a 3-month period) and

influent and effluent water samples from both a drinking-water treatment plant (collected over a 3-

month period) and a wastewater treatment plant (collected over a 2-year period) in Finland.

All samples were collected in their respective countries and extracted by established methods. The

mutagenic potential of the food extracts was first determined by the standard plate incorporation

assay (Ames test), using two strains of Salmonella enterica sv. Typhimurium (TA 100 and TA 98)

in the presence and absence of metabolic activation (S9 mix), and subsequently by a

methylcellulose overlay, as well as treat-and-wash assays, while the oestrogenicity of the water and

food samples, as well as food packaging materials, was determined by a yeast bioluminescent assay,

using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ER and S. cerevisiae

BMA64/luc). The cytotoxicity of the food extracts was measured by the trypan blue and lactate

dehydrogenase tests, using the HepG2 cell line, as well as by the boar sperm motility assay, while

possible DNA damage was assessed by the comet assay.

The mutagenicity of commercially processed food items in Finland was generally low: 60% or 73%

were non-mutagenic in S. Typhimurium strains TA 100 and TA 98, respectively. While the majority

of the initially positive samples proved negative in the complementary assays, cold cuts of cold-

smoked beef, grilled turkey and smoked chicken (a single batch of each) were also mutagenic in all

three assays with the TA 100 strain, with and without metabolic activation, indicating that the

mutagenic effect was not secondary to histidine release from the food products. The low

mutagenicity outcome of the Finnish food items was further confirmed by independent chemical

analyses of similar food products for four polycyclic aromatic hydrocarbons.

In contrast to the outcome in Finland, the majority of food items from Nigeria (75%) were

mutagenic in the Ames test, either in the presence or absence of the S9 mix and in either of the

strains. Chin-chin, hamburger, suya and bean cake were mutagenic in all three assays with the

Salmonella TA 100 strain, either in the presence or absence of the S9 mix. However, none of the

food samples caused DNA damage in the comet assay. They were also not cytotoxic in any of the

three assays measuring this aspect.

In all, 31% of the sachet-packed water samples in Nigeria were oestrogenic, with concentrations

ranging from 0.79 to 44.0 ng/l oestradiol equivalent concentrations (EEQs), while the tap and

bottled water samples from Finland showed no signs of oestrogenicity in the in vitro test. Similarly,

the oestrogenic activity of the influent samples from the wastewater treatment plant in Helsinki

were generally low (from below the limit of detection to 0.7 ng/l EEQ), except in March and

August 2011, when relatively high levels (14.0 and 7.8 ng/l EEQ, respectively) were obtained. No

oestrogenic activity was recorded in any of the treated effluent samples from the wastewater

treatment plant, nor was any in the influent and effluent samples from the drinking-water plant. The

outcome of this study implies that Nigerian food items and drinking-water sources are more likely

to contain mutagenic and oestrogenic chemicals than their Finnish counterparts, and efforts should

be made to reduce the level of human exposure to these chemicals in the diet.

Acknowledgements

Firstly, I wish to thank the Almighty God for seeing me through the period of my

doctoral studies, and for His provision of sound health and guidance. May His name be praised

forevermore, Amen.

This study was conducted at the Department of Food Hygiene and Environmental

Health, Faculty of Veterinary Medicine, University of Helsinki, Finland. Financial support from the

Research Foundation of the University of Helsinki, Walter Ehrström Foundation, Finland, Benson

Idahosa University (BIU), Nigeria and the doctoral programme in Food Chain and Health (formally

ABS Graduate School) of the University of Helsinki is gratefully acknowledged.

I wish to express my deepest gratitude to my supervisor, Professor Raimo Pohjanvirta,

for giving me the opportunity to do my doctoral studies under his kind supervision, for his patience,

insightful comments, suggestions and discussions all through the years of my studies and for the

confidence he has shown in my ability. I am grateful to the immediate past Head of the Department

(HOD), Professor Hannu Korkeala, and current HOD, Dr. Mari Nevas, for providing an enabling

environment for me to conduct my doctoral research.

The departmental secretary, Johanna Seppälä, and coordinator of the doctoral

programme in Food Chain and Health, Laila Huumonen, are acknowledged for their immense

administrative assistance. Special thanks to Susanna Lukkarinen, Mirja Hokkanen, Maria Anderson,

Heimo Tasanen, Grit Kabiersch and Derek Ahamioje for technical and laboratory assistance, and

my colleagues, especially Selma Mahiout and the staff of the Department of Food Hygiene and

Environmental Health, University of Helsinki, for meaningful discussions during my studies.

I am also grateful to the President of BIU, Bishop F.E.B. Idahosa II, the current Vice-

Chancellor, Professor Ernest Izevbhigie, and Professor Johnson O. Oyedeji (one-time acting Vice-

Chancellor of BIU) for the opportunity and support they have given me all through my studies.

Prof. H.S.A. Aluyi, Prof. (Mrs) C.L. Igeleke, Dr. Stephen Uzoekwe, Dr. Taidi Ekrakene, Dr. Peter

Ekunwe, Dr. (Mrs) M.O. Ehigiator, Mr. Tim Diagi, Mrs Roseline Ojiobor, Deacon Favour

Omorogbe, Mr. Osarobo Odeh and Mr. Maxwell Osagie, all of BIU, Nigeria are gratefully

acknowledged for the roles they played in the pursuit of my doctoral degree.

I am indebted to my parents, Mr. and Mrs. D.A. Omoruyi, for their prayers, love and

support, and to my inlaw, Mrs. Helen Osayamwen, siblings (Efosa Omoruyi, Bright Omoruyi,

Iyobor Ajayi, Osemwonyemwen Osaro and Gift Omoruyi), Prof. and Prof. (Mrs.) N.O. Eghafona,

Prof. I.N. Ibeh, Pastor and Pastor (Mrs) Tony Ekilisie-Uzor, Rev. Ben Akhigbe II, Pst. Kay

Akhigbe II, Mr. Wilson Ahanor, Mr. Godwin Edobor, Mr. Festus Osayamwen and Mr. Martins

Ediale for their continued prayers.

Special thanks go to you, my teacher, friend and personal life coach, Ms. Destiny

Edoh-Osunde, for believing in me, and for your encouragement during difficult times. Mr. Charles

Uwagboe, Mr. Aike Orhue and Mr. Philip Alari are gratefully acknowledged for making my stay in

Finland memorable.

Finally, I appreciate and would also like to dedicate this work to my beautiful and

lovely wife, Joy Omoruyi (a.k.a. My Nosarieme) and to our children, Destiny and Osarumese

Omoruyi, for their prayers, love, encouragement, understanding and sacrifice all through the period

of this study, and for the time I had to be away.

God bless you all.

In loving memories of my father in-law, Deacon Wilfred Ajayi Osayamwen

&

Professor Anthony Uyiekpen Osagie

May your gentle soul continue to rest in perfect peace, Amen.

Table of contents

Abstract 3

Acknowledgements 5

Table of contents 8

List of original publications 11

Abbreviations 12

1.0 Introduction 13

2.0 Literature review 15

2.1 Food and food processing 15

2.2 Toxicological safety of processed food 15

2.3 Sources of toxic chemicals in processed food 16

2.3.1 Substances deliberately added to food (food additives) 17

2.3.2 Substances inadvertently contaminating food 18

2.3.2i Pesticides 18

2.3.2ii Heavy metals and other environmental pollutants 20

2.3.2iii FCMs 21

2.3.3 Process-generated contaminants 21

2.3.3i Polyaromatic hydrocarbons (PAHs) 21

2.3.3ii Heterocyclic aromatic amines (HAAs) 25

2.3.3iii N-nitrosoamines (NAs) 25

2.3.3iv Acrylamide (AA) 26

2.4 Health effects of mutagens in food 28

2.5 Mechanism of activity of mutagens 29

2.6 Methods for determining the presence of mutagens in food 30

2.7 Xenoestrogens and EDC exposure: An overview 30

2.8 Xenoestrogen exposure via the food chain 32

2.8.1 Phytoestrogens 32

2.8.1a Health effects of phytoestrogens 33

2.8.1ai Negative health effects of phytoestrogens 33

2.8.1aii Positive health effects of phytoestrogens 34

2.8.2 FCMs and unintentionally added substances 35

2.9 Xenoestrogens in the environment 36

2.10 Health effects of synthetic xenoestrogens 37

2.10.1 Effects of xenoestrogens in males 37

2.10.2 Effects of xenoestrogens in females 39

2.11 Mechanism of activity of EDCs 39

3.0 Aims of the study 42

4.0 Materials and methods 43

4.1 Materials 43

4.2 Microorganisms 43

4.3 Cell line 44

4.4 Food samples 44

4.5 Drinking-water samples 45

4.6 Wastewater samples 45

4.7 Sample preparation: Food samples 46

4.8 Sample preparation: Water samples 47

4.9 Sample preparation: Food packaging material 47

4.10 Cytotoxicity assays 47

4.10.1 Trypan blue test 48

4.10.2 LDH assay 48

4.10.3 Boar sperm motility inhibition bioassay 48

4.11 Mutagenicity assay 49

4.11.1 Standard plate incorporation assay 49

4.11.2 Treat-and-wash assay 50

4.11.3 Methylcellulose overlay assay 50

4.12 Comet assay 51

4.13 Yeast bioluminescent assay 51

4.14 Chemical analysis: GC-MS/MS 52

4.15 Statistical analysis/Interpretation of data 52

5.0 Results 54

5.1 Recovery experiment 54

5.2 Mutagenicity test results 54

5.2i Standard plate incorporation assays 54

5.2ii Modified Ames test 56

5.3 Cytotoxicity assay: Trypan blue exclusion assay 58

5.4 Cytotoxicity assay: LDH assay 58

5.5 Cytotoxicity assay: Boar sperm motility assay 59

5.6 Comet assay 59

5.7 GC-MS/MS 59

5.8 Oestrogenic activity assay: Commercially processed food 61

5.9 Oestrogenic activity assay: Bottled water, mineral water, tap and sachet-pure water 61

5.10 Oestrogenic activity assay: Food packaging material 62

5.11 Oestrogenic activity assay: Influent and effluent water samples 62

6.0 Discussion 63

6.1 Recoveries and sample representativeness 63

6.2 Mutagenicity studies of commercially processed food 64

6.3 Genotoxicity of water samples 68

6.4 Oestrogenic activities of commercially processed food items and drinking water 69

6.5 Oestrogenic activity of food packaging material 71

6.6 Oestrogenic activity of waste water samples 72

7.0 Conclusions 74

8.0 References 75

List of original publications

This thesis is based on the following publications referred to in the text by their Roman numerals:

I Omoruyi, I.M., Kabiersch, G. and Pohjanvirta, R. (2013). Commercial processed

food may have endocrine-disrupting potential: soy-based ingredients making the

difference. Food Additives and Contaminants: Part A. 30(10): 1722-1727.

II Omoruyi, I.M. and Pohjanvirta, R. (2014). Genotoxicity of processed food items and

ready-to-eat snacks in Finland. Food Chemistry. 162: 206-214.

III Omoruyi, I.M., Ahamioje, D. and Pohjanvirta, R. (2014). Dietary exposure of

Nigerians to mutagens and estrogen-like chemicals. International Journal of

Environmental Research and Public Health. 11: 8347-8367.

IV Omoruyi, I.M. and Pohjanvirta, R. (2015). Estrogenic activity of wastewater, bottled

waters and tap water in Finland as assessed by a yeast bio-reporter assay.

Scandinavian Journal of Public Health. 43(7): 770-775.

These articles have been reprinted with the kind permission of their copyright holders.

Abbreviations

AA Acrylamide

aPAD Acute population-adjusted reference dose

BaA Benzo[a]anthracene

BbF Benzo[b]fluoranthene

BaP Benzo[a]pyrene

BPA Bisphenol A

Ch Chrysene

DEHP di(2-ethylhexyl)phthalate

DES Diethylstilboestrol

DMSO Dimethylsulphoxide

DNA Deoxyribonucleic acid

EDC Endocrine-disrupting chemical

EEQ Oestradiol equivalent concentration

EFSA European Food Safety Authority

ER Oestrogen receptor

ETU Ethylenethiourea

EU European Union

FCM Food contact material

FIC Fold induction-corrected

HAA Heterocyclic aromatic amine

ITX Isopropylthioxanthone

LOD Limit of detection

LOQ Limit of quantification

MC Methyl cellulose

MeIQ 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline

MeIQx 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline

MeOH Methanol

NA N-nitrosoamine

NADP Nicotinamide adenine dinucleotide phosphate

NDMA N-nitrosodimethylamine

NPRO N-nitrosoproline

NPYR N-nitrosopyrrolidine

NSAR N-nitrososarcosine

NVNA Nonvolatile N-nitrosoamine

PAH Polycyclic aromatic hydrocarbon

PCB Polychlorinated biphenyl

PCDDs Polychlorinated dibenzodioxins

PCDE Polychlorinated diphenyl ether

PCDFs Polychlorinated dibenzofurans

PET Polyethylene terephthalate

PVC Polyvinylchloride

PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

ROS Reactive oxygen species

VNA Volatile N-nitrosoamine

WHO World Health Organization

WWTP Wastewater treatment plant

1.0 Introduction

Recently, there has been increased debate on the effects of chemicals on humans,

especially following long-term exposure and exposures at critical windows of life. Two major

classes of chemicals involved in this debate are mutagens and oestrogens, and possible sources of

human exposure to these chemicals include processed food, drinking-water sources and effluent

waters discharged from wastewater treatment plants (WWTPs).

Processed food items may contain mutagens such as polycyclic aromatic

hydrocarbons (PAHs) and heterocyclic aromatic amines (HAAs) (Tikkanen et al., 1993; Sinha and

Rothman, 1999; Wretling et al., 2010; Chung et al., 2011; Essumang et al., 2012), along with

several other compounds (pesticides, food contact materials (FCMs) etc.) inadvertently

contaminating the food. In Sweden, Finland and The Netherlands for example, product groups

including potato crisps, French fries, coffee, bread, biscuits and breakfast cereals reportedly

contribute more than 90% to the total daily intake of mutagenic acrylamide (AA) (Konings et al.,

2003; Svensson et al., 2003; Eerola et al., 2007; Hirvonen et al., 2011). Epidemiological studies

have substantially furthered our understanding of the links between diet, genetic toxicity and cancer

(Doll and Peto, 1981; Akintowa et al., 2007; Sharif et al., 2008; Sinha et al., 2009; Francois et al.,

2010; Hirvonen et al., 2010). These studies have revealed that diet is a key contributor to human

cancer, with approximately 32% of cancers estimated as being attributable to dietary factors

(Willett, 1998). In Finland, dietary AA intake by male smokers is positively associated with

increased risk of lung cancer (Hirvonen et al., 2010). In Argentina, Hawaii and The Netherlands,

process-generated contaminants in food are associated with increased risk of different types of

cancer (Marchand et al., 2002; Voskuil et al., 2002; Navarro et al., 2004).

Regarding endocrine-disrupting chemicals (EDCs), most information is available on

compounds possessing oestrogen-like activity. Examples of such EDCs include bisphenol A (BPA),

polyvinylchloride (PVC), di(2-ethylhexyl)phthalate (DEHP), polychlorinated biphenyls (PCBs),

dioxin and dioxin-like chemicals. These chemicals have been reported in most of the daily products

used by humans, including food, FCMs and plastic bottles (Stroheker et al., 2003; ter Veld et al.,

2006; Behr et al., 2011; Plotan et al., 2013). The presence of EDCs in the human diet is worrisome,

also due to an increase in certain cancer types (breast, stomach and colon cancer), especially in the

industrialized countries, and the established linkage between hormonal disorder and EDC exposure

(Fisher, 2004; Meeker, 2010; Walvoord, 2010). In Finland, for example, cancers of the oesophagus,

stomach, lungs and colon appear to be on the increase (Weiderpass and Pukkala, 2006; Hirvonen et

al., 2010). In Nigeria, there is a paucity of data on the incidence of various cancer types. However,

the major forms of cancer in Nigeria that have been linked with EDC exposure (breast and prostate

cancers) are on the increase (Jedy-Agba et al., 2012).

From the point of view of risk assessment, EDCs present a particular challenge,

because they may have non-monotonous dose-response curves not adequately covered by

conventional toxicological experimentation and may be capable of causing untoward impacts at

environmentally prevailing, low concentrations (Fagin, 2012). The overall presence of these

chemical compounds in processed food, drinking-water sources and the environment is alarming, in

view of the fact that these items are a prerequisite for human life. In addition, most toxicological

studies on processed food and drinking-water sources are targeted at only one or a specific class of

compounds, neglecting the possibilities of unintentionally added substances and formation of new

substances in the final product as a result of processing.

2.0 Literature review

2.1 Food and food processing

Food is any substance, usually of plant or animal origin that is consumed to provide

nutritional support for the body. While most food can, at least in principle, be eaten in its raw form,

many also undergo some form of processing for reasons ranging from palatability, safety, shelf life

extension and texture to flavour (Ramaswamy and Marcotte, 2006). The methods used in food

processing are largely dependent on the type of food being processed, the quantity, desired product

and available resources/technology. The processing methods employed in most developed countries

have evolved over the years, from the traditional use of coal to more sophisticated techniques such

as electroheating, high-pressure processing, cold/liquid smoking, ultrasound, use of modified

atmospheres and microfiltration. Despite current advances in food-processing techniques

worldwide, these crude methods are still the methods of choice in most developing countries

(Essumang et al., 2012). In addition, the major aims considered in most modified methods of food

processing are on improving and preserving the flavour, texture and nutrients of food, as well as

killing vegetative cells contaminating it (Farber, 1991).

2.2 Toxicological safety of processed food

Food safety issues are increasing in importance globally, due to significant increases

in the number and outbreaks of foodborne diseases, as well as increased incidences of cancer. The

outbreaks of foodborne diseases are well documented (Bean et al., 1990; O’Mahony et al., 1990;

Mead et al., 1999; Maruzumi et al., 2005; Cleary et al., 2010; Rowlands et al., 2010; Centre for

Disease Control (CDC), 2013; Coldea et al., 2013; Gould et al., 2014), and this has further

strengthened strategies and awareness of microbiological food safety. On the other hand,

toxicological food safety has suffered some setbacks with respect to awareness, especially among

consumers. This may be related to the fact that most of the biological endpoints (e.g. cancer) of

chemicals in processed food are observed after lengthy periods of time and often from long-term

exposure. Epidemiological studies have furthered our understanding of the links between diet and

cancer (Akintowa et al., 2007; Sharif et al., 2008; Sinha et al., 2009; Francois et al., 2010). Recent

comprehensive analysis of the available literature data shows convincing evidence for consumption

of processed meat as a cause of increased risk of colorectal, prostate, breast, pancreatic and colon

cancer (Anderson et al., 2002; Navarro et al., 2004; Corpet, 2011; Berjia et al., 2014). In the case of

cancers of the oesophagus, stomach and lung, the causal relationship is suggestive (World Cancer

Research Fund (WCRF)/American Institute for Cancer Research (AICR), 2007). It is also

noteworthy that the causes of cancer are complex, and in addition to diet, general lifestyle habits

and individual genetic make-up are important factors (King et al., 2000; WCRF and AICR, 2007;

Alaejos et al., 2008).

2.3 Sources of toxic chemicals in processed food

Toxic chemicals/compounds reported in processed food are present and/or formed in

different ways, as outlined in Figure 2.1 below. These sources can be divided into deliberate

addition and unintentional contamination.

Figure 2.1. Sources of human exposure to both mutagens and oestrogens.

2.3.1 Substances deliberately added to food (food additives)

Food additives are substances added intentionally to food to improve or modify its

taste, colour and shelf life (di Sotto et al., 2014). There are 363 different additives, with 23

recognized uses, permitted in food in the European Union (EU) (EU, 2008; EU, 2013). Over the

years, the safety of many of these food additives has come into question, either as a result of their

toxic properties or their ability to interact with electron-rich biological macromolecules, resulting in

adverse health effects, including reactive genotoxicity and carcinogenicity (di Sotto et al., 2014). To

this end, controversy exists both on the toxicological safety of food additives and the use of these

additives in different countries. For example, while food additives such as amaranth and vegetable

carbon (carbon black) have been banned in the United States of America (USA), these substances

are still in use within the EU, Australia and New Zealand (European Food Safety Authority EFSA,

2010; EFSA, 2012a). Similarly, azo dyes such as blue 1, blue 2, yellow 5 and yellow 6, which are

banned within the EU for their hyperactive effects, are still heavily used in the USA.

Similar controversy also exists on the toxicity of food additives. While the

genotoxicity of some food additives (amaranth, allura red, new coccine, tartrazine, erythrosine,

phloxine and rose Bengal) has been reported at low doses (Tsuda et al., 2001; Sasaki et al., 2002;

Shimada et al., 2010), more recent reports show that these food additives are not a source of worry

(Poul et al., 2009; EFSA, 2010; EFSA, 2012a; di Sotto et al., 2014).

2.3.2 Substances inadvertently contaminating food

In addition to substances deliberately added to food during processing, a number of

unwanted substances have also been reported in food products, with some of them having genotoxic

and oestrogenic effects. Examples of such substances are included below.

2.3.2i Pesticides

Global reliance on pest-control chemicals (pesticides) in agriculture is an essential

component of food production, and this process often leaves toxic residual compounds in food

products. The presence of pesticide residues in processed food items is dependent on factors such as

the persistent nature of the pesticide, degradation product, type and portion of food material,

processing technique and other environmental factors. Some pesticides (e.g. malathion, ethion,

profenofos, fenpropathrin, chlorpyrifos, cypermethrin, 1,1-bis(p-chlorophenyl)-2,2,2-

trichloroethane (DDT), lindane, aldrin and diazinon), the majority of which are known to have long-

term toxicological effects, have been reported in a large variety of raw and processed food products

(Saeed et al., 2001; Fenske et al., 2002; Bai et al., 2006; Lorenzin, 2007; Maver et al., 2007;

Gonzalez-Curbelo et al., 2012; Ahmed et al., 2014; Lozowicka et al., 2014; Mohebbi-Nozar et al.,

2014; Wong et al., 2014), years after their prohibition. For example, residues of chlorpyrifos and

lindane pesticides have been reported in ready-to-eat foods in the USA (Fenske et al., 2002), Italy

(Lorenzin, 2007), Ghana (Johnson and Yawson, 2000) and in trace amounts in Spain (Gonzalez-

Curbelo et al., 2012). These pesticides impact the central nervous, cardiovascular and respiratory

systems in humans (International Agency for Research on Cancer IARC, 1979; Agency for Toxic

Substances and Disease Registry ATSDR 1989). They are also carcinogenic and teratogenic in

mouse (IARC, 1979; Tian et al., 2005). A number of publications have reported increased and/or

unchanged levels of pesticides during processing (Pietrino, 1991; El-Hoshy, 1997). However, recent

evidence shows that the levels of pesticide residues in food decrease during processing (Lewis et

al., 1996; Nagayami, 1996; Nagesh and Verma, 1997; Abou-Arab, 1999; Dikshit, 2002; El-

Nabarawy et al., 2002), but the levels/quantities detected in the final products are still of concern.

In 2002, Fenske et al. reported a chlorpyrifos pesticide exposure through diet in preschool children

from Washington at 2.5 μg kg-1 day-1, exceeding the recommended acute population-adjusted

reference dose (aPAD) exposure of 1.7 μg kg-1 day-1. Similarly, varying levels of pesticide residues

have been reported in human serum globally (Glynn et al., 2003; Heudorf et al., 2003; Axmon et al.,

2008; Bachlet et al., 2011; Saoudi et al., 2014). Some pesticides also undergo various forms of

transformation during processing, possibly due to heat reaction or reactions with other chemical

agents in food, thereby leading to the formation of new compounds, usually with even more toxic

potential. The fungicides ethylenebisdithiocarbamates, for example, are suspected endocrine

disruptors, and when taken up by plants, can be transformed into carcinogenic ethylenethiourea

(ETU) during processing, thereby rendering them of concern to human health (IARC, 2001; Kontou

et al., 2004; Geetanjali and Santosh, 2009).

2.3.2ii Heavy metals and other environmental pollutants

Heavy metals such as arsenic, mercury and lead, as well as environmental pollutants

(e.g. dioxins, polychlorinated dibenzodioxins PCDDs/polychlorinated dibenzofurans PCDFs, PCBs

and dioxin-like PCBs) have become a public health issue worldwide, especially during recent

decades (Rose et al., 2001; Thorpe et al., 2001; Foran et al., 2004; Hites et al., 2004; Ashizuka et

al., 2005; Chen et al., 2008; Loran et al., 2010; Ibrahim et al., 2011). These toxic environmental

contaminants have been reported in fresh animal products and in a number of commercially

processed foods and drinking water (Kim et al., 2001; Mayer, 2001; Amakura et al., 2003; Schecter

et al., 2003; Watanabe et al., 2003; Yang et al., 2004; Yoshida et al., 2004; Chen et al., 2008;

Perello et al., 2010; Nostbakken et al., 2015). Despite their presence in food, the actual potential

risk to humans is influenced by dietary practices, such as consumption patterns, food packaging and

preparation methods. The long-term effect of repeated exposure, especially since childhood, could

also be of concern (Vogt et al., 2012). Most heavy metals act as co-mutagens, increasing the risk

and formation of cancer (Fischer et al., 2005). For example, co-exposure of mouse hepatoma Hepa-

1 cells to low concentrations of arsenic and BaP increases BaP-deoxyribonucleic acid (DNA)

adduct levels by as much as 18-fold (Maier et al., 2002). There are two divergent views as to the

effect of processing on the levels of environmental pollutants in processed food. While Rose et al.

(2001) reported that processing techniques such as frying, grilling and barbecuing significantly

reduce the levels of selected PCDD/PCDFs in processed beef, Perello et al. (2010) argued that

cooking processes are only of limited value as a means of reducing the concentrations of

PCDD/PCDFs, PCBs and polychlorinated diphenyl ethers (PCDEs) in food. Another independent

study showed that techniques such as the use of firewood considerably increase the levels of these

contaminants in processed food (Yang et al., 2004).

2.3.2iii FCMs

FCMs are commonly used in the packaging and food industries. They are complex

formulas of various substances, such as polymers, antioxidants, solvents, plasticizers, adhesion

promoters etc. that provide specialized functions to FCMs (Canellas et al., 2015). In addition,

FCMs can also contain impurities from raw materials or by-products from side reactions among

various ingredients, which are often unknown to FCM producers. Volatile and nonvolatile

compounds in FCMs are underestimated sources of chemical food contaminants and potentially

relevant sources of human exposure to both mutagens and EDCs (Lionti et al., 2014; Mertl et al.,

2014). These compounds can also migrate from FCMs to food through the various layers (Canellas

et al., 2010; Aznar et al., 2011; Vera et al., 2011; Sendon et al., 2012; Mertl et al., 2014), and under

normal conditions of use (ter Veld et al., 2006; Vandenberg et al., 2007; Le et al., 2008). The

migration of these substances from FCMs into food is dependent on factors such as temperature,

type of food and the nature of the packaging materials (Dabrowska et al., 2003; Pocas and Hogg,

2007; Westerhoff et al., 2008). Moreover, the constant introduction of novel packaging materials

has increased the number of specific hazards to which humans are exposed via migration from

packaging into food (Arvanitoyannis and Bosnea, 2004).

2.3.3 Process-generated contaminants

2.3.3i Polyaromatic hydrocarbons (PAHs)

PAHs are condensed compounds of linked aromatic rings and are formed by

incomplete combustion of organic materials (Samanta et al., 2002; Farhadian et al., 2011). These

chemicals have oestrogenic properties (Santodonato, 1997), are known carcinogens in humans

(Samanta et al., 2002) and cause mammary tumours in laboratory animals (el-Bayoumy et al.,

1995; Hecht, 2002). Exposure to PAHs in the general population occurs primarily through charred,

smoked and broiled foods (Djinovic et al., 2008; Farhadian et al., 2010; Wretling et al., 2010;

Alomirah et al., 2011; Chung et al., 2011; Essumang et al., 2012; Aaslyng et al., 2013), leafy

vegetables (Phillips, 1999), wood- and coal-burning stoves (Lewis et al., 1999), air pollution (Lioy

and Greenberg, 1990) and tobacco smoke (Besaratinia et al., 2002). The deposition of PAHs in

processed food items mainly occurs through processing techniques, such as grilling, barbecuing,

smoking and frying (Djinovic et al., 2008; Farhadian et al., 2010; Wretling et al., 2010; Alomirah et

al., 2011; Essumang et al., 2012), and their formation and concentration are dependent on the

type/method of processing, processing time and the type of food being processed. For example,

heating highly fatty food directly by smoking is known to produce high levels of PAHs (Djinovic et

al., 2008; Chung et al., 2011; John et al., 2011). Of the known PAHs, 15 (Figure 2.2 and Table 2.1)

are genotoxic and carcinogenic (Scientific Committee on Food (SCF), 2002; EFSA, 2008). Of the

known genotoxic and carcinogenic PAHs, benzo[a]pyrene (BaP) is the most commonly studied and

has shown various toxicological effects in experimental animals (SCF, 2002; Schneider et al.,

2002). For this reason, BaP has been used as a marker of carcinogenic PAHs in food since 2002.

However, this compound is not always detectable in foods containing PAHs, and in 2008, EFSA

suggested the use of the sum of four carcinogenic PAHs (PAH4), which included BaP,

benzo[b]fluoranthene (BbF), benzo[a]anthracene (BaA) and chrysene (Ch), as markers for PAH

concentration (EFSA, 2008). This has recently been implemented in EU regulations (European

Commission, 2011). The presence of these PAHs in processed food is worrisome, because

epidemiological studies show an increased risk of intestinal, breast, bladder, prostate, stomach,

oesophageal and pancreatic cancers after high levels of consumption of processed meat, particularly

well-done fried and barbecued red meat (Navarro et al., 2004; Norat et al., 2005; Sinha et al., 2009;

Ferguson, 2010; John et al., 2011; Berjia et al., 2014).

Benz[a]anthracene Benzo[b]fluoranthene Benzo[j]fluoranthene

Benzo[k]fluoranthene Benzo[g,h,i]perylene Benzo[a]pyrene

Chrysene Cyclopenta[c,d]pyrene Dibenzo[a,h]anthracene

Dibenzo[a,e]pyrene Dibenzo[a,h]pyrene Dibenzo[a,i]pyrene

Dibenzo[a,l]pyrene Indeno[1,2,3-c,d]pyrene 5-methylchrysene

Figure 2.2. Structures of the 15 principal carcinogenic PAHs.

Tab

le 2

.1. F

ifte

en p

rinc

ipal

PA

Hs

and

thei

r ph

ysic

oche

mic

al p

rope

rtie

s.

PAH

s M

F M

W (

g/m

ol)

MP

(°C

) B

P (°

C)

L

og K

ow

Sol.

at 2

5 °C

V

P at

25

°C

(g/L

)

(mm

/Hg)

B[a

]A

C18

H12

22

8.3

160

437.

6

5.79

9.

4 x

10-6

2.

1 x

10-7

B[b

]F

C20

H12

25

2.3

168

NR

5.78

1.

2 x

10-6

5.

0 x

10-7

B[j

]F

C20

H12

25

2.3

166

NR

6.11

2.

5 x

10-6

2.

62 x

10-8

B[k

]F

C20

H12

25

2.3

217

480

6.

84

8.0

x 10

-7

9.7

x 10

-10

B[g

,h,i]

P C

22H

12

276.

3 27

3 54

5

6.78

2.

6 X

10-7

1.

0 x

10-1

0

B[a

]P

C20

H12

25

2.3

179-

179.

3 31

0-31

2

5.97

1.

62 x

10-6

5.

49 x

10-9

Chr

ysen

e C

18H

12

228.

3 25

5 44

8

5.5

2.0

x 10

-6

7.8

x 10

-9

CP[

c,d]

P C

18H

10

226.

3 N

R

438

(at 7

60 m

mH

g)

6.70

3.

7 x

10-6

1.

4 x

10-1

0

DB

[a,h

]A

C22

H14

27

8.3

266

524

6.

5 2.

49 x

10-6

1.

0 x

10-1

0

DB

[a,e

]P

C24

H14

30

2.4

233.

5 N

R

7.

28

8.02

x 1

0-8

7.03

x 1

0-11

DB

[a.h

]P

C22

H14

30

2.4

317

NR

7.28

3.

5 x

10-8

6.

41 x

10-1

2

DB

[a.i]

P C

24H

14

302.

4 28

0 27

5 (a

t 0.0

5 m

mH

g)

7.28

5.

54 x

10-7

1.

78 x

10-1

1

DB

[a,l]

P C

24H

14

302.

4 16

2.4

NR

7.71

3.

6 x

10-7

4.

8 x

10-1

0

I[1,

2,3-

c,d]

P C

22H

12

276.

3 16

3.6

536

6.

7 1.

9 x

10-7

1.

25 x

10-1

0

5-M

C

C19

H14

24

2.3

117.

5 N

R

6.

07

6.2

x 10

-5

2.53

x 1

0-7

MF:

Mol

ecul

ar f

orm

ula;

MW

: M

olec

ular

wei

ght;

MP:

Mel

ting

poin

t; B

P:

Boi

ling

poin

t; So

l.: W

ater

sol

ubili

ty;

VP:

Vap

our

pres

sure

; N

R:

Not

repo

rted

.

2.3.3ii Heterocyclic aromatic amines (HAAs)

HAAs are formed in food through the condensation of creatine/creatinine and

Strecker degradation radicals (pyridines and pyrazines) generated from the reaction of sugars and

amino acids during the Maillard reaction (Jägerstad et al., 1998). The formation of these

compounds (heterocyclic amines HCAs) in food is influenced by factors such as temperature,

type of processing, cooking time, pH, precursor concentrations, fat, moisture content and types

of amino acid present (Knize et al., 1994; Nagao and Sugimura, 1995; Pais et al., 1999). In

general, cooking at high temperature (usually above 150 °C) and for longer periods of time

increases HAA formation (Knize et al., 1994; Skog et al., 1995; Jägerstad et al., 1998; Persson et

al., 2002; Solyakov and Skog, 2002). 2-Amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-

amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-

phenylimidazo[4,5-b]pyridine (PhIP) are the most potent HAAs and are classified as possible

human carcinogens (IARC, 1993). They have been reported in processed meat and fish products

(Nagao et al., 1977; Gross and Fay, 1995; Knize et al., 1997, 1998; Sanz et al., 2008) and are

said to be over 100- and 2000-fold more mutagenic than the toxic aflatoxin B1 and BaP,

respectively (Stavric, 1994). Among the known HAAs, some are not directly mutagenic, but co-

mutagenic (e.g. 9H-pyrido[3,4-b]indole and 2-methyl- -carboline), and thus enhance the

mutagenicity of other HAAs in food (Jägerstad et al., 1998; Skog and Solyakov, 2002; Sugimura

et al., 2004).

2.3.3iii N-nitrosamines (NAs)

N-nitroso compounds (NAs and N-nitrosamides) are formed in food by the reaction

of nitrite with amines or amides. Dietary exposure of humans to these compounds mainly occurs

from consumption of cured meat and fish products (Dietrich et al., 2005). Curing of meat inhibits

the growth of Clostridium botulinum, thereby decreasing the risk of this organism of producing

toxins and heat-resistant spores (Herrmann et al., 2015). It also modifies the taste and preserves

the colour of meat (Lijinsky, 1999). However, the curing agents (usually sodium nitrite) can

nitrosate the amines in meat, resulting in the formation of NAs. This is favoured by factors such

as heat, meat quality, fat content, storage and maturation conditions etc. NAs are divided into

two forms: volatile (VNA) and nonvolatile (NVNA), with VNAs (e.g. N-nitrosodimethylamine

(NDMA) and N-nitrosopyrrolidine (NPYR)) being the most widely studied, and are often

referred to as the most potent carcinogens of the two. However, there is a recent report that the

NVNAs (e.g. N-nitrosoproline (NPRO) and N-nitrososarcosine (NSAR)) could undergo

decarboxylation into their carcinogenic counterparts, either during heat treatment or by microbial

activity in the large intestine (Herrmann et al., 2015). The carcinogenic properties of NAs have

been extensively investigated. They induce tumours of the liver, lung, oesophagus, bladder,

pancreas and other sites in various animal species (Bogovski and Bogovski, 1981; Preussmann

and Stewart, 1984; Lijinsky, 1992; Hecht, 1997). Endogenous NAs account for 45 75% of the

total exposure and can primarily be formed by bacteria in the oral cavity after ingestion of nitrate

(Dubrow et al., 2010) or in the acidic environment of the stomach, where nitrite reacts with the

degradation products of amino acids (Habermeyer and Eisenbrand, 2009).

2.3.3iv Acrylamide (AA)

AA is a colourless and odourless crystalline solid, used in its polymerized form as

a flocculant in water purification, paper and fabric manufacturing and as a sealing adjuvant in

tunnel construction (Curtis et al., 2014; Pedreschi et al., 2014). It shows both neurological and

reproductive effects at high doses (Friedman, 2003; Tyl and Friedman, 2003), and it decreases

litter size at low doses in rodents (Tyl and Friedman, 2003).

The presence of AA in processed food was propelled into the spotlight in 2002,

following investigation of AA exposure resulting from an industrial accident in Sweden (Tareke

et al., 2002). Since then, high concentrations of this genotoxic compound have been reported in

processed carbohydrate-rich food globally (Rufian-Henares et al., 2007; Wenzl et al., 2007;

Chen et al., 2012; Cheng et al., 2012; Shamla and Nisha, 2014; Omar et al., 2015; Pacetti et al.,

2015; Wyka et al., 2015). AA is formed in high-carbohydrate food from the reaction of a free

amino acid (asparagine) with reducing sugars during high-temperature cooking and processing,

usually via the Maillard reaction (Figure 2.3). Considering the toxic nature of AA, the European

Chemical Agency, in March 2010, added AA to its list of “very high concern” (Linback et al.,

2012). Modification of processing conditions, such as lowering time, temperature, pH,

presoaking in water, addition of antioxidants and use of asparaginase to reduce the asparagine

concentration prior to cooking are some recommendations for possible reduction of AA in food

products (Hendriksen et al., 2009; Food Drink Europe, 2011). However, their application in the

food industry remains a pending issue.

Figure 2.3. Acrylamide formation from asparagine and glucose.

2.4 Health effects of mutagens in food

Process-generated contaminants in food, such as PAHs and HAAs, are all well-

known human carcinogens. The PAH-induced contaminant BaP is known to induce oral cancer

in humans, disrupt embryo development and induce significant DNA damage in the mouse

(Walle et al., 2006; Einaudi et al., 2014; Zhan et al., 2014), while the HAA-induced contaminant

PhIP causes lung, bladder and stomach cancer in humans, as well as colon and mammary cancer

in other animals (IARC, 1993).

Recent comprehensive analysis of available literature data show convincing

evidence for consumption of processed meat as a cause of increased risk of colorectal cancer; in

the case of cancers of the oesophagus, stomach and lung, the causal relationship is suggestive

(WCRF/AICR, 2007). Cooking meat at a relatively high temperature and close to the cooking

source has been linked with increased incidence of colorectal cancer in Argentina, Hawaii and

The Netherlands (Marchand et al., 2002; Voskuil et al., 2002; Navarro et al., 2004). Findings

from a prospective European cohort study link HAA intake with increased risk of colorectal

adenoma (Rohrmann et al., 2009). A number of process-generated contaminants (PAHs and

HAAs) have been reported in varying concentrations in different types of smoked meat in

several parts of Europe (Eerola et al., 2007; Stumpe-Viksna et al., 2008; Wretling et al., 2010)

and Africa (Akpambang et al., 2009; Essumang et al., 2012; Amos-Tautua et al., 2013).

However, no nexus has been established in relation to the increased incidence of cancer in these

countries. In Finland, cancers of the oesophagus, lungs, stomach and colon are on the increase

(Weiderpass and Pukkala, 2006), some of which have been linked with dietary AA intake

(Hirvonen et al., 2010). In Nigeria, there is a paucity of data on the incidence of various cancer

types. However, the major forms of cancer in Nigeria, breast and prostate cancers, may be

increasing (Jedy-Agba et al., 2012). Both have been associated with meat-cooking habits

(Grover and Martin, 2002).

2.5 Mechanism of activity of mutagens

A mutagen is a biological, chemical or physical agent that alters or causes a

permanent change (mutation) in the genetic material (usually DNA) of an organism. Such

mutations are usually the first step in a sequence of events that ultimately leads to the

development of cancer. The mechanisms of activity of mutagens in relation to cancer are

complex and largely unclear. However, most mutagens generate reactive oxygen species (ROS),

suggesting that oxidation of DNA may play a role in carcinogenesis (Fischer et al., 2005).

Metabolic activation of PAHs and HAAs to reactive metabolites and the DNA adducts formed

are postulated to be central to the carcinogenesis of PAH- and HAA-induced cancers (Hecht,

2002). PAHs and HAAs are metabolized through activation and detoxification pathways

(Mordukhovich et al., 2010). When exposure is high or detoxification is insufficient, PAH-DNA

or HAA-DNA adducts are formed in body tissues (Santella 1999; Gammon and Santella, 2008).

These adducts persist when repair mechanisms are inadequate (Braithwaite et al., 1999).

Mutation in tumour suppressor genes, such as p53, caused by PAHs or HAAs

induces cells to grow uncontrollably, thereby leading to cancer. The p53 gene is one of the most

commonly mutated genes identified in various types of human tumours (Nakanishi et al., 2000).

Codons with p53 genes are major binding sites for PAHs (Chakravarti et al., 1998). In addition

to p53, metabolically activated PAH (PAH-diol-epoxides) often target oncogenic sequences

within the ras family of proto-oncogenes, including Ha-ras, Ki-ras and N-ras (codons 12, 13

and 61). Mutation of ras at these particular codons converts it into an oncogene, capable of

transforming cells into a malignant phenotype.

2.6 Methods for determining the presence of mutagens in food

The presence of mutagens in commercially processed food is mainly determined by

chromatographic techniques (high-performance liquid chromatography HPLC, gas

chromatography-mass spectrometry GC-MS etc.). Although these methods are effective, they are

time-consuming, expensive and highly specific, identifying only targeted mutagens, which are

further used for risk assessment, and often requiring additional toxicological studies. Most of the

toxicological studies and risk assessments are carried out, based on studies with exposure to one

chemical at a time or between one and four chemicals (in the case of PAHs). This is particularly

challenging, considering that exposure to a mixture of different chemicals, some of which are co-

mutagens, increase the mutagenic effects of already known mutagens. One way of elucidating

the possible potential biologic effect of a mixture of compounds is by using the bacterial reverse-

mutation assay (Ames test). The Ames test is over 90% accurate in predicting genotoxicity

(Weisburger, 2001; Gatehouse, 2012). The principle of this bacterial reverse mutation test is that

it detects mutations that revert to mutations present in the test strains (usually Salmonella) and

restores the functional capability of the bacteria to synthesize an essential amino acid (usually

histidine). The revertant bacteria are detected by their ability to grow in the absence of the amino

acid required by the parent test strain (Maron and Ames, 1983; Gatehouse, 2012). This test is

rapid, inexpensive, specific and relatively easy to perform. The specificity of the test strains

provides useful information on the types of mutations that are induced by genotoxic agents and

gives insight into further studies.

2.7 Xenoestrogens and EDC exposure: An overview

Xenoestrogens are natural (e.g. phytoestrogens) and synthetic (e.g. PCBs, BPA and

phthalates) chemical compounds that have oestrogenic effects on living organisms via the

endocrine system. The endocrine systems regulate metabolic processes, including nutritional,

behavioural, reproductive, growth, intestinal, cardiovascular and kidney functions and also

maintain homeostasis (World Health Organization WHO, 2002). For many endocrine systems,

homeostasis programming is established during fetal/neonatal development, so that an imbalance

at this stage of life, e.g. from exposure to a xenoestrogen, may result in permanent

misprogramming (Seckl and Meaney, 2004; Owen et al., 2005). Hormones are the chemical

messengers of the endocrine system and are released into the bloodstream from numerous glands

and tissues (Figure 2.4). Hormonal imbalances are usually due to the effects of xenoestrogens.

Figure 2.4. Hormone-producing glands and tissues of the endocrine system, with sample

hormones [in colour] (Modified from Norris, 1996).

EDCs are defined as exogenous substances that alter the functions of the endocrine

system and thereby cause adverse health effects in an intact organism or its progeny

(WHO/International Programme on Chemical Safety IPCS, 2002; Guo et al., 2013). EDCs are

differentiated from other groups of substances with various modes of action by three criteria: the

presence of i) an adverse effect in an intact organism or a (sub)population, ii) endocrine activity

and iii) a plausible causal relationship between the two (EFSA, 2013). Controversies exist on the

effects and ways of assessing EDC exposure, both within the scientific community and among

regulators worldwide. Currently, no formal criteria have been established, internationally or at

the EU level, for identifying EDCs (European Commission, 2014).

2.8 Xenoestrogen exposure via the food chain

Xenoestrogens are ubiquitous and heterogeneous and have gained notoriety, due to

their association with certain reproductive disorders, developmental abnormalities and other

adverse physiological effects in both humans and wildlife (Fisher, 2004; Bourguignon and

Parent, 2010; Zama and Uzumcu, 2010). In addition, oestrogen mimics have also been reported

among chemicals used for processing and preserving food (Sinha et al., 2009; Connolly et al.,

2011; Zhang et al., 2012a), as well as in soy-based food products (Takamura-Enya et al., 2003;

Behr et al., 2011). The sources of human exposure to xenoestrogens include phytoestrogens and

synthetic xenoestrogens.

2.8.1 Phytoestrogens

Phytoestrogens are naturally occurring plant compounds found in numerous fruits

and vegetables (Cederroth et al., 2012). They are nonsteroidal compounds that bind to and

activate oestrogen receptors (ERs) and by mimicking the conformational structure of 17 -

hydroxyoestradiol (Kuiper et al., 1997, 1998). Phytoestrogens are categorized into three classes:

the isoflavones, lignans and coumestans. Genistein and daidzin are the predominant isoflavones

found in soybeans and make up the most important dietary source of phytoestrogens for humans

(Cederroth et al., 2012).

2.8.1a Health effects of phytoestrogens

The safety of phytoestrogens is mainly controversial. A critical look into the

available literature also shows inconsistent results.

2.8.1ai Negative health effects of phytoestrogens

Exposures to phytoestrogens have a number of negative health effects in both

humans and experimental animals. Examples of such effects include enhancement of

proliferation or metastasis of some cancer types (Helferich et al., 2008; Martínez-Montemayer et

al., 2010; de la Parra et al., 2012), altered fertility and disruption of several aspects of

reproduction [e.g. sexual development, timing of puberty, sex-dependent behaviours, testicular

and ovarian endocrine functions, gamete production, pregnancy and lactation] (Cederroth et al.,

2012). The first reported case of the potential effect of phytoestrogens on reproduction was in the

1940s, when ewes grazing in clover pastures developed an infertility syndrome, due to exposure

to high levels of formononetin, an isoflavone present in red clover (Trifolium pratense; Bennetts

et al., 1946). This syndrome (later referred to as `clover disease´), resulted in reduced ovulation,

low lambing and structural defects in the reproductive tract (Adams, 1995). Furthermore,

phytoestrogens taken at high doses, or at critical stages of development in rodents, result in

severe reproductive tract disorders (Lamartiniere et al., 1995; Strauss et al., 1998; Tou et al.,

1999), impair fertility in female mice (Nagao et al., 2001; Jefferson et al., 2005) and enhance

vaginal cell maturation in female human infants (Bernbaum et al., 2008). In male marmoset

monkeys, infants fed on isoflavone (soy)-.containing formula milk for 1–1.5 months showed

serum testosterone decreased by 50–70%, compared with co-twin counterparts fed on cow

formula milk (Sharpe et al., 2002). In humans, epidemiological data showed an increase in the

incidence of hypospadias in sons of women consuming vegetarian diets during pregnancy (North

and Golding, 2000), possibly due to an increased exposure to phytoestrogens. However, this was

not substantiated with blood levels of phytoestrogens for vegetarian versus non-vegetarian

mothers (North and Golding, 2000).

2.8.1aii Positive health effects of phytoestrogens

One of the notable beneficial impacts of phytoestrogens reported most often in the

literature is their anticancer properties. The anticancer properties of phytoestrogens are a result

of the presence of peptides such as lunasin and the Bowman-Birk inhibitor, a 43- and 71- amino-

acid protease inhibitor (Galvez et al., 2001; Armstrong et al., 2003). These biological peptides

are released during gastrointestinal digestion or during the fermentation of soy proteins by

enzymatic proteolysis. For example, bioactive peptides encrypted in the amino-acid sequence of

soy proteins exert anticancer, antihypertensive, hypocholesterolaemic, antiobesity and

antioxidant activities (Martinez-Villaluenga et al., 2008, 2009, 2010; Wang et al., 2008).

Epidemiological studies have shown that phytoestrogens may protect against breast cancer and

cardiovascular diseases (Pilsakova et al., 2010; Zhang et al., 2012b). In addition, they possess

antioxidant properties and influence malignant cell proliferation, differentiation and

angiogenesis, making them potential anticancer agents (Adlercreutz, 1995). Studies amongst

Western populations have consistently shown null or inverse relationships between increasing

intakes of phytoestrogens and the risk of different cancer types and reproductive health (Shultz et

al., 1991; Mitchell et al., 2001; Linseisen et al., 2004; Zhang et al., 2004; Chang et al., 2007;

Rossi et al., 2008; Bandera et al., 2011; Hedelin et al., 2011; Ollberding et al., 2012).

2.8.2 FCMs and unintentionally added substances

FCMs are unintentionally added substances in food, and their presence usually

occurs from leaching of food packaging materials (Brotons et al., 1995; Stroheker et al., 2003;

ter-Veld et al., 2006). Examples of chemicals in FCMs include BPA, 4-nonylphenol and several

phthalates. The leaching of chemical substances into food and drinking water occurs under

normal use conditions (ter-Veld et al., 2006; Vandenberg et al., 2007; Le et al., 2008) and could

be influenced by factors such as storage conditions, sunlight exposure and ambient temperature

(Dabrowska et al., 2003; Westerhoff et al., 2008). There are over 50 chemical compounds

authorized for use in FCMs that have oestrogenic potential (Muncke, 2009). Interestingly, when

FCMs are assessed for their health risk, they are not routinely tested for their endocrine-

disrupting potential (Muncke, 2011). Currently, a number of substances used in FCMs are still

being evaluated for their safety. However, the Endocrine Society has expressed its concern about

the widespread exposure of humans to these chemicals, because they are capable of affecting

multiple endpoints within a living system (Diamanti-Kandarakis et al., 2009). Drinking-water

sources, as well as bottled mineral and flavoured waters, contain oestrogenic substances, largely

due to leaching of several phthalates and other plasticizers, including BPA used in packaging

materials (Wagner and Oehlmann, 2009, 2011; Li et al., 2010; Plotan et al., 2013). The use of

BPA in FCMs generated intense debate over the past decade, leading to its ban for use in FCMs.

Recently, the Food and Drug Administration (FDA) and EFSA reported that the use of BPA in

FCM is of no toxicological concern (FDA, 2014; EFSA 2014a). However, although not directly

on BPA, EFSA has expressed concern over chronic dietary exposure to FCMs and other

contaminants in food, particularly among the most vulnerable groups (EFSA, 2012; 2014a). This

is also the opinion presented in the scientific literature (Schonfelder et al., 2002; Markey et al.,

2005; Benachour and Aris, 2009; Angle et al., 2013; Cabaton et al., 2013; Weber et al., 2015).

Similarly, polybrominated biphenyl ether (PBDE) flame retardants, which are applied in plastics

used in FCMs, certain phthalate plasticizers, as well as isopropylthioxanthone (ITX),

benzophenone and 4-methylbenzophenone, used in printing inks for (non-plastic) FCMs, are of

concern, due to their possible oestrogenic effects and widespread occurrence in food (EFSA,

2014b).

2.9 Xenoestrogens in the environment

It has been widely recognized that treated and untreated effluents discharged from

WWTPs are major sources of EDCs in the environment (Ahn et al., 2012; Bazin et al., 2012;

Ferguson et al., 2013; Brockmeier et al., 2014; Long et al., 2014; Xu et al., 2014). 4-

Nonylphenol, the main degradation product of alkylphenol polyethoxylates, is widely used as a

surfactant in household, agriculture and industrial processes, and BPA, an industrial raw material

mainly used in the plastic, rubber, adhesive and cable industries, are the two major contributors

to the endocrine-disrupting activities in aquatic environments (Auriol et al., 2006). Pesticides

from agricultural runoff have also been indicted for their endocrine-disrupting potentials (Liu et

al., 2005; Zhao et al., 2008, 2009; Jin et al., 2009; Zhang et al., 2010a, b; Yaglova and Yaglov,

2014; Lu et al., 2015; Pandey and Mohanty, 2015). Moreover, studies have shown that

metabolites of these pesticides sometimes possess more potent oestrogenic activity than their

parent compounds (Lao et al., 2010; Zhang et al., 2010a; Lu et al., 2015), making them more of a

potential threat. Considering the rates and levels of these possible oestrogenic compounds in the

environment and their reported effects on nonhuman species, it is, of course, more worrisome,

because it is totally impossible to remove these contaminants from the environment within a

short time.

2.10 Health effects of synthetic xenoestrogens

The health effects of xenoestrogens in rodents, reptiles and laboratory animals are

well documented (Guillette and Iguchi, 2003; Kidd et al., 2007; Coe et al., 2010; Pollock et al.,

2010; Marmugi et al., 2014; Sobolewski et al., 2014; van Esterik et al., 2014). In humans, the

evidence is less compelling.

2.10.1 Effects of xenoestrogens in males

Xenoestrogens have profound effects on developmental functions and reproductive

organs in both males and females. They have been implicated in declining human sperm count

and quality, fertility impairment and in abnormalities of the male reproductive tract (Toppari et

al., 1996). EDCs stimulate the proliferation of epithelial cells in the reproductive tract and

mammary gland of females and in the prostate of males (Kushner et al., 2003). They interfere

with various sperm functions and, therefore, could impair human fertilization (Schiffer et al.,

2014), leading to low sperm count and declining male reproductive performance (Delbes et al.,

2006). Moreover, xenoestrogenic performance PBDEs cause DNA damage and affect

neurodevelopmental behaviour at the critical endpoint (EFSA 2014b).

Epidemiological studies have continuously linked exposure to chemicals (e.g. to

phthalates, PCBs, dioxins and nonpersistent pesticides) with reduced semen quality. In a USA-

based study, Duty et al. (2003) found links between monobutyl phthalate exposure and poor

sperm motility and concentrations. Exposure to persistent organochlorine pollutants (e.g. PCBs

and p,p-dichlorodiphenyldichloroethylene [p,p-DDE]), which are notable EDCs, also has a

negative impact on human sperm chromatin integrity (Spano et al., 2005).

Mocarelli et al. (2008) also furthered our understanding of the role timing plays in end-point

effects, since exposure to dioxin at specific stages in life significantly impacted semen quality.

This study was based on men exposed to high levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), the most toxic dioxin, as a result of a chemical plant explosion in 1976 in Seveso, Italy.

Men exposed prepubertally (1–9 years of age) demonstrated poor semen quality as adults.

Interestingly, men exposed between 10–17 and 18–27 years of age showed slightly positive or

no differences in semen quality, respectively. Several occupational studies have also found

associations between pesticide exposure and reduced semen quality (Whorton et al., 1979;

Larsen et al., 1998; Juhler et al., 1999; Abell et al., 2000; Padungtod et al., 2000; Oliva et al.,

2001; Lifeng et al., 2006).

The onset of puberty is regulated by gonadotropins that stimulate increases in

oestrogen levels in both boys and girls, resulting in the pubertal growth spurt. The average age of

puberty has decreased over the last century, presumably due to improved nutrition, but perhaps

also due to exposure to EDCs (Teilmann et al., 2002). Environmental chemicals, including

phthalate plasticizers, p,p’-DDE and polybrominated biphenyls, have been suggested as potential

causative agents for precocious puberty (Blanck et al., 2000; Colon et al., 2000; Krstevska-

Konstantinova et al., 2001).

2.10.2 Effects of xenoestrogens in females

Human studies in 1999 showed a possible link between maternal dietary exposure

to fish from the contaminated Great Lakes and reduced fertility (Buck et al., 1999). Increased

risk of infertility and spontaneous abortion for women working in the agricultural industry has

also been reported (Rupta et al., 1991; Fuortes et al., 1997). This implies that exposure to PCBs

and pesticides via agricultural activities could account for this reduced fertility and spontaneous

abortion. In another independent study, blood samples from 2000 American and 50 Southeast

Asian women found a link between PCB and p,p’-DDE levels and altered menstrual cycles in

women (Cooper et al., 2005; Windham et al., 2005). The suggestive implication is that such

alterations may influence other endpoints (e.g. fertility, pregnancy and reproductive cancers). In

a case-control study, women with ovarian diseases showed higher levels of BPA in their sera

than did normal healthy women (Takeuchi and Tsutsumi, 2002; Takeuchi et al., 2004). A study

of 1188 women found a significant relationship between diethylstilboestrol (DES) exposure and

uterine fibroids (Baird and Newbold, 2005). In analysis of these studies, Baird and Newbold

(2005) concluded that there was a definitive increase in uterine fibroids in DES-exposed

daughters, and the discrepancies between the studies were due to the differences and sensitivities

of the methods used to detect the tumours. Increase in hormone-related disorders, especially in

the industrialized countries, is also linked to EDC exposure (Fisher, 2004; Meeker, 2010).

2.11 Mechanism of activity of EDCs

EDCs can impart adverse effects by a number of different mechanisms, including,

but not limited to, the following:

1. Activating or suppressing expression of ERs via a genomic or non-genomic pathway

2. Activating or suppressing expression of the androgen receptor

3. Activating the aryl hydrocarbon (AH) receptor

4. Inhibition of steroid hormone synthesis, transport or metabolism

5. Modulation of neurotransmitter receptors

6. Other less-defined mechanisms (WHO, 2002; SCOPE/IUPAC, 2003).

A single EDC may also affect multiple target cells by multiple mechanisms.

Activation or suppression of the ER (ER or ER ) is the most widely studied mechanism of

EDC effect. ERs are nuclear receptors present in many cell types. They are large protein

molecules with a specific binding site that has conformational specificity for the 17 -oestradiol

molecule (Thomson, 2005). Despite the specificity of the binding site, it is sufficiently generic to

allow binding by a wide range of natural and synthetic compounds. Oestrogenic compounds act

by diffusing into the cell nucleus and binding there to the ligand-binding domain of the receptor,

whereupon the ER-ligand undergoes a conformational change and dimerizes. The dimers, with

cofactors, form a complex that binds to specific sequences of DNA, known as oestrogen-

response elements (EREs), of oestrogen-responsive genes. Binding to the ERE stimulates

transcription and messenger-ribonucleic acid (mRNA) synthesis, leading to increased or

decreased expression of oestrogen-responsive genes that encode specific proteins (Gruber et al.,

2002; WHO, 2002). This is the classic pathway of oestrogenicity at the cellular and whole-body

levels and is shown schematically in Figure 2.5.

Figure 2.5 Schematic of the (xeno)estrogen nuclear receptor-mediated mechanism (modified

from Schug et al., 2011). 1. Xenoestrogens circulate in the plasma and enter the cells. 2. The

oestrogenic compound diffuses into the nucleus and binds to the ER located in the nucleus (3). 4.

The receptor occupied undergoes conformational change, dimerizes and binds with cofactors to

specific sequences on oestrogen-responsive genes. 5. mRNA is transcribed. 6. The mRNA is

transported from the nucleus to the cytoplasm to act as a template for protein synthesis.

3.0 Aims of the study

Several independent lines of evidence indicate that heat-processed food and

drinking-water sources contribute to human cancer risk through the ingestion of genotoxic and

oestrogenic compounds, thereby causing a number of adverse effects (Jägerstad and Skog, 2005;

Sinha et al., 2009). Pollution of bodies of water through discharge of inadequately treated

sewage waters has also been linked to oestrogenic effects in fish, with man being the final

recipient of such pollution. Most of the toxicological studies carried out on food and water

sources are based on exposure to one chemical at a time, neglecting the possibility of co-

exposure to a mixture of chemicals, as well as the formation of new complex and even more

toxic compounds during processing. This is usually the case in reality, since exposure from food

is to a mixture of different chemicals.

This research work therefore was primarily aimed at the following:

i. To investigate to what degree commercially employed methods in the processing of foods and

ready-to-eat snacks in Finland and Nigeria can inadvertently result in the occurrence of

genotoxic substances in the final products.

ii. To determine whether commercially processed food items possess oestrogenic potential, using

a yeast bioreporter assay.

iii. To determine whether drinking-water sources from both Finland and Nigeria contain

oestrogenic chemicals, using an assay similar to that in ii above.

iv. To determine the oestrogenic activity of treated and untreated water from both a drinking-

water plant and WWTP in Helsinki.

4.0 Materials and methods

4.1 Materials

All chemicals used in this study were of analytical grade. The -nicotinamide

adenine dinucleotide phosphate (NADP) and glucose-6-phosphate used were obtained from

Roche Biochem (Stockholm, Sweden). The Aroclor-induced S9 from rat liver was purchased

from Trinova Biochem (Giessen, Germany). The histidine, potassium chloride, magnesium

sulphate, Celite 545, potassium phosphate dibasic anhydrous and sodium ammonium phosphate

were purchased from Merck AG (Darmstadt, Germany). The magnesium chloride hexahydrate,

ethyl acetate and citric acid monohydrate were obtained from VWR International (Leuven,

Belgium). The dichloromethane, hexane, acetone, cyclohexane, methanol (MeOH) and toluene

were purchased from J.T. Baker (Deventer, The Netherlands). The biotin, tryptophan,

methylcellulose (MC), dimethyl sulphoxide (DMSO), BaP, 2-aminoanthracene, sodium azide,

oestradiol, BPA, Florisil, progesterone and testosterone were purchased from Sigma-Aldrich

(Steinheim, Germany). The D-luciferin was obtained from Biotherma (Handen, Sweden). The

yeast nitrogen base medium without amino acids was obtained from Becton Dickinson and

Company (East Rutherford, NJ, USA). The Supelclean ENVI-Chrom P (6-ml, 500-mg) solid-

phase extraction (SPE) cartridges were purchased from Supelco (Bellefonte, PA, USA), while

the strata-X 33u polymeric reversed-phase (500 mg/5 ml) was obtained from Phenomenex

(Aschaffenburg, Germany).

4.2 Microorganisms

The bacteria, Salmonella enterica sv. Typhimurium strains TA 100 and TA 98,

were obtained from the Pasteur Institute (Paris Cedex, France). Two recombinant yeast strains

Saccharomyces cerevisiae BMAEREluc/ER and S. cerevisiae BMA64/luc (Leskinen et al.,

2005) were used in this study. In the yeast bioluminescent assay, the BMAEREluc/ER served

as a reporter strain in which the ER is expressed. In ligand binding, the dimerized receptor

binds the EREs in the promoter region of the luc reporter gene. In S. cerevisiae BMA64/luc, the

luciferase is expressed constitutively, and this strain was used for determination of cytotoxicity

in the test samples. Both yeast strains were kind gift donations by Dr. Johanna Rajasärkkä of the

Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry,

University of Helsinki. The yeasts were grown on Difco Yeast Nitrogen Base Medium (Difco

Laboratories Inc. (Becton Dickinson), Franklin Lakes, NJ, USA) without amino acids,

supplemented with 40% glucose and their respective amino acids.

4.3 Cell line

The human hepatocellular carcinoma-derived cell line (HepG2) was obtained from

the American Type Culture Collection through LGC Standards (Boras, Sweden) and cultured in

Eagle’s Minimum Essential Medium (LGC Standards) containing 10% heat-inactivated fetal

bovine serum (Sigma-Aldrich). The cells were maintained at 37 °C in a humidified atmosphere

of 5% CO2 in an air atmosphere incubator (NuAire Inc., Plymouth, MN, USA).

4.4 Food samples

A total of 116 food samples, representing 39 varieties of three lots each, obtained

from both Finland and Nigeria, were used in this study. The food samples from Finland were

classified as commercially processed food products and ready-to-eat snacks (II). Here, 45

samples of industrially processed and packaged food products were purchased from a popular

supermarket in Helsinki (Prisma, Viikki), while the ready-to-eat food samples were acquired

from a local representative (at SOKOS City Centre) of a global chain of hamburger restaurants

also located in Helsinki. All samples were collected at separate times, and the cooking conditions

such as time and temperature were not available.

A total of 36 samples (three lots of 12 varieties), representing commonly consumed,

commercially processed food items in Nigeria, were also evaluated for their mutagenic potential.

All products were extracted before the expiry date shown on the packages. Twenty food samples

were also obtained from Prisma for confirmatory testing by chemical analysis.

4.5 Drinking-water samples

A total of 60 water samples, representing tap water (hot and cold) collected

bimonthly in Finland, both from the premises of the University of Helsinki, Viikki campus and a

residential building in Vantaa, Finland, over a 3-month (March–May) period, 10 different brands

each of bottled still and mineral waters, purchased from a local grocery store (Prisma, Viikki)

and 16 sachet-pure water samples sold in Benin City metropolis, Edo State, Nigeria were used in

this study.

4.6 Wastewater samples

Treated and untreated water samples, taken bimonthly, were obtained from the

WWTP in Viikinmäki, Helsinki over the preceding 20-month period on two occasions in 2011

and 2014. The Viikinmäki WWTP is the largest of such plant in Finland, serving over 1

million inhabitants and processing both household (85%) and industrial (15%) wastewater from

five different districts (Helsinki, Kerava, Tuusula, Järvenpää, Sipoo and of the central and

eastern districts of Vantaa) in Finland. It receives approximately 270 000 m3 of wastewater per

day and an average of 100 million m3 each year. Equivalent samples from a household water

purification plant (located in the same region) were also obtained in March, April and June of

2014.

4.7 Sample preparation: Food samples

Possible mutagenic compounds in Finnish food products were extracted, using the

method described by Peters et al. (2004), with slight modifications. Briefly, 20 g of food sample

were homogenized with 80 ml of 1-M NaOH at 24 000 revolutions per minute (rpm). The

homogenate was mixed with 20 g of Extrelut refill material (VWR International, Helsinki,

Finland) and then poured into an empty Extrelut 20 column. The organics were eluted from the

Extrelut column with 40 ml of dichloromethane/toluene solution (95:5 v/v) into a cartridge. The

organics were finally eluted with 5 ml of MeOH-NH4OH (9:1) solution and evaporated to

dryness under a gentle stream of nitrogen in a fume hood.

The Nigerian food samples were extracted in Nigeria (Tikkanen, 1991), and approximately 2 ml

of the final extracts were shipped on ice to the Department of Food Hygiene and Environmental

Health, Faculty of Veterinary Medicine, University of Helsinki for analysis. For determination of

recovery, fresh meat samples were spiked with 250 μg of BaP and 2-aminoanthracene in two

different cases and extracted in the same way as above. The food samples for confirmatory

chemical analysis were extracted by accelerated solvent extraction and SPE (Jira et al., 2008),

using super-clean ENVI-Chrom P (6- ml, 500-mg) cartridges.

4.8 Sample preparation: Water samples

All water samples (500 ml) obtained from Finland were extracted by the SPE

method, as described by Kopperi et al. (2013), using Phenomenex strata-X 33u polymeric

reversed phase 500 mg/5 ml (Phenomenex), while pure water samples from Nigeria (1000 ml

each) were extracted by the liquid-liquid extraction (LLE) method, as described by Barber et al.

(2000). The percentage recovery of both extraction methods was determined by spiking distilled

water samples with 2.5 mg of BPA and extracting them in the same way as the test samples.

4.9 Sample preparation: Food packaging material

Samples that were oestrogenic were unwrapped and their packaging materials

reused for negative samples to disclose possible leaching of xenoestrogens. The repackaging was

done in such a way as to maintain close contact between the food sample and packaging

material. The repackaged food samples were kept in an oven at 80 °C for 30 min and then at 4 °C

for 7 days before extraction to maximize the leaching effect without spoiling the foodstuffs. For

extraction, these packaging materials were embedded in MeOH in a mechanical shaker at 60 °C

overnight. This is a slight modification of a previous method used with success to extract

oestrogenic compounds from food samples (Behr et al., 2011).

4.10 Cytotoxicity assays

The cytotoxic effect of the food extract concentrations used in this study was

investigated, using three independent assays measuring trypan blue exclusion, lactate

dehydrogenase (LDH) activity and boar sperm motility.

4.10.1 Trypan blue test

HepG2 cells were grown in 24-well plates (VWR, Finland) until semi-confluent

cells were obtained (48 h). This was followed by exposure of the cells to different concentrations

of food extracts for 4, 24 or 48 h. After exposure, the cells were trypsinized, using a 0.25% (w/v)

trypsin–0.53 mM ethylenediaminetetraacetic acid (EDTA) solution (LGC Standards). The

trypsinized cells were transferred to 1.5-ml Eppendorf tubes and centrifuged for 5 min at 2500

rpm. The pellets were then resuspended in phosphate-buffered saline (PBS), after which 10 μl of

the cells were mixed with 5 μl (0.8 mM) of trypan blue dye before microscopic observation. The

lysis solution served as positive controls for both the trypan blue test and LDH assay.

4.10.2 LDH assay

The activity of LDH was determined in HepG2 cells exposed to the same

concentrations of the food extracts used in the genotoxicity assays. The LDH test measures

plasma membrane integrity and was performed according to the instructions provided in the

Cytotoxicity Detection KitPLUS (LDH), version 6 (Roche Biochem).

4.10.3 Boar sperm motility inhibition bioassay

Extracts of selected food samples were assessed for their mitochondrial toxicity,

using the boar sperm motility assay (Andersson et al., 2010). Briefly, 2 ml of boar semen in

screw-capped exposure vials were exposed to 10 μl of food extracts for 30 min, 24 or 48 h at 20

°C. Vehicle (DMSO) exposure was prepared simultaneously with the test samples for each time

point. After exposure, the vials were shaken gently to disperse the sperm cells, and 200 μl of the

suspension were drawn into a warmed test tube and placed in a heating block (30 °C) for

approximately 5 min to activate sperm motility. Sperm motility was assessed by dispensing the

warmed sperm suspension onto a microscopic slide, using a pre-warmed capillary tube, and

immediately observed with a 40-x inverted phase-contrast objective.

4.11 Mutagenicity assay

The mutagenic potential of the food extracts was determined initially by the

standard plate incorporation assay. Samples showing mutagenic potential in this assay were

subjected to treat-and-wash as well as MC overlay assays to ascertain to what degree a localized

release of proteins, peptides or histidine from the samples contributed to the outcome.

4.11.1 Standard plate incorporation assay

The standard plate incorporation assay was performed as described by Maron and

Ames (1983), using Salmonella strains TA 100 and TA 98 with and without metabolic activation

(S9 mix). The amount of S9 used in the S9 mix was 10%. Water and DMSO were used as

negative controls for both strains, while sodium azide (0.04 mg/ml) and 2-aminoanthracene (0.02

mg/ml) served as positive controls for TA 100 and TA 98, respectively. BaP (0.1 mg/ml) was

also used as a positive control for both strains. The volume of the controls used was 50 μl per

plate in triplicate plates. Sodium azide is a known direct mutagen in Salmonella TA 100

(Mortelmans and Zeiger, 2000), whereas 2-aminoanthracene is metabolically activated by

monooxygenases of the cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A

family) in rat liver (Carrière et al., 1992). Likewise, BaP requires metabolic activation for

mutagenicity (Gabbani et al., 1998).

For all samples, four different concentrations of the food extracts (25, 50, 100 and 200 mg/ml)

were tested in triplicate plates (50 μl per plate). The highest concentration (200 mg/ml) was

equivalent to 1 g of the food sample. The plates were incubated at 37 °C for 48 h.

4.11.2 Treat-and-wash assay

The treat-and-wash assay was conducted according to the method described by

Thompson et al. (2005). The protocol applied was as per the standard plate incorporation assay,

with the exception that the S9 mix, bacteria and sample extract were incubated for 90 min prior

to the addition of molten top agar. Briefly, a 500 μl aliquot of S9 mix/phosphate buffer (0.2 M,

pH 7.4) was combined with 100 μl each of late-log bacterial culture and sample extract solution

in a sterile 15 ml tube. The mixture was incubated for 90 min in a mechanical shaker (180 rpm)

at 37 °C. The extended duration of the bacterial exposure compensated for the absence of

bacterial exposure on the plates, since the test sample was washed away prior to plating. After a

90 min pre-incubation, 10 ml of washing solution (Oxoid No. 2 nutrient broth; Oxoid

(ThermoFisher Scientific Inc.), Waltham, MA, USA in PBS [1:7 v/v]) was added, and the

washed bacteria were collected by centrifugation at 2000 g for 30 min. All but approximately

700 μl of the supernatant was removed and discarded, and the bacteria were resuspended in the

residual supernatant prior to plating via the top agar.

4.11.3 Methylcellulose overlay assay

The MC overlay assay was performed, as previously described (Thompson et al.,

2005). Briefly, a 500 μl aliquot of S9 mix/phosphate buffer (0.2 M, pH 7.4) was combined with

100 μl of late-log bacterial culture in a sterile 15 ml tube. A 2 ml aliquot of the MC overlay

suspension was added to the tube, and a 100 μl aliquot of the sample extract solution was added

immediately afterward. The mixture was overlaid on a pre-warmed (37 °C) minimal glucose

plate. The plates were held at 4 °C for 1 h after plating to ensure gelling of the MC overlay, and

subsequently incubated (not inverted) at 37 °C for 48 72 h. The MC overlay was prepared on

the day of the test (Thompson et al., 2005), and the mixture was stirred at 50–60 °C throughout

use.

4.12 Comet assay

The comet assay (single-cell gel electrophoresis) was used to evaluate possible

breakage in the single-stranded DNA of HepG2 cells, following treatment with extracts of food

samples showing mutagenic potential. The comet assay was performed under alkaline conditions

(pH > 13), as described previously (Singh et al., 1998). Hydrogen peroxide and 0.7% DMSO

served as positive and negative controls, respectively.

4.13 Yeast bioluminescent assay

The yeast bioluminescent assay was performed, as previously described (Leskinen

et al., 2005; Rajasärkkä and Virta, 2011). The optimized parameters for the 384 well plates that

were used in this study have also been reported (Rajasärkkä and Virta, 2011), except that 5%

ethanol was used as the vehicle, because the 10% DMSO reported by Rajasärkkä and Virta

(2011) was cytotoxic in the test system.

4.14 Chemical analysis: GC-MS/MS

Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis was

performed, using a gas chromatograph (Agilent, 6890N; Agilent Technologies, Santa Clara, CA,

USA) coupled with a Micromass Quattro Micro GC triple-quadrupole analyser (Waters,

Micromass; Waters Corp., Milford, MA, USA), using an Agilent J and W Select PAH (30 m x

0.25 mm x 0.15 μm) column, helium as the carrier gas, at a flow rate of 1.0 ml/min, with the

following conditions:

Injection: Splitless injection, injection volume, 1 μl. Electron ionization (EI). Injector

temperature 300 ˚C, transfer-line temperature 300 °C, ion source temperature 275 °C. The

column temperature programme was as follows: Initial temperature 110 °C (0.7 min), 85 °C/min

to 180 °C, 3 °C/min to 230 °C (7 min), 28 °C/min to 280 °C (15 min), 14 °C/min to 350 °C (5

min).

4.15 Statistical analysis/Interpretation of data

The mutagenic potency of each food sample was determined from the slope of the

linear portion of the dose-response curve by linear-regression analysis, using the software

program Prisma 4.0 (GraphPad Software Inc., San Diego, CA, USA). In addition to the

requirement for a statistically significant (P < 0.05) dose-response effect, only those samples

were considered mutagenic whose highest test concentration generated at least twice as many

revertants as the negative control (DMSO). For proper interpretation and clarity, the number of

revertants obtained was compared with both their experiment-specific controls and aggregate

controls across all experiments. The P values of these comparisons in the tables, figures and

supplementary tables are derived from the regression analyses. In the oestrogenic activity assays,

the fold induction, fold induction-corrected (FIC) and limit of detection (LOD) were calculated,

as described previously (Leskinen et al., 2005). The sigmoidal dose-response curves for

increasing concentrations of oestradiol and BPA were obtained, using Prisma 4.0. The oestradiol

and BPA equivalents of the food samples showing oestrogenic activity were calculated from the

probit transformation of the curves.

5.0 RESULTS

5.1 Recovery experiment

The percentage recoveries of the chemicals added in the spiking experiments were

relatively high, ranging from 68 % to 89 % (Table 5.1). They were also comparably high

between the alternative methods employed.

Table 5.1 Percentage recoveries from the spiking experiments.

Extraction method Test substance % recovery

SPE (food samples) BaP 73

SPE (food samples) 2-aminoanthracene 71

SE (food samples) BaP 89

SE (food samples) 2-aminoanthracene 77

SPE (water samples) BPA 72

LLE (water samples) BPA 68

Key: SPE: solid-phase extraction; SE: soxlet extraction; LLE: liquid-liquid extraction

5.2 Mutagenicity test results

5.2i Standard plate incorporation assays

The mutagenic activity of the commercially processed food items expressed as the

number of revertants per gram original product, obtained by the standard plate incorporation

assay, are presented in Tables 2a, b, 3a, b (II) and Tables 2 and 3 (III) for the Finnish and

Nigerian food samples, respectively. Both commercially processed food items and ready-to-eat

snacks in Finland generally exhibited fairly low levels of mutagenic activity, in both Salmonella

TA 100 and TA 98 (II). Extracts of industrially processed grilled chicken, smoked fish, cold-

smoked fish, grilled beef, French fries, mashed potato, hamburger (beef) and hamburger

(chicken) showed no discernible mutagenic responses in any of the three batches examined in

either strain of Salmonella, regardless of the S9 status. Meanwhile, extracts from seven food

products (smoked chicken, honey-roasted chicken, grilled turkey, cold-smoked beef, cold-

smoked fish, sausage and pepper salami) generated revertants at least twice as high as those of

the negative control (DMSO) in at least one of the three batches investigated, and in one or both

strains of Salmonella. Of interest was the extract of cold cuts of grilled turkey, which showed

clear mutagenic activity in all three batches examined with Salmonella TA 98 (Table 2b; II). In

two cases, this occurred without metabolic activation (S9 mix), and once in its presence. One of

these lots (batch 1) also proved mutagenic in TA 100, both in the presence and absence of the S9

mix. This particular batch also yielded a drastic mutagenic response in a modified Ames test (see

below). With the ready-to-eat snacks, three of the snack products examined generated a more

than twofold higher number of revertants than the control compounds, and produced a significant

dose response. Chicken nuggets were particularly mutagenic in all the batches examined with

Salmonella TA 100, mainly in the presence of the S9 mix. In Salmonella TA 98, the numbers of

revertants produced by chicken nuggets were either marginally elevated or, if they were over

twice as high as the control, lacked a significant dose response (Table 3b; II).

The results from commercially processed food items in Nigeria were more

alarming and revealing, since the majority of food samples investigated (75 %) exhibited fairly

high mutagenic activity (the maximal response being comparable to those elicited by the positive

control, BaP), mainly in the Salmonella TA 100 strain (III). Chin-chin, hamburger, suya and

bean cake were the most mutagenic. For all of these food products, the number of revertants

generated was over two-fold as that of DMSO in all the batches analysed and mostly

independent of metabolic activation. A somewhat surprising result was found with extracts of the

potato products (French fries and potato chips) from Nigeria, since at least one of the lots of both

products proved directly mutagenic with the TA 100 strain (III). This was certainly not the case

with similar food products in Finland (II). Meanwhile, roasted maize, plantain chips and coconut

candy were the only food products without mutagenic activity in the TA 100 strain (Table 2; III).

In Salmonella TA 98, only three food or snack varieties (potato chips, peanut and suya)

exhibited mutagenic potency in at least one of the batches investigated (Table 3; III). Suya

displayed the most consistent outcome, with all its batches being mutagenic in the presence of

the S9 mix. In support of the result with TA 100, the same batch of potato chips (number 3) also

exhibited direct mutagenicity in TA 98.

5.2ii Modified Ames test

To ascertain to what degree a localized release of proteins, peptides or histidine

contributed to the mutagenicity test results obtained with the standard plate incorporation assay,

‘treat-and-wash’ as well as MC overlay assays were performed. The outcome was dependent on

the bacterial strain, type of food, S9 status and assay. For some food extracts initially mutagenic

in the standard plate incorporation assay, the number of revertants decreased below the two-fold

limit level in comparison with the negative control. Hence, the original Ames test result was, in

these cases, interpreted to be of secondary nature and not due to genuine mutations. For some

commercially processed Finnish food items and ready-to-eat snacks, the complementary assays

tended to reduce the number of revertants per gram obtained, compared with the results obtained

with the standard plate incorporation assay (II). Several surprises also emerged in these

complementary assays with the Finnish food samples, since extracts of industrially processed

food (cold cuts of smoked chicken, honey-roasted chicken and cold-smoked beef) that initially

required metabolic activation for their mutagenicity in the standard plate incorporation assay

were, unexpectedly, directly mutagenic with the Salmonella TA 100 strain in the MC overlay

assay (Supplementary Table 2; II). One of these samples (cold-smoked beef) also showed similar

results in the treat-and-wash assay (Supplementary Table 6; II). Interestingly, none of the ready-

to-eat food samples exhibited any form of mutagenicity in either of these auxiliary assays with

Salmonella TA 100 (Supplementary Tables 4 and 8; II) in contrast to their outcome in the

standard plate incorporation assay.

The most striking result was obtained with an extract of industrially processed cold

cuts of grilled turkey (batch 1). Although this item showed clear mutagenic activity in all three

assays both in the presence and absence of the S9 mix with the Salmonella TA 100 strain, both

the MC overlay and treat-and-wash procedures tended to diminish the revertant number,

compared with the standard assay (Figure 1B; II). With the Salmonella TA 98 strain, however,

by far the most conspicuous mutagenic response was recorded in the treat-and-wash assay, in

which it produced revertants over 13- and 5-fold higher than its control with and without the S9

mix, respectively (Supplementary Table 7 and Figure 2; II). The two other batches of grilled

turkey were also mutagenic with the TA 98 strain in this assay, both in the presence and absence

of the S9 mix, but no such drastic enhancement by the treat-and-wash procedure was seen

(Supplementary Table 7; II).

In contrast to the situation in Finland, a large number of food items from Nigeria

were mutagenic in all three assays, both in the presence and absence of the S9 mix. For some

food items (hamburger, suya and bean cake), a single lot was mutagenic in all three assays, but

only in the presence of the S9 mix. In the absence of S9, the outcome with these three products

varied. In contrast to this pattern, a single batch of hamburger (batch 2) was mutagenic in all

three assays, both in the presence and absence of the S9 mix. Meanwhile, extracts of fried

chicken (batch 2) and bean cake (batch 2) that required metabolic activation for their

mutagenicity in the standard plate incorporation assay were, unexpectedly, directly mutagenic in

the treat-and-wash assay in the Salmonella TA 100 strain (Table 4; III). One of these samples

(bean cake, batch 2) also showed similar results in the MC overlay assay (Table 4; III).

5.3 Cytotoxicity assay: Trypan blue exclusion assay

The percentage viability of the HepG2 cells in the trypan blue exclusion test

exhibited an inverse correlation with the concentration of some food extracts (smoked chicken,

grilled turkey and cold-smoked beef) from Finland (II), following a 48-h exposure. Meanwhile,

the non-survival percentage of the HepG2 cells following treatment with extracts of

commercially processed food items from Finland and Nigeria in all cases did not exceed 50% (II

and III) following 4-, 24- or 48-h exposures. Therefore, they were considered non-cytotoxic in

this assay at all time points.

5.4 Cytotoxicity assay: LDH assay

In both studies (II and III), there was a direct correlation between the test samples

and LDH release. However, the maximum released never exceeded 25% of that induced by the

positive control substance applied (lysis solution). Hence, the extracts were classified as non-

cytotoxic in this assay, following exposure for 4, 24 or 48 h.

5.5 Cytotoxicity assay: Boar sperm motility assay

The effect of the food extracts on boar sperm motility was also investigated,

following a 4-h exposure. However, none of the food extracts tested affected boar sperm motility

in a statistically significant manner.

5.6 Comet assay

As a follow-up of the results obtained in the conventional Ames test, samples

showing mutagenic potential in at least one strain of Salmonella were screened for single-strand

breaks, DNA-DNA/DNA-protein cross-linking and alkali-labile sites by the comet assay. None

of the samples investigated tested positive when compared with both positive and negative

controls. In the positive controls (treatment with hydrogen peroxide), the expected DNA

migration towards the anode was observed by microscopic examination.

5.7 GC-MS/MS

The majority of Finnish food samples produced low levels of PAHs (BaP, BaA, Ch

and BbF), except for a single batch of smoked fish (Table 5.2). In this lot, all four PAHs,

exceeded the acceptable limit of 5 μg/kg. The concentration of BaP was 8.2 μg/kg, while those

of BaA and Ch were three-fold greater than the acceptable limit. For another lot of this same

product, the levels of all PAHs were less striking, slightly lower than the acceptable limit.

Tab

le 5

.2 L

evel

s of

PA

Hs

in c

omm

erci

ally

pro

cess

ed F

inni

sh f

oods

and

thei

r m

utag

enic

pot

entia

l onl

y in

the

pres

ence

of

the

S9 m

ix.

P

AH

s (μ

g/kg

)

A

mes

test

(re

vt/g

)

Food

item

BaP

B

aA

Ch

BbF

Su

m

TA

100

T

A98

Sm

oked

ham

N

D

< L

OQ

<

LO

Q

ND

0

201

± 9.

1 34

±1.

5

N

D

ND

<

LO

Q

ND

0

174

±12

.5

32 ±

0.8

N

D

ND

<

LO

Q

ND

0

189

±7.

9 37

±4.

1 H

oney

-roa

sted

chi

cken

N

D

< L

OQ

<

LO

Q

ND

0

247

±11

.0

25 ±

2.1

ND

N

D

< L

OQ

N

D

0 19

8 ±

15.0

41

±6.

4

<

LO

Q

< L

OQ

<

LO

Q

ND

0

258

±9.

3 34

±4.

0

Gri

lled

turk

ey

ND

0.

81

0.84

N

D

1.60

29

7 ±

19.8

45

±0.

0

N

D

ND

N

D

ND

0

225

±0.

0 39

± 3

.4

ND

N

D

< L

OQ

N

D

0 18

8 ±

10.1

27

± 1

.5

Pepp

er s

alam

i N

D

< L

OQ

0.

88

ND

0.

88

241

± 14

.6

32 ±

4.9

N

D

< L

OQ

<

LO

Q

ND

0

168

± 8.

7 30

± 0

.6

ND

<

LO

Q

< L

OQ

N

D

0 20

0 ±

4.9

34 ±

3.2

C

old-

smok

ed b

eef

ND

<

LO

Q

< L

OQ

N

D

0 20

9 ±

5.1

39 ±

5.0

<

LO

Q

< L

OQ

<

LO

Q

ND

0

188

± 8.

7 28

±0.

0

N

D

ND

<

LO

Q

ND

0

188

± 0.

0 29

±2.

1 S

auna

-sm

oked

ham

N

A

NA

N

A

NA

N

A

NA

N

A

ND

N

D

< L

OQ

N

D

0 26

8 ±

11.2

42

± 1

.2

ND

N

D

< L

OQ

N

D

0 15

8 ±

3.8

28 ±

1.2

Smok

ed f

ish

4.

7 4.

5 4.

7 4.

5 18

.40

392

±12

.0*

51 ±

4.7

*

8.

2 15

15

5.

8 44

47

8 ±

41.2

* 64

± 4

.9*

1.

0 3.

9 3.

0 0.

8 8.

7 40

1 ±

22.8

* 45

± 2

.0*

Key

: N

D:

Not

de

tect

ed;

LO

Q:

Lim

it of

qu

antif

icat

ion

(0.7

8 μ

g/kg

);

LO

D:

Lim

it of

de

tect

ion

(0.2

6 μ

g/kg

);

NA

: N

ot

appl

icab

le;

aste

risk

(*):

Sig

nifi

cant

ly d

iffe

rent

fro

m c

ontr

ol (

P <

0.0

5). S

um: T

he to

tal s

um o

f be

nzo[

a]py

rene

, ben

zo[a

]ant

hrac

ene,

chr

ysen

e an

d be

nzo[

b]fl

uora

nthe

ne.

5.8 Oestrogenic activity assay: Commercially processed food

The majority of commercially processed food samples and ready-to-eat snacks in

Finland failed to exhibit detectable oestrogenic activity. However, extracts of industrially processed

and packaged hamburgers (beef and chicken) and of pepper salami were consistently oestrogenic in

all batches examined (Table 2; I). Pepper salami showed the most potent oestrogenic activity, with

oestradiol equivalents varying between 1.6 and 443 pg/g. Extracts of chicken hamburger were the

least oestrogenic of the positive samples (0.2--0.6 pg/g), while the oestradiol equivalents of beef

hamburger fell between these two. There was wide variation in the oestrogenic activities among

different batches of the same products, amounting to almost 1000-fold for pepper salami.

Meanwhile, equivalent burger products obtained from a hamburger restaurant showed no

oestrogenic activity in the test system. When it was revealed that all the positive food samples

contained a soy-based ingredient (Table 3; I), while none of the negative samples purchased from

the supermarket harboured it, two different brands of soy sauces were examined to verify soy

oestrogenicity in this assay system; both generated an intense signal (Table 4; I).

5.9 Oestrogenic activity assay: Bottled water, mineral water, tap and sachet-

pure water

In all, 31% of the sachet-pure water samples from Nigeria were oestrogenic in this

assay. The oestrogenicity of the positive samples ranged from 0.79 to 44.0 ng/l (median: 23.0 ng/l)

oestradiol equivalent concentrations (EEQs). In contrast, no brands of bottled still and mineral

waters, as well as tap water, from Finland yielded any oestrogenic activity (IV).

5.10 Oestrogenic activity assay: Food packaging material

Commercially processed food samples that were non-oestrogenic in the assay were

repackaged in the packaging materials of the positive oestrogenic samples of equivalent products to

ascertain whether the oestrogenic activities in these foodstuffs actually originated from the

wrappers. To complement this approach, the oestrogenic activities of the wrappers of all positive

food samples were also directly determined. Both the repackaged Finnish food items and the

wrappers exhibited no oestrogenic activity in our test system (I). Meanwhile, 40% of the sachet-

pure water samples generated positive signals in the yeast-based assay (Table 8; III).

5.11 Oestrogenic activity assay: Influent and effluent samples

The oestrogenic activities of the influent samples from the WWTP were low

(approximately 0.5 ng/l EEQ) throughout this period, except for 2 months in 2011, March and

August, when they peaked at 14 and 7.8 ng/l, respectively (Table 2; IV). The influent and effluent

samples from an equivalent household water purification plant, as well as all treated effluent waters

from the WWTP, likewise showed no oestrogenic activity in this test system.

6.0 Discussion

Commercially processed food and drinking-water sources are prerequisites for human

life and are consumed in increasing amounts globally. Therefore, it is of the utmost importance to

ensure that, in addition to their microbiological safety, they do not contain chemicals that may pose

a toxicological risk to consumer health. A conceivable potential risk in this regard is the presence of

genotoxic compounds deliberately added to, inadvertently contaminating or arising in the

processing of food. Environmental exposure to xenoestrogens also occurs via indiscriminate

discharge of inadequately treated sewage water into lakes and streams, and the terrestrial

environment. Regular screening studies are necessary to verify that the methods used by food

vendors, bottled/sachet water companies and in the treatment of influent and drinking water are also

appropriate and sound from this point of view. The present investigation was aimed at exploring the

current situation in Finland and Nigeria.

6.1 Recoveries and sample representativeness

As observed in 4.7 and 4.8 above, two different methods each were used for the

extraction of food and water samples in both countries, due to differences in the types of laboratory

material available. However, the recoveries of potentially mutagenic and oestrogenic compounds in

the two assays were comparable. Both methods have also previously been used for the extraction of

mutagenic and oestrogenic compounds in food and water samples.

The carefully selected Finnish food products investigated represent those commonly

consumed in Finland and probably in Europe. The sampling covered food products processed by

various methods (grilled, smoked, cold-smoked and fried) and of differing origins (fish, meat and

plant). Ready-to-eat snacks from a fast-food centre were also examined to cover a broader range of

commonly consumed food products (II). In Nigeria, the situation is different from that in Finland

regarding the types of food consumed, and Nigerian foodstuffs do not faithfully represent the

varieties commonly consumed elsewhere in Africa. The food products analysed encompassed

snacks, quick meals and delicacies, which are widely consumed across all social classes. Bean cake,

for example, is a popular quick meal among most Nigerians, and its preparation is time-consuming.

Ready-to-eat bean cake vendors are preferably patronized, based on the customer’s perceived

hygienic standards of the food handlers, and not on the processing technique, although this food

item can only be prepared by deep frying. The same also holds for the special delicacy suya (red

meat processed by direct smoking with wood or coal).

6.2 Mutagenicity studies of commercially processed food

The Ames test is a rapid in vitro assay for detection of the genotoxic potential of

chemicals (Maron and Ames, 1983) and has a high predictability for rodent carcinogens (McCann

et al., 1975; Zeiger, 1998; Mortelmans and Zeiger, 2000; Hakura et al., 2005). Although there is

evidence that this test system does not detect some genotoxic chemicals, such as AA, either in the

presence or absence of metabolic activation (Knaap et al., 1988; Dearfield et al., 1995), it is still

considered reliable for the detection of most mutagens and potentially genotoxic chemicals (Hakura

et al., 2005). Metabolic activation is also regarded as a critical step in mutation (Guengerich, 2000),

since many potential carcinogens remain inactive until they are enzymatically transformed into

electrophilic species that are capable of covalently binding to DNA and thereby leading to mutation.

To this end, the rat Arochlor-induced liver S9 fraction offers a more complete representation of the

metabolic profile than other S9 fractions (Hakura et al., 2005), because it contains both phase I and

phase II activities (Brandon et al., 2003).

The mutagenic activity observed in this study with some extracts of commercially

processed food items in Finland and Nigeria showed that such products cannot be completely

ignored as health hazards. The levels/number of revertants per gram obtained for some food items

may be of toxicological significance, due to the magnitude of mutagenic activities (2.5 13-fold

over those obtained with negative controls). For some food products (both in Finland and Nigeria),

there was a notable (to over 10-fold) variation in mutagenic potencies between batches and among

equivalent products and various products processed in the same way. There was also variation in

mutagenic activity among equivalent food products with the three assays (standard plate

incorporation, MC overlay and treat-and-wash assays), casting doubt on the significance of some

findings. Only a fraction of the commercially processed food items in Finland (smoked chicken,

grilled turkey and cold-smoked beef), initially mutagenic in the standard plate incorporation assay,

were also mutagenic in the complementary assays. In these cases, the mutagenic effect was

considered genuine (i.e. not secondary to histidine release from the food products) and thus of

concern. Meanwhile, the mutagenicity test results of processed food items from Nigeria gave

insight into the probable differences in processing techniques used in the developed and developing

countries. A total of 75% of commercially processed food items in Nigeria were mutagenic in the

standard plate incorporation assay with the TA 100 strain; likewise, this was almost the case in the

complementary assays as well. This is in contrast to the study of commercial food items in Finland,

where generally only a single batch of each product was mutagenic.

Considered together with a study published in Finland over 20 years ago (Tikkanen,

1991), the current findings imply that processing techniques in Finland have improved remarkably,

because the majority of food items investigated in that study were mutagenic, which is in contrast

to that reported here (II). It is also noteworthy that Tikkanen (1991) did not consider the possible

release of histidine from the food products. In that study and in others, only Salmonella TA 98 was

used, and sometimes only in the presence of the S9 mix. Based on findings from the present study,

TA 98 is far less sensitive to food mutagens than is TA 100.

The low levels of mutagenic activities observed in Finnish food samples were

surprising and somewhat unexpected, compared with the results of Nigerian food items, foodstuffs

from elsewhere (Stavric et al., 1995; Sharif et al., 2008) and previously in Finland (Tikkanen,

1991). To this end, an independent chemical analysis of the four principal PAHs was carried out on

similar food products (three lots of each) previously examined in the mutagenicity assays, albeit

using a slightly different method of extraction. Interestingly, the results of the chemical analysis

lend further credence to the mutagenicity test results, because the majority of food items contained

non-detectable levels of PAHs. This finding reinforces the view that the food industry in Finland

has recognized its responsibility and taken appropriate steps towards producing products that do not

pose genotoxic hazards to consumers. However, much still needs to be done with the various

smoking techniques. In accordance with the results of the mutagenicity assays, the majority of food

items did not contain significant levels of PAHs, while those harbouring any of the mutagenic

PAHs showed variation among batches of the same and equivalent products. All meat products

(smoked ham, honey-roasted chicken, grilled turkey, pepper salami, cold-smoked beef and sauna-

smoked pork) mostly contained PAHs below the limit of quantification (LOQ) and LOD of 0.78

and 0.26 μg/kg, respectively. Djinovic et al. (2008) reported similar findings from Serbia, while in

Sweden a significant number of processed meat and meat products (9 out of 38 ) contained BaP,

ranging from 6.6 to 36.9 μg/kg, exceeding the 5.0 μg/kg maximum level established by the EU

(Wretling et al., 2010). Elsewhere, varying concentrations of PAHs have been reported in

commercially processed meat, meat products, fish and fish products (Farhadian et al., 2010;

Alomirah et al., 2011; Chung et al., 2011; Essumang et al., 2012; Aaslyng et al., 2013). The results

of both the Ames test and GC-MS/MS analysis in Finland are in agreement with two recent

independent studies (Jägerstad and Skog, 2005; EFSA, 2008). Jägerstad and Skog (2005) reported

that the daily intake of nitrosodimethylamine was significantly lower in Finland (0.08 μg/day) than

in other European countries (0.12–0.38 μg/day). A report submitted to EFSA by 16 participating

EU countries and compiled/released in 2008 showed that the total dietary exposure to BaP was also

lower (185 ng/day) in Finland than in other participating countries (188 255 ng/day). For PAH2,

PAH4 and PAH8, the levels obtained in Finland were slightly higher than those recorded in the

United Kingdom (EU country with the lowest levels of PAH2, PAH4 and PAH8).

As for the Nigerian food samples, the cause of the high number of revertants obtained

with most food products is suggestive. The processing techniques (charcoal-grilled, deep-frying)

commonly used in Nigeria have been implicated in generating high levels of both PAHs and HAAs

in the final products (Iwasaki et al., 2010; Liao et al., 2010, 2012; Chung et al., 2011; Essumang et

al., 2012). Double-heat treatment/reuse of cooking oil, which is always the case in Nigeria and

probably in most African countries, results in an increase in the genotoxic activity of food products

(Isidor and Parrella, 2009; Srivastava et al., 2010). During frying, cooking oil undergoes

deterioration through various chemical and physical processes such as oxidation, polymerization,

hydrolysis and cyclization, leading to the formation of both volatile and nonvolatile undesirable by-

products (Isidor and Parrella, 2009). These derivatives are partially absorbed by the fried food,

which thus becomes carcinogenic (Alomirah et al., 2010). For example, the PAH compounds BaP,

BaA and Ch are all well-known human carcinogens that have been detected in different types of

cooking oil (Alomirah et al., 2010). Meat and fish products grilled and/or smoked traditionally are

also usually heavily contaminated with the PAH compound BaP (Akpambang et al., 2009).

Most of the food items (except suya) investigated in this study have not been

previously screened for either PAHs or HAAs in Nigeria, and the PAH compound BaP was first

reported in suya (8.5 μg/kg) from Nigeria in 1982 (Bababunmi et al., 1982). The probable reason

for the high level in that case, in addition to the processing method, was the use of car tyres for

smoking. This method also increased the levels of PAHs in food products in Ghana, compared with

other methods (Abdul et al., 2014). After the initial study by Bababunmi et al. (1982), even higher

levels of PAHs have been reported. Duke and Albert (2007) found BaP contents ranging from 6.5 to

21.5 μg/kg in suya meat from four different selling points in the Niger Delta axis of Nigeria, while

Akpambang et al. (2009) reported the levels of BaP, Ch, BbF and BaA in commercially processed

suya in Nigeria to be 10.1, 25.6, 12.3 and 15.4 μg/kg dry weight, respectively. More recently,

Amos-Tauta et al. (2013) reported a mean concentration of BaA in suya of 7.23 μg/kg. In all of

these studies, the concentrations of all PAHs were greater than the acceptable limit of 5.0 μg/kg.

6.3 Genotoxicity of drinking water

Considerable mutagenicity in drinking water in Finland has previously been reported

(Vartiainen and Liimatainen, 1986; Vartiainen et al., 1988). This was largely attributed to high

levels of the by-products of disinfection (mainly chlorination) stemming from chemical reactions

with humic substances. For example, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and

several other chlorinated hydroxyl furanones were found in 71% of Finnish drinking-water samples

with mutagenic outcomes (Kronberg and Vartianien, 1988; Smeds et. al., 1997). Consequently, a

linear relationship was reported between exposure to Finnish drinking-water mutagenicity and the

risk of bladder, kidney, stomach and pancreatic cancers, as well as lymphomas in people consuming

the water (Koivusalo et al., 1994, 1995). Realization of these ensuing health risks led to substantial

changes in water treatment practices by increased use of artificial groundwater, chloramine

disinfection and ozonation, use of chlorine dioxide as post-disinfectant, as well as improved

coagulation/flocculation techniques, thereby rapidly decreasing mutagenicity (Nissinen et al.,

2002). Two recent studies in Nigeria also reported drinking-water sources to be genotoxic

(Adelanwa et al., 2011; Olorunfemi et al., 2014). Thus, household water can also be a genotoxic

risk factor, but it was not specifically addressed in this thesis.

6.4 Oestrogenic activities of commercially processed food items and drinking

water

We investigated the presence of xenoestrogens in commercially processed food items

(mainly meat and fish products) arising from substances deliberately added to or inadvertently

contaminating the food, formed as a result of food processing or leaching from food packaging

materials. Overall, food packaging materials do not seem likely to be a notable source of human

exposure to xenoestrogens through intake/consumption of commercially processed Finnish foods.

In addition, no significant oestrogenic activity emerged as a result of processing. However, three

varieties of commercially processed Finnish food items tested positive in this assay. Interestingly,

they all shared one property: a soy-based ingredient. The oestrogenic activity of soy as well as of

soy-based products has been reported previously (Takamura-Enya et al., 2003; Behr et al., 2011).

Since the two soy sauces analysed in this study were also potentially oestrogenic (I), It was

concluded that the soy-based components are likely to be at least a contributing factor for the

oestrogenic activity in these food products (pepper salami, chicken and beef hamburger). The

calculated oestradiol and genistein equivalents of the soy-containing products investigated present

pepper salami as the most oestrogenic of the three, with its oestradiol equivalents ranging from 1.6

to 443 pg/g. Although no information is available on the oestrogenic activity of pepper salami, its

oestradiol equivalent recorded here is almost of the same magnitude (150 1544 ng/kg original

product) obtained for related products (Behr et al., 2011). Soy-free products were also reported to

be non-oestrogenic in their test system. As for soy-based hamburgers, the oestradiol equivalent

levels obtained here are similar to (although slightly lower than) those found by Behr et al. (2011).

In Nigeria, drinking water is mainly sold in small polyethylene bags and is usually

referred to by the populace as sachet-pure water. To the best of my knowledge, this is the first study

to investigate the possible oestrogenic activity of sachet-packed drinking water in Nigeria. The

microbiological and physiochemical quality of sachet-packed water in Nigeria has been studied

quite extensively (Orisakwe et al., 2006; Olaoye and Onilude, 2009; Oyedeji et al., 2010; Edema et

al., 2011; Adebayo et al., 2012; Muazu et al., 2012; Omalu et al., 2012; Onilude et al., 2013). All of

these studies reported that the majority of sachet-packed pure waters sold in Nigeria were heavily

tainted by pathogens and, therefore, unsafe for consumption. A similar situation has also been

reported in sachet waters from Ghana (Addo et al., 2009; Ackah et al., 2012). Two studies from

both countries particularly reported high levels of lead, far above the recommended limit (Orisakwe

et al., 2006; Ackah et al., 2012).

The outcome of this study shows that sachet-packed pure water may also contain

oestrogen-like chemicals. A total of 31% of such water samples investigated were oestrogenic, with

EEQs ranging from 0.79 to 44.0 ng/l. However, both the frequency of positive samples and their

concentrations were actually lower than was feared. In recent studies carried out in Europe, using a

comparable in vitro yeast assay, Pinto and Reali (2009) analysed mineral waters packed in

polyethylene terephthalate (PET) bottles in Italy. The levels they detected varied from 0.03 to 23.1

ng/l (mode 9.5 ng/l) EEQs. Somewhat surprisingly, tap water comprising either surface water or

groundwater contained approximately 15 ng/l EEQ. In another study, Wagner and Oehlmann

(2009) determined the oestrogenic activities in 20 major brands of bottled water in Germany.

Twelve of these samples proved positive, with the levels ranging from 2.64 to 75.2 ng EEQ/l (mean

18.0 ng/l). More recently, Real et al. (2015) reported that 79.3% of the bottled water (including

water in plastic and glass) in southern Spain was oestrogenic (mean: 0.113 ± 0.07 pmol/l oestradiol-

17 equivalent E2EQ). Thus, substances exhibiting oestrogen-like activity are common in water

samples in both the industrialized and developing countries. Despite the above information, water

samples taken from drinking-water sources (tap water, bottled still and mineral water) and drinking-

water plants (treated and untreated) in Finland again showed no notable oestrogenic activity. This

outcome is similar to three recent findings in South America, Europe and Asia. Using a similar in

vitro assay and by complimentary chemical analysis, no oestrogenic activity was reported in tap

water in Brazil (Bergamasco et al., 2011). Similarly, Maggioni et al. (2013) observed no

oestrogenic activity in five different brands of PET-bottled mineral water in Italy. In contrast,

Zheng et al. (2013) reported oestrogenic activity in tap water collected over a 1-year period in

China, ranging from 35.2 to 1511 pg/l EEQ. The negative outcome of the Finnish study stems from

legislation, favourable administration, extensive research and follow-up approaches aimed at

sustainable use of clean water resources (Ministry of Agriculture and Forestry, Finland, 2009). In

2002, Finnish drinking-water quality was ranked by the United Nations as the best in the world

(World Water Council, 2003), which is in line with the current findings.

The contamination of drinking water by oestrogenic chemicals is multifaceted. These

chemicals may arise from plastic or glass bottles, bottle caps, transport pipelines, disinfection

agents, the bottling process itself or from environmental pollution of water sources (Real et al.,

2015). A recent study in the southeastern USA showed that drinking-water sources may be

contaminated by EDCs, with average total concentrations of pharmaceutical and personal care

products and EDCs averaging 360 and 98 ng/l in source water and drinking water, respectively

(Padhye et al., 2014). A similar situation was also reported in China (Hu et al., 2013). Exposure of

PET-bottled water to sunlight and high temperature (60 °C) in the presence of carbon dioxide

increases the migration of substances (formaldehyde, acetaldehyde and antimony) from PET bottles

into drinking water (Bach et al., 2014, 2013). Antimony was previously reported in water from PET

bottles (Shotyk et al., 2006) and is said to be oestrogenic in vitro (Sax, 2010).

6.5 Oestrogenic activity of food packaging material

Food packaging materials are a putative and controversial source of human exposure

to xenoestrogens globally (Brotons et al., 1995; Stroheker et al., 2003; ter Veld et al., 2006). The

oestrogenic activity of packaging materials results from the leaching of plasticizers used in FCMs.

Several of these plasticizers, such as a tris(2-ethylhexyl)trimellitate and benzoate mixture, are

oestrogenic (ter Veld et al., 2006). However, in the material consisting of commercially processed

and packaged food items in Finland, no evidence of this was obtained (I), except for sachet

materials used in packaging pure water in Nigeria (III). The latter finding suggests the presence of

oestrogenic compounds in the packaging material. These compounds usually leach under normal

use conditions (ter Veld et al., 2006; Vandenberg et al., 2007; Le et al., 2008).

6.6 Oestrogenic activity of wastewater samples

The concentration rates found in influent samples from the Viikinmäki WWTP were

lower than those reported elsewhere. Using a similar in vitro bioreporter assay, influent wastewater

samples showed oestrogenic activity ranging from below the detection limit to 25 ng/l EEQ in

France (Bellet et al. 2012), while a recent report revealed strikingly high levels [1136 ± 269 ng/l

EEQ] in China (Zhao et al., 2015). In the latter study, the value increased (1417 ± 320 ng/l EEQ)

after primary treatment. In Spain, Germany and China, the concentrations measured by MS were

also far higher than those determined in the present study (Petrovic et al., 2002; Andersen et al.,

2003; Zhou et al., 2012). Varying levels of EDCs and oestrogenicity have also been reported in

effluent samples globally (Petrovic et al., 2002; Andersen et al., 2003; Smit et al., 2011; Ahn et al.,

2012; Bazin et al., 2012; Jarosova et al., 2012; Schiliro et al., 2012; Ferguson et al., 2013).

However, this is in contrast to the case in Finland. The oestrogenic outcome of the effluent samples

reported here is in accordance with a recent study from 16 European countries, including Finland, in

which effluent samples were screened for possible oestrogenic activity (Jarosova et al., 2014). This

implies that the treatment method (activated sludge with mechanical, chemical and biological

purification) currently employed in Helsinki is effective in removing oestrogenic compounds from

wastewater during treatment. Activated sludge and/or an upflow anaerobic sludge blanket reactor,

followed by chlorination steps, were also effective in removing EDCs from wastewater (Rahman et

al., 2009; Pessoa et al., 2014).

In Nigeria, there are no working sewage treatment plants (Daily Trust, 2014).

Industrial and domestic wastewater collections are decentralized, with each individual building

having its respective septic tank for the collection of its waste. When such septic tanks are emptied,

the contents are often discharged untreated into bodies of water. These bodies of water serve as

water supplies for drinking and irrigation purposes. In addition to untreated waste discharged into

bodies of water, high levels of rainfall, agricultural runoff and increased use of pesticides in Nigeria

further increase exposure to toxic chemicals from bodies of water. Studies have shown varying

concentrations of insecticides and herbicides in surface and river waters (Behfar et al., 2013; Scott

et al., 2014; Stone et al., 2014; Agbohessi et al., 2015; Rose et al., 2015), which in Nigeria are

crude drinking-water sources and are also used for fishing. In particular, fish from such polluted

bodies of water were recently reported to contain high levels of pesticides (Agbohessi et al., 2015;

Rose et al., 2015), some of which are known to have oestrogenic potential (Agbohessi et al., 2015).

This implies that rivers in Nigeria could be a potential reason for concern of exposure to both

pathogenic organisms and oestrogenic chemicals.

7.0 Conclusions

We arrived at the following conclusions from the current studies:

1. Commercially processed foods are potential sources of human exposure to genotoxic chemicals.

These chemicals are often difficult to regulate/control, because they are formed in food as a result

of food processing. However, appreciable progress has been made in Finland towards reducing the

levels of these contaminants in commercial foodstuffs.

2. In Nigeria, much still needs to be done, since the majority of food items (chin-chin, hamburger,

suya and bean cake) investigated were proven to be mutagenic. The dissimilar mutagenic outcome

in the two countries may largely be due to differences in processing techniques.

3. Drinking-water sources (tap water, bottled still and mineral waters) and water from drinking-

water treatment plants in Finland are not sources of concern, with respect to their oestrogenic

potentials.

4. Meanwhile, sachet-pure water samples from Nigeria, as well as packaging materials, could pose

grave problems for consumers, because 31% of the samples were oestrogenic, of which 40% were

attributed to FCMs.

5. A 2-year study of both influent and effluent wastewater samples from Viikinmäki WWTP in

Finland showed that the treatment process (activated sludge coupled with mechanical, chemical and

biological purification) used in the treatment of wastewater is effective in removing oestrogenic

chemicals. In Nigeria, there are no centralized WWTPs. This may impair proper waste treatment

and also increases exposure to EDCs.

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Commercial processed food may have endocrine-disrupting potential: soy-based ingredients making thedifferenceIyekhoetin Matthew Omoruyia, Grit Kabierschb & Raimo Pohjanvirtaa

a Department of Food and Environmental Hygiene (Food and Environmental ToxicologyUnit), Faculty of Veterinary Medicine, University of Helsinki, F-00014 Helsinki, Finlandb Division of Microbiology, Department of Food and Environmental Sciences, Faculty ofAgriculture and Forestry, University of Helsinki, F-00014 Helsinki, FinlandPublished online: 26 Jul 2013.

To cite this article: Iyekhoetin Matthew Omoruyi, Grit Kabiersch & Raimo Pohjanvirta (2013) Commercial processed food mayhave endocrine-disrupting potential: soy-based ingredients making the difference, Food Additives & Contaminants: Part A,30:10, 1722-1727, DOI: 10.1080/19440049.2013.817025

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Commercial processed food may have endocrine-disrupting potential:soy-based ingredients making the difference

Iyekhoetin Matthew Omoruyia*, Grit Kabierschb and Raimo Pohjanvirtaa

aDepartment of Food and Environmental Hygiene (Food and Environmental Toxicology Unit), Faculty of Veterinary Medicine,University of Helsinki, F-00014 Helsinki, Finland; bDivision of Microbiology, Department of Food and Environmental Sciences,Faculty of Agriculture and Forestry, University of Helsinki, F-00014 Helsinki, Finland

(Received 7 April 2013; final version received 14 June 2013)

Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly characterised sources of humanexposure to endocrine-disrupting chemicals (EDCs). In this study we investigated the presence of xenoestrogens incommercially processed and packaged Finnish foods, arising from substances deliberately added or inadvertently contam-inating the food, substances formed as a result of food processing, or substances leaching from food packaging materials.Samples were obtained in three separate batches of equivalent products from both a supermarket and a local representativeof a global chain of hamburger restaurants and extracted by a solid-phase extraction method. Their endocrine-disruptingpotential was determined by yeast bioluminescent assay, using two recombinant yeast strains Saccharomyces cerevisiaeBMAEREluc/ERα and S. cerevisiae BMA64/luc. In this test system, the majority of samples (both foodstuffs and wrappers)analysed proved negative. However, all batches of industrially prepared hamburgers (but not those obtained from ahamburger restaurant) as well as pepper salami significantly induced luciferase activity in the BMAEREluc/ERα yeaststrain indicating the presence of xenoestrogens, with estradiol equivalents of these products ranging from 0.2 to 443 pg g–1.All three products contained soy-based ingredients, which apparently accounted for, or at least contributed to, their highestrogenic activity, since no signal in the assay was observed with extracts of the packaging material, while two differentsoy sauces tested yielded an intense signal (28 and 54 pg ml–1 estradiol-equivalent). These findings imply that by and largechemicals arising in the processing or packaging of foodstuffs in Finland constitute an insignificant source of xenoestrogensto consumers. However, soy-derived ingredients in certain food items might render the entire products highly estrogenic.The estrogenic activity of soy is attributed to isoflavones whose health effects – though widely considered beneficial – arecontroversial. As hamburgers are a popular type of food among children, the findings are noteworthy and possibly ofconcern.

Keywords: endocrine-disrupting compounds; processed food; ready-to-eat snacks; soy; xenoestrogens

Introduction

Certain compounds known to interfere adversely with thehuman endocrine system have been reported in theimmediate environment, especially in natural water bodies(Eggen et al. 2003; Sumpter 2005). Such substances areusually referred to as endocrine-disrupting chemicals(EDCs). EDCs that mimic the activity of estrogen withinthe human organism are ubiquitous, and have gainednotoriety due to their association with certain reproductivedisorders, developmental abnormalities and other adversephysiological effects in both humans and wildlife (Fisher2004; Bourguignon & Parent 2010; Zama & Uzumcu2010). Incidentally, estrogen mimics have also beenreported among chemicals used for processing and preser-ving food (Sinha et al. 2009; Connolly et al. 2011; Zhang,Jia et al. 2012) as well as in soy-based food products(Takamura-Enya et al. 2003; Behr et al. 2011). Foodpackaging materials are also reported sources of humanexposure to EDCs (Brotons et al. 1995; Stroheker et al.2003; ter-Veld et al. 2006). Known EDCs from food

packaging materials include bisphenol A, nonylphenoland several phthalates. Similarly, bottled mineral and fla-voured waters are also sources of exposure to EDCs(Plotan et al. 2012).

The overall presence of EDCs in the human diet isworrisome due to an increase in certain cancer types (breast,stomach, colon), especially in industrialised countries, andthe associated linkage between cancer and EDC exposure(Fisher 2004; Meeker 2010; Walvoord 2010). In Finland,for example, cancers of the oesophagus, stomach and colonappear to be on the increase (Weiderpass & Pukkala 2006).From the point of view of risk assessment, EDCs present aparticular challenge because they may have non-monoto-nous dose–response curves not adequately covered by con-ventional toxicological experimentation, and because oftheir capability of causing untoward impacts at environ-mentally prevailing low concentrations (Fagin 2012).

Despite the global concern and awareness of humanexposure to EDCs, processed food items and ready-to-eatsnacks are yet a poorly delineated source of such

*Corresponding author. Email: matthew.omoruyi@helsinki.fi

Food Additives & Contaminants: Part A, 2013Vol. 30, No. 10, 1722–1727, http://dx.doi.org/10.1080/19440049.2013.817025

© 2013 Taylor & Francis

exposure. This is evidenced by the limited number ofstudies done on the presence of EDCs in processedfoods. Moreover, such studies are totally lacking inFinland. Hence, the present work sought to investigatethe abundance of xenoestrogens in commercially pro-cessed Finnish foods (mainly meat and fish products),arising from substances deliberately added to or inadver-tently contaminating the food, substances formed as aresult of food processing, or substances leaching fromfood packaging materials.

Materials and methods

Chemicals and medium

Estradiol, progesterone, testosterone and bisphenol Awerepurchased from Sigma-Aldrich (Steinheim, Germany).D-luciferin was obtained from Biotherma (Handen,Sweden). Yeast nitrogen base medium without aminoacids was obtained from Becton Dickinson (FranklinLanes, NJ, USA) while genistein was obtained from LCLaboratories (Woburn, MA, USA).

Microorganisms

Two recombinant yeast strains Saccharomyces cerevi-siae BMAEREluc/ERα and S. cerevisiae BMA64/luc(Leskinen et al. 2005) were used. BMAEREluc/ERαserved as a reporter strain, in which the ERα isexpressed. Upon ligand binding, the dimerised receptorbinds the estrogen response elements in the promoterregion of the luc reporter gene. In BMA64/luc, lucifer-ase is expressed constitutively, and this strain was usedfor the determination of cytotoxicity of the samples.Both yeast strains were kindly provided by JohannaRajasärkkä of the Department of Food andEnvironmental Sciences, Faculty of Agriculture andForestry, University of Helsinki. Yeasts were grown onDifco Yeast Nitrogen Base medium without aminoacids, supplemented with glucose and the amino acidsalanine, histidine and leucine.

Samples

Forty-five samples (three lots of 15 different products) ofindustrially processed and packaged food products and 15samples of ready-to-eat snacks (three lots of five differentsnack varieties) were evaluated for their possible estro-genic activities. The industrially processed and packagedfood products were purchased from a popular supermarketin Helsinki (Prisma, Viikki), while the ready-to-eat snackswere acquired from a local representative (Sokos CityCentre) of a global chain of hamburger restaurants(McDonald’s) also located in Helsinki. The sample typesand manufacturers are listed in Table 1.

We carefully ensured that all products were extractedbefore the expiry date shown on the packages.

Extraction of food samples

Possible estrogenic compounds were extracted from foodsamples using the method described by Peters et al. (2004)with slight modifications. Briefly, 20 g of food samplewere homogenised with 80 ml of 1 M NaOH at24,000 rpm for approximately 10 min. The homogenatewas mixed with 20 g of Extrelut refill material (VWRinternational, Helsinki, Finland) and then poured into anempty Extrelut 20 column. The organics were eluted fromthe Extrelut column with 40 ml of dichloromethane/toluene solution (95:5 v/v) into a cartridge. The organicswere finally eluted with 5 ml of MeOH-NH4OH (9:1)solution and evaporated to dryness under a gentle streamof nitrogen. For determination of recovery, fresh meat wasspiked with 250 ng of estradiol and extracted in the sameway as above. The percentage recovery was approxi-mately 78%.

Repackaging and extraction of packaging materials

The samples found to be estrogenic were unwrappedand their packaging materials reused for negative sam-ples to disclose possible leaching of xenoestrogens. Therepackaging was done in such a way that there was aclose contact between the food sample and packagingmaterial. The repackaged food samples were kept in anoven at 80°C for 30 min and then at 4°C for 7 daysbefore extraction to maximise the leaching effect with-out spoiling the foodstuffs. For extraction, these packa-ging materials were embedded in methanol in a

Table 1. Industrially processed food items analysed and theirmanufacturers.

Product Manufacturer

Smoked chicken Company AHoney roasted chicken Company BGrilled chicken Company CGrilled turkey Company BSmoked fish Company DCold-smoked fish Company EFried fish Company ECold-smoked beef Company BGrilled beef Company CPepper salami Company FSausage Company GFrench fries Company GMashed potatoes Company CHamburger (beef) Company HHamburger (chicken) Company A

Food Additives & Contaminants: Part A 1723

mechanical shaker at 60°C overnight. This is a slightmodification of a previous method used with success toextract estrogenic compounds from food samples (Behret al. 2011).

Bioassay

The yeast bioluminescent assay was performed as pre-viously described (Leskinen et al. 2005; Rajasärkkä &Virta 2011). The optimised parameters for 384-well platesused in this study have also been reported (Rajasärkkä &Virta 2011), except that 5% ethanol was used as thevehicle because the 10% DMSO reported by Rajasärkkäand Virta (2011) was cytotoxic in the test system.Estradiol, genistein and bisphenol A were used as positivecontrols, while progesterone and testosterone served asnegative controls.

Data analysis

The fold induction, fold induction corrected (FIC) andLOD were calculated as described previously (Leskinenet al. 2005). The sigmoidal dose–response curves forincreasing concentrations of estradiol, genistein andbisphenol A were obtained using the software programPrism 4.0 (GraphPad Software, Inc., San Diego, CA,USA). The estradiol and genistein-equivalents of foodsamples showing estrogenic activity were calculatedfrom probit transformation of the curves.

Results

With S. cerevisiae BMAEREluc/ERα, all positive controls(estradiol, genistein and bisphenol A) produced sigmoidaldose–response curves (Figure 1). In this test system,

estradiol proved to be 103–105-fold as potent as bisphenolA and genistein. This is in agreement with data gatheredfrom other assays (Leskinen et al. 2005; ter Veld et al.2006; Rajasärkkä & Virta 2011). Meanwhile, there was noluciferase activity detectable in the BMAEREluc/ERαyeast strain when it was treated with increasing concentra-tions of either progesterone or testosterone (data notshown). The LOD in the estrogenic assay was 2.4 FICand corresponded to 76 fM, 1.8 pM and 1.2 nM ofestradiol, genistein and bisphenol A, respectively.

The majority of food samples analysed failed to exhi-bit detectable estrogenic activity. However, extracts ofindustrially processed and packaged hamburgers (beefand chicken) and of pepper salami were consistently estro-genic in all their batches examined (Table 2). Peppersalami showed the most potent activities, with its estra-diol-equivalent varying between 1.6 and 443 pg g–1.Extracts of chicken hamburger were the least estrogenicof the positive samples (0.2–0.6 pg g–1), while the estra-diol-equivalent of beef hamburger fell between the two.An important observation was the wide variation in theestrogenic activities among different batches of the sameproducts amounting to almost 1000-fold for pepper sal-ami. Meanwhile, equivalent burger products obtainedfrom a hamburger restaurant did not show any estrogenicactivity in the test system.

Samples previously found to be non-estrogenic in theassay where repackaged in the packaging materials ofpositive estrogenic samples of equivalent products toascertain if the estrogenic activities in these foodstuffsactually originated from the wrappers. To complementthis approach, the estrogenic activities of the wrappers ofall positive samples were also directly determined. Bothrepackaged food samples and the wrappers exhibited noestrogenic activity in the test system. When it turned outthat all the positive food samples contained a soy-basedingredient (Table 3) while none of the negative samplespurchased from the supermarket harboured it, two differ-ent brands of soy sauces were examined to verify soyestrogenicity in this assay system. They both generatedan intense signal (Table 4).

The recombinant yeast strain, S. cerevisiae BMA64/luc, produced an intense signal with all samples in theassay conducted, indicating the viability of the cells. Thisimplies that neither the vehicle (5% ethanol) used in thestudy nor the test samples were cytotoxic to the cells.

Discussion

The present study investigated the presence of xenoestro-gens in commercially processed Finnish foods (mainlymeat and fish products), arising from substances deliber-ately added to or inadvertently contaminating the food,substances formed as a result of food processing, or sub-stances leaching from food packaging materials. The

60

Estradiol

Bisphenol A

Genisten

50

40

30

20

10

00.1 1 10 100 1000 10,000

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d

100,000 1,000,000 1.0×1007

Figure 1. Dose–response curve of increasing concentrations ofestradiol, genistein and bisphenol A.

1724 I.M. Omoruyi et al.

results show that, overall, food packaging materials arenot likely to be a notable source of xenoestrogens incommercially processed Finnish foods. Also, we observedthat no significant estrogenic activity may have emergedas a result of processing. However, three different varietiesof foodstuffs tested positive in this assay. Interestingly,they all shared one property: a soy-based ingredient. Asthe two soy sauces analysed also proved potently estro-genic, the soy-based components are likely to be at least acontributing factor for the estrogenic activity in these foodproducts (pepper salami, chicken and beef hamburger)

obtained from a supermarket. The calculated estradioland genistein-equivalents of the soy-containing productsinvestigated present pepper salami as the most estrogenicof the three, with its estradiol-equivalents ranging from 1.6to 443 pg g–1. The estrogenic activity of soy as well as ofsoy-based products has been reported previously(Takamura-Enya et al. 2003; Behr et al. 2011), andstems from its isoflavones (mainly genistein) which arealso considered antioxidants. At present, controversyremains as to the health impacts of isoflavones. Whileepidemiological studies have shown that soy and soy-foods may protect against breast cancer and cardiovasculardiseases (Pilsakova et al. 2010; Zhang, Kang et al. 2012),experimental studies have suggested that soy isoflavonescould actually enhance the proliferation or metastasis ofsome types of cancer (Helferich et al. 2008; Martínez-Montemayer et al. 2010; de la Parra et al. 2012).Moreover, soy-based isoflavones have been reported toimpair fertility in female mice by neonatal exposure(Nagao et al. 2001; Jefferson et al. 2005), and enhancevaginal cell maturation in female human infants(Bernbaum et al. 2008). In male marmoset monkeys,feeding of infants with soy formula milk for 1.0–1.5 months decreased serum testosterone by 50–70% com-pared with co-twin counterparts fed on cow’s formulamilk (Sharpe et al. 2002).

Estradiol and several other sex hormones are widelyused as growth enhancers in cattle, especially in theUnited States, Australia and Canada (Preston 1999).Because estradiol is thousands of times more potent thanestrogenic pesticides and other industrial food contami-nants, its use as an implant in animals has largely beencondemned. Studies have shown that estradiol may pose arisk of serious negative effects at very low concentrations,especially in infants and young children (Saenz deRodriguez et al. 1985; Andersson & Skakkebaek 1999).This reinforces the view that the estradiol equivalencelevels found in this study may be of concern. TheUSFDA has established a maximum safe tissue level ofestradiol in muscle, liver, kidney and animal fat at 0.12,

Table 2. Estradiol and genistein-equivalent concentrations of samples showing estrogenic activity.

Samples

Estradiol-equivalent concentrations (pg g–1 original product)

Batch 1 Batch 2 Batch 3

Hamburger (beef) 0.8 12.9 0.7Hamburger (chicken) 0.2 0.6 0.3Pepper salami 13.6 1.6 443

Genistein-equivalent concentrations (ng g–1 original product)

Hamburger (beef) 12.8 3.6 × 105 5.8Hamburger (chicken) 0.09 3.4 0.2Pepper salami 4.4 × 105 1.6 × 102 n.a.

Note: n.a., Not applicable (>109).

Table 3. Ingredients present in food products showing estro-genic activities.

Beef hamburger Chicken hamburgerPeppersalami

Beef Broiler meat PorkPork Breaded chicken patty Horse meatWater Water LardSoy protein product Soy protein

preparationsSoy protein

Salt Salt SaltSpices and flavourings Spices SpicesBread crumbs Bread crumbs PreservativePotato Potato flakes AromaHalogenated vegetableoil

Potato flour Antioxidant

Stabiliser Canola oil Yeast extractGlucose Chicken fat Gelatin

Egg ThickenerOnion MaltodextrinLemon juiceconcentrate

Glucose

Table 4. Estradiol and genistein-equivalent concentrations ofsoy sauces.

Soy sauceEstradiol-equivalent

(pg ml–1 original product)Genistein-equivalent

(ng ml–1 original product)

Soy king 28.0 6.3 × 103

Aroi 54.0 7.4 × 107

Food Additives & Contaminants: Part A 1725

0.48, 0.36 and 0.24 μg kg–1, respectively (Doyle 2000).However, two dissenting reviews (Andersson &Skakkebaek 1999; SCVMRPH 1999) have expressed con-cern about the safety of such concentrations. Although theestradiol-equivalent concentrations detected in the presentstudy were mostly (except for a single batch of peppersalami) lower than the residual limits set by the USFDA,they suggest that for people favouring soy-based foodproducts in their nutrition the exposure to compoundswith estrogenic activity may be high.

In contrast to the hamburgers purchased from a super-market, the equivalent products (cheese, beef and chickenhamburgers) obtained from a hamburger restaurantproved, however, not to be estrogenic with theBMAEREluc/ERα yeast strain. Surprisingly, both cheeseburgers and beef hamburgers obtained from the restaurantwere informed to contain soy. A possible explanation forthe divergent outcome with soy-based burger productsobtained from a supermarket and a restaurant is a differentconcentration of the soy ingredients (this was not revealedin the product labels). The soy content might be so low inthe restaurant burgers that the estrogenic activity fellbelow the detection limit. In addition to the soy-basedingredient, pepper salami (which exhibited the highestestrogenic activity of all) contained an unidentified anti-oxidant (Table 4). This may further have contributed to theoutcome, since some antioxidants (propyl gallate, propa-nediolphosphite and butylated hydroxyanisole) have beenreported to be estrogenic in both ERα and ERβ reportergene cell lines (ter Veld et al. 2006). In agreement withthis, butylated hydroxytoluene and hydroxyanisole as wellas propyl gallate (compounds extensively use as antiox-idants) were recently demonstrated to be estrogenic inWistar prepubescent rats (Pop et al. 2013).

The findings of the present study are in keeping withpreviously published data on similar food products.Although no information exists on the estrogenic activityof pepper salami, its estradiol-equivalent recorded here isalmost of the same magnitude (150–1544 ng kg–1 origi-nal product) obtained for related products (Behr et al.2011). Soy-free products were also reported to be non-estrogenic in their test system. As to soy-based hambur-gers, the estradiol-equivalent levels obtained here aresimilar to (although slightly lower than) those found byBehr et al. (2011).

Food packaging materials are a putative and contro-versial source of human exposure to xenoestrogens(Brotons et al. 1995; Stroheker et al. 2003; ter Veld et al.2006). The estrogenic activity of packaging materialsmight result from leaching of plasticisers used in softeningthe wrappers. Several of these plasticisers such as tris(2-ethylhexyl)trimellitate and benzoate mixture have beenreported to be estrogenic (ter Veld et al. 2006). However,in our material consisting of commercially processed andpackaged Finnish foods, no evidence of this was obtained.

Food packaging materials of estrogenic samples as well asnegative food items repackaged in wrappers of positivesamples were not estrogenic in the test system.

In conclusion, the findings imply that, by and large,chemicals arising in the processing or packaging of food-stuffs in Finland constitute an insignificant source of xenoes-trogens to consumers. However, soy-derived ingredients andantioxidants in certain food items may render the entireproducts highly estrogenic. The estrogenic activity of soy isattributed to isoflavones whose health effects – thoughwidely considered beneficial – are controversial. As hambur-gers are a popular type of food among children, the findingsare noteworthy and possibly of concern.

AcknowledgementsThis research was supported by grants from the ResearchFoundation of the University of Helsinki and the WalterEhrström Foundation. The authors are grateful to JohannaRajasärkkä of the Department of Food and EnvironmentalSciences, University of Helsinki, for providing the yeaststrains. Benson Idahosa University in Benin City, Nigeria, isalso acknowledged for a fellowship given to IyekhoetinMatthew Omoruyi. The authors declare there are no conflictsof interest.

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Analytical Methods

Genotoxicity of processed food items and ready-to-eat snacks in Finland

Iyekhoetin Matthew Omoruyi ⇑, Raimo PohjanvirtaDepartment of Food Hygiene and Environmental Health (Food and Environmental Toxicology Unit), Faculty of Veterinary Medicine, P.O. Box 66, FI-00014, University ofHelsinki, Finland

a r t i c l e i n f o

Article history:Received 31 August 2013Received in revised form 6 March 2014Accepted 13 April 2014Available online 24 April 2014

Keywords:Ames testFood processingReady-to-eat foodIndustrially processed foodGenotoxicityMutagenicityCytotoxicity

a b s t r a c t

Processed foods are an insufficiently characterized source of chemical mutagens for consumers. Here, weevaluated the genotoxicity of selected food products in Finland. Mutagenicity was determined by thestandard plate incorporation assay followed by methylcellulose overlay and treat-and-wash assays, usingthe Salmonella strains TA 100 and 98 with and without metabolic activation. Generally, the mutagenicactivity of food samples was low, but exhibited lot-wise variation. Cold cuts of cold-smoked beef, grilledturkey, and smoked chicken (a single batch of each) were mutagenic in all three assays with the TA 100strain with and without metabolic activation, indicating the mutagenic effect was not secondary tohistidine release from the food products. However, none of the food extracts showing mutagenicpotential induced DNA damage in vitro using the Comet Assay. Our findings imply that in Finland today,there are still products the production methods of which should be refined to reduce the potential risk ofmutagenicity to consumers.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Food processing is the set of methods and techniques used totransform raw ingredients into food or to modify food propertiesfor consumption by humans or animals. There has been a growingdemand for produced foods globally due to changes in the lifestyleof the world population (Akintowa, Awodele, Emeka, & Osajare,2007). The majority of these foods is processed and preserved usingmethods (physical and chemical) that have the potential to triggerformation of mutagenic, genotoxic and carcinogenic substances. Ithas been estimated that 1–2 g of potentially mutagenic substancesare consumed by humans every day from food and beverages alone(Ames & Gold, 1990). Previous epidemiological studies have sub-stantially furthered our understanding of the links between dietand genetic toxicity, and cancer (Akintowa et al., 2007; Francoiset al., 2010; Sharif, Ghazali, Rajab, Haron, & Osman, 2008; Sinhaet al., 2009). These studies have revealed that diet is a key contribu-tor tohumancancerwith approximately32%of cancers estimatedasbeing attributable to dietary factors as a whole (Willet, 1998),although the contribution of dietary genotoxic substances is appar-ently far less prominent (World Cancer Research Fund/AmericanInstitute for Cancer Research, 2007).

According to a recent comprehensive analysis of the available lit-eraturedata, the evidence is convincing for consumptionofprocessedmeat as a cause of increased risk of colorectal cancer; in the case of

cancers of the oesophagus, stomach and lung, the causal relationshipis suggestive (World Cancer Research Fund/American Institute forCancer Research, 2007). High temperature cooking methods such aspan-frying and grilling/barbecuing produce compounds such as het-erocyclic amines and polycyclic aromatic hydrocarbons (PAHs),which are well-known animal carcinogens (Ferguson, 2010). Highlycarcinogenic N-nitroso compounds are also known to be formedduring frying of nitrite-treated bacon and meat (Cross & Sinha,2004), althoughhumans aremainly exposed toN-nitroso compoundsvia endogenous synthesis in the stomach (Lutz, 1990).

Acrylamide, a compound that has gained considerable attentionin recent years due to its high toxicity (Erkekoglu & Baydar, 2010),foremost its carcinogenicity (Hogervorst et al., 2010), and commonoccurrence in, for example, a variety of snacks, has been shown toform in carbohydrate-rich foods as a result of heat processing(Hogervorst et al., 2010). In the daily diet of the Swedes and theDutch, product groups including potato crisps, French fries, coffee,bread, biscuits and breakfast cereals contribute more than 90% tothe total intake of acrylamide (Konings et al., 2003; Svenssonet al., 2003). This has also been reported for Finnish foodstuffs(Eerola, Hollebekkers, Hallikainen, & Peltonen, 2007). On the otherhand, heterocyclic aromatic amines are often present in hamburg-ers (Knize et al., 1998), and commercially sold hamburgers havebeen reported to possess variable levels of mutagenic activity(Gabbani et al., 1998; Stavric, Matula, Klassen, & Downie, 1995).There are also reports of mutagenic activity in urine of subjectswho have consumed foodstuffs processed at a high temperature(Gabbani et al., 1998; Peters et al., 2004).

http://dx.doi.org/10.1016/j.foodchem.2014.04.0550308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +358 919157095.E-mail address: matthew.omoruyi@helsinki.fi (I.M. Omoruyi).

Food Chemistry 162 (2014) 206–214

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Food Chemistry

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The mutagenic potential of commercially processed food prod-ucts depends on a number of factors related to cooking conditions,such as the equipment used, ingredients, temperature and cookingtime, as attested by considerable differences in the mutagenicactivity among equivalent products from different manufacturersor restaurants (Knize, Dolbeare, Carroll, Moore, & Felton, 1994;Peters et al., 2004; Tikkanen, 1991; Tikkanen, Sauri, & Latva-Kala,1993). For example, the Ames/Salmonella test shows a correlationbetween meat-processing temperature and the number of revert-ant colonies per gram of meat (Peters et al., 2004; Tikkanen,1991). Also, industrial processing of food has a marked effect onthe mutagenic activity of the final product, based on the variationsrecorded in equivalent products from various manufacturers(Tikkanen et al., 1993).

The presence of mutagenic compounds in commercially heat-processed foods such as meat, fish and poultry products wasobserved in Finland over twenty years ago (Tikkanen, 1991). There-after, a number of toxic compounds have been extracted at varyingconcentrations from such products in Finland (Eerola et al., 2007;Tikkanen et al., 1993). However, since the methods of preparingthese products have probably undergone marked changes overthe years, screening of the overall mutagenic potential of commer-cially produced foodstuffs is warranted to ensure that they do notrepresent a genotoxic hazard to consumers. To this end, the pres-ent study set out to use the Ames test together with complemen-tary assays to investigate the mutagenicity of processed andpreserved foodstuffs as well as some ready-to-eat snacks inFinland.

2. Materials and methods

2.1. Materials

All materials used in this study were of analytical grade. TheNADP and glucose-6-phosphate used were obtained from RocheBiochem (Stockholm, Sweden). Aroclor-induced S9 from rat liverwas purchased from Trinova Biochem (Giessen, Germany). Salmo-nella enterica sv. typhimurium strains TA 100 and TA 98 wereobtained from Pasteur’s Institute (Paris Cedex, France). Histidine,potassium chloride, magnesium sulphate, potassium phosphatedibasic anhydrous and sodium ammonium phosphate werepurchased from Merck AG (Darmstadt, Germany). Magnesiumchloride hexahydrate and citric acid monohydrate were acquiredfrom VWR international (Leuven, Belgium). Biotin, tryptophan,methylcellulose (MC), dimethyl sulfoxide, benzo[a]pyrene,2-aminoanthracene and sodium azide were obtained fromSigma–Aldrich (Steinheim, Germany).

2.2. Cell line

Human hepatocellular carcinoma-derived cell line (HepG2) wasobtained from American Type Culture Collection through LGC stan-dards (Boras, Sweden) and cultured in Eagle’s Minimum EssentialMedium (LGC standards, Boras, Sweden) containing 10% heat-inac-tivated fetal bovine serum (Sigma–Aldrich, Steinheim, Germany).The cells were maintained at 37 �C in a humidified atmosphere of5% CO2 in air atmosphere incubator (NuAire Inc., Plymouth, USA).

2.3. Sampling

Fourty-five samples of industrially processed and packagedfood products and 15 samples of ready-to-eat snacks were evalu-ated for their mutagenic potential. The industrially processed andpackaged food products were purchased from a popular supermar-ket in Helsinki (Prisma, Viikki), while the ready-to-eat food

samples were acquired from a local representative (at SOKOS CityCentre) of a global chain of hamburger restaurants also located inHelsinki, Finland. The sample types and manufacturers are listedin Supplementary Table 1. A total of three batches were collectedat separate times. The cooking conditions such as time and temper-ature were not available. We carefully ensured that all productswere extracted before the expiry date shown on the packages.

2.4. Extraction

Possible mutagenic compounds were extracted from the foodsamples using the method described by Peters et al. (2004) withslight modifications. Briefly, 20 g of food sample was homogenizedwith 80 ml of 1 mol/l NaOH at 24,000 rpm. The homogenate wasmixed with 20 g of Extrelut refill material (VWR international, Hel-sinki, Finland) and then poured into an empty Extrelut 20 column.The organics were eluted from the Extrelut column with 40 ml ofdichloromethane/toluene solution (95:5 v/v) into a cartridge. Theorganics were finally eluted with 5 ml of MeOH–NH4OH (9:1) solu-tion and evaporated to dryness under a gentle stream of nitrogenin a fume hood. For determination of recovery, fresh meat wasspiked with 50 ll of benzo[a]pyrene and 250 ng of 2-aminoanthra-cene in two different cases and extracted in the same way as above.

2.5. Cytotoxicity assays

The cytotoxic effect of the concentrations of food extracts usedin this study was investigated by three independent assays mea-suring trypan blue exclusion, lactate dehydrogenase activity, andboar sperm motility.

2.5.1. Trypan blue testHepG2 cells were grown in 24-well plates (VWR, Finland) until

semi-confluent cells were obtained (48 h). This was followed byexposure of the cells to different concentrations of food extractsfor 4, 24 or 48 h. After exposure, the cells were trypsinised using0.25% (w/v) trypsin–0.53 mmol/l EDTA solution (LGC standards,Boras, Sweden). Trypsinised cells were transferred to 1.5 ml Eppen-dorf tubes and centrifuged for 5 min at 2,500 rpm. Pellets were thenre-suspended in PBS, after which 10 ll of the cells were mixed with5 ll (0.8 mmol/l ) trypan blue dye before microscopic observation.

2.5.2. Lactate dehydrogenase (LDH) assayThe activity of lactate dehydrogenase (LDH) was determined in

HepG2 cells exposed to the same concentrations of the foodextracts used in genotoxicity assays. The LDH test measuresplasma membrane integrity and it was performed according tothe instructions provided in the Cytotoxicity Detection KitPLUS

(LDH), version 6 (Roche Biochem, Stockholm, Sweden).

2.5.3. Boar sperm motility inhibition bioassayExtracts of selected food samples were assessed for their mito-

chondrial toxicity using the boar sperm motility assay (Anderssonet al., 2010). Briefly, 2 ml of boar semen in screw-capped exposurevials was exposed to 10 ll of food extracts for 30 min, 24 or 48 h at20 �C. Vehicle (DMSO) exposure was prepared simultaneously withthe test samples for each time point. After exposure, the vials wereshaken gently to disperse the sperm cells, and 200 ll of the sus-pension was drawn into a warmed test tube and placed in a heat-ing block (30 �C) for approximately 5 min to activate spermmotility. Sperm motility was assessed by dispensing warmedsperm suspension onto a microscopic slide using a pre-warmedcapillary tube, and immediately observed with a 40-� invertedphase contrast objective.

I.M. Omoruyi, R. Pohjanvirta / Food Chemistry 162 (2014) 206–214 207

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2.6. Mutagenicity assay

The mutagenic potential of food extracts was determined ini-tially by the standard plate incorporation assay. Samples showingmutagenic potential in this assay were subjected to treat-and-wash as well as methylcellulose overlay assays to ascertainto what degree a localized release of proteins, peptides or histidinefrom the samples contributed to the outcome.

2.6.1. Standard plate incorporation assayThe standard plate incorporation assay was performed as

described by Maron and Ames (1983) using Salmonella strains TA100 and TA 98 with and without metabolic activation (S9 mix).The amount of S9 used in the S9 mix was 10%. Water and DMSOwere used as negative controls for both strains while sodium azide(0.04 mg/ml) and 2-aminoanthracene (0.02 mg/ml) served as posi-tive controls for TA 100 and TA 98, respectively. Benzo[a]pyrene(0.1 mg/ml) was also used as a positive control for both strains.The volume of controls used was 50 ll/plate in triplicate plates.Sodium azide is a known direct mutagen in Salmonella TA 100(Mortelmans & Zeiger, 2000), whereas 2-aminoantracene ismetabolically activated by mono-oxygenases of the CYP1A familyin rat liver (Carrière, de Waziers, Courtois, Leroux, & Beaune,1992). Likewise, benzo[a]pyrene requires metabolic activation formutagenicity (Gabbani et al., 1998).

For all samples, four different concentrations of the foodextracts (25, 50, 100 and 200 mg/ml) were tested in triplicateplates (50 ll/plate). The highest concentration (200 mg/ml) wasequivalent to 1 g of the food sample. The plates were incubatedat 37 �C for 48 h.

The results of the mutagenic activities are presented as thenumber of revertant colonies per gram of food sample. Only themean and standard deviation of the highest concentration for allfood extracts, and the dose–response curve for extracts showingmutagenic activity are shown.

2.6.2. Treat-and-wash assayThe treat-and-wash assay was conducted according to the

method described by Thompson, Morley, Kirkland, and Proudlock(2005). The protocol applied was as per the standard plate incorpo-ration assay with the exception that the S9 mix, bacteria and sam-ple extract were incubated for 90 min prior to the addition ofmolten top agar. Briefly, a 500-ll aliquot of S9 mix/phosphate buf-fer (0.2 mol/l, pH 7.4) was combined with 100 ll each of late-logbacterial culture and sample extract solution in a sterile 15 mltube. The mixture was incubated for 90 min in a mechanical shaker(180 rpm) at 37 �C. The extended duration of bacterial exposurecompensated for the absence of bacterial exposure on plates, asthe test sample was washed away prior to plating. After a 90-min preincubation, 10 ml of wash solution (Oxoid No. 2 nutrientbroth in phosphate-buffered saline [1:7 v/v]) was added, and thewashed bacteria were collected by centrifugation at 2000g for30 min. All but approximately 700 ll of the supernatant wasremoved and discarded, and the bacteria were re-suspended inthe residual supernatant prior to plating via top agar.

2.6.3. Methylcellulose overlay assayMethylcellulose overlay assay was performed as previously

described (Thompson et al., 2005). Briefly, a 500-ll aliquot of S9mix/phosphate buffer (0.2 mol/l, pH 7.4) was combined with100 ll of late-log bacterial culture in a sterile 15 ml tube. A 2-mlaliquot of the MC overlay suspension was added to the tube, anda 100-ll aliquot of the sample extract solution was added immedi-ately afterward. The mixture was overlaid on a pre-warmed (37 �C)minimal glucose plate. Plates were held at 4 �C for 1 h after platingto ensure gelling of the MC overlay, and subsequently incubated

(not inverted) at 37 �C for 48–72 h. The MC overlay was preparedon the day of the test (Thompson et al., 2005), and the mixturewas stirred at 50–60 �C throughout use.

2.7. Comet Assay

The Comet Assay (single-cell gel electrophoresis) was used toevaluate possible breakage of single- or double-stranded DNA inHepG2 cells following treatment with extracts of food samplesshowing mutagenic potential. The Comet Assay was performed inalkaline conditions (pH >13) as described previously (Singh et al.,1988). Hydrogen peroxide and 0.7% DMSO served as positive andnegative controls, respectively.

2.8. Statistical analysis/interpretation of data

The mutagenic potency of each food sample was determinedfrom the linear slope of the dose-response curve by linear regres-sion analysis using Prisma 4.0 (GraphPad software Inc. San Diego,CA). In addition to a statistically significant (p < 0.05) dose–response effect, samples were only considered mutagenic wherethe highest test concentration generated at least twice as manyrevertants as the negative control (DMSO). For proper interpreta-tion and clarity, the number of revertants obtained was comparedwith both experiment-specific controls and aggregate controlsacross all experiments. The p values of these comparisons pre-sented in Tables, Figs. and Supplementary Tables are derived fromthe regression analyses.

3. Results

3.1. Plate incorporation assay: Control substances

In the conventional Ames test, the negative control substancesused elicited 5–6 times as many colonies in the TA 100 strainas in the TA 98 strain (Table 1). In the presence of S9 mix, benzo[a]-pyrene elevated colony number from this level to 2.5- (TA 100) or4-fold (TA 98) higher. In TA 100, benzo[a]pyrene caused an almost2-fold increase even in the absence of S9, which did not occur in TA98. Also, sodium azide instigated a larger increase in the colonynumber with S9 (7-fold) than without (4-fold), despite being therecommended direct control mutagen for TA 100 (Mortelmans &Zeiger, 2000). On the other hand, 2-aminoanthracene performedas expected in TA 98 generating 15- and 2.5-fold increments incolony abundances with and without S9, respectively.

3.2. Plate incorporation assay: Industrially processed food items

The mutagenic activity of industrially processed food productsexpressed as the number of revertants per gram original product,obtained using the standard plate incorporation assay, is presentedin Tables 2a and 2b. Overall, the products exhibited fairly lowmutagenic activity in both Salmonella strains. However, the resultsshowed some variation in the mutagenic potency both amongequivalent products processed differently and among differentproducts processed in the same way.

Extracts of industrially processed grilled chicken, smoked fish,cold-smoked fish, grilled beef, French fries, mashed potatoes,hamburger (beef) and hamburger (chicken) did not show dis-cernible mutagenic potential in any of the three batches exam-ined in either strain of Salmonella, disregard of the S9 status(Tables 2a and 2b). Seven of the other products examined pro-duced revertants at least two-fold higher than negative control(DMSO) in at least one of the three batches screened, and inone or both strains. The number of revertants produced by

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cold-smoked beef on Salmonella TA 100 (with S9) ranged from420.3 to 583.3 revt/g, showing clear mutagenic activity in twoof the batches (Table 2a). In the third batch investigated, thenumber of revertants (420.3 ± 14.6) was more than twice thatyielded by negative controls, but there was no dose–response,so it was not considered mutagenic. The extract of cold cuts ofgrilled turkey showed clear mutagenic activity in all the threebatches examined with Salmonella TA 98 (Table 2b). In twocases, this occurred without S9 mix and once in its presence.One of these lots (batch 1) proved mutagenic also in TA 100,both in the presence and absence of S9 mix. This particularbatch also yielded a significant mutagenic response in the mod-ified Ames test (see below). Some batches of pepper salami andsausage were mutagenic only in the TA 100 strain, and one lot offried fish only in the TA 98 strain.

Supplementary Fig. 1 presents the dose-response curves ofthose industrially processed food products, which displayedsignificant mutagenic activity. The number of revertants increased,in most cases in a similar fashion, as a function of extract concen-tration in both strains and irrespective of S9 status.

3.3. Plate incorporation assay: Ready-to-eat snacks

Tables 3a and 3b show the results of the mutagenic potential ofready-to-eat snacks from a popular hamburger retailer. At thehighest concentration, three out of five snack products examinedgenerated a more than twice the number of revertants found incontrol, and produced a significant dose–response. Extracts ofchicken nuggets were mutagenic in all the batches examined withSalmonella TA 100, mainly in the presence of S9 mix (Table 3a). InSalmonella TA 98, the numbers of revertants produced by chickennuggets were either marginally elevated or, if they were greaterthan two-fold compared with the control, they lacked a significantdose–response (Table 3b).

Similarly, extracts of beef hamburger obtained from hamburgerretailer were repeatedly mutagenic (Tables 3a and 3b) whereasindustrially-processed beef burgers were negative (Tables 2a and2b). All potato products (French fries and mashed potatoes) as wellas burgers containing chicken, from a supermarket or thehamburger retailer, proved not to be mutagenic.

Table 1Ranges for revertant colonies obtained with control substances in the standard plate incorporation assay.

Controls Number of revertant colonies

Salmonella TA 100

Respective controls* Aggregate controls**

+S9 �S9 +S9 �S9

Water 166.7–178.7 128.0–151.7 172.7 ± 22.9 139.8 ± 20.0DMSO 145.7–185.7 120.7–179.3 173.6 ± 22.7 147.3 ± 27.3Sodium azide 1000.0–1104.0 553.0–569.7 1052.0 ± 164.2 561.0 ± 75.9Benzo[a]pyrene 327.3–442.3 239.0–247.3 384.8 ± 67.4 241.7 ± 71.1

Salmonella TA 98Water 25.3–34.3 19.7–27.7 29.8 ± 4.9 21.8 ± 4.8DMSO 22.0–34.0 19.7–24.0 28.5 ± 6.7 21.2 ± 3.32-Aminoanthracene 400.3–449.3 54.0–58.0 424.8 ± 82.1 55.6 ± 16.1Benzo[a]pyrene 82.6–116.7 18.0–33.3 98.8 ± 31.1 25.8 ± 7.3

* Range.** Mean ± SD.

Table 2aNumber of revertants generated by the highest concentrations (1.0 per g of food sample) of extracts of industrially processed food products on Salmonella TA 100 (mean ± SD) inthe standard plate incorporation assay.

Food product Revertants per gram

Batch 1 Batch 2 Batch 3

+S9 �S9 +S9 �S9 +S9 �S9

Chicken1 524.7 ± 99.6�,a 239.3 ± 20.8 266.7 ± 27.4 258.0 ± 8.5 260.0 ± 16.0 279.7 ± 17.2Chicken2 544.7 ± 43.1�,a 255.0 ± 18.5 398.7 ± 9.0 274.7 ± 23.5 244.0 ± 16.7 262.3 ± 7.8Chicken3 241.7 ± 23.7 201.0 ± 13.3 272.7 ± 20.1 224.0 ± 23.1 227.7 ± 38.5 263.0 ± 12.2Turkey3 709.7 ± 68.0�,a 394.0 ± 18.4�,a 272.7 ± 20.1 224.0 ± 23.1 292.7 ± 54.1 270.7 ± 32.6Fish1 243.7 ± 10.2 198.3 ± 6.7 223.0 ± 10.4 230.7 ± 18.0 201.0 ± 15.9 194.3 ± 21.1Fish4 291.7 ± 52.5 256.0 ± 22.7 236.3 ± 12.0 234.3 ± 21.4 214.3 ± 14.6 215.7 ± 9.1Fish5 648.0 ± 91.5 281.0 ± 58.3 306.7 ± 25.1 254.0 ± 23.6 268.0 ± 20.1 327.0 ± 25.1Beef3 294.3 ± 29.0 211.7 ± 22.8 318.0 ± 25.2 257.7 ± 42.6 176.0 ± 10.0 179.0 ± 14.0Beef4 583.3 ± 153.4�,a 291.3 ± 54.7 420.3 ± 14.6 376.3 ± 13.4 454.3 ± 42.7�,a 381.3 ± 13.4�,a

Sausage 337.3 ± 19.0 310.7 ± 22.8�,a 221.3 ± 22.5 172.7 ± 15.9 238.3 ± 14.6 215.7 ± 2.1Pepper salami 398.3 ± 68.1 282.0 ± 20.1 371.0 ± 4.6�,a 289.7 ± 8.5 433.0 ± 30.1�,a 391.7 ± 3.2�,a

French fries 260.0 ± 23.5 200.7 ± 15.0 197.3 ± 8.1 177.0 ± 26.0 155.7 ± 17.8 146.0 ± 23.5MashedPotatoes 219.7 ± 14.5 154.0 ± 9.4 274.3 ± 8.7 145.3 ± 16.7 163.3 ± 19.5 140.7 ± 22.5Hamburger* 244.3 ± 11.6 200.7 ± 11.2 232.7 ± 6.1 192.0 ± 7.2 189.7 ± 24.2 184.3 ± 24.9Hamburger** 272.0 ± 22.9 198.0 ± 10.4 220.3 ± 5.1 188.0 ± 29.6 183.7 ± 18.9 181.3 ± 6.5

Key: 1: smoked; 2: honey-roasted; 3: grilled; 4: cold-smoked; 5: fried.* Chicken** Beef� Significantly different from respective controls (P < 0.05).a Significantly different from aggregate control (P < 0.05).

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Table 2bNumber of revertants generated by the highest concentrations (200 mg/ml) of extracts of industrially processed food products on Salmonella TA 98 (mean ± SD) in the standardplate incorporation assay.

Food product Revertants per gram

Batch 1 Batch 2 Batch 3

+S9 �S9 +S9 �S9 +S9 �S9

Chicken1 62.7 ± 5.0 41.3 ± 8.1 46.3 ± 3.2 34.0 ± 7.9 41.0 ± 3.6 34.7 ± 9.6Chicken2 68.3 ± 3.2 47.3 ± 2.9 38.3 ± 3.2 37.3 ± 1.5 39.7 ± 6.0 34.0 ± 5.2Chicken3 46.3 ± 8.3 29.7 ± 3.5 45.0 ± 7.9 39.3 ± 9.0 40.7 ± 15.5 26.7 ± 5.0Turkey3 71.7 ± 24.8 57.3 ± 13.7�,a 45.7 ± 3.1 47.3 ± 7.6�,a 53.7 ± 9.3� 36.3 ± 11.9Fish1 36.3 ± 0.6 29.3 ± 1.2 33.3 ± 3.1 28.7 ± 2.5 32.0 ± 4.4 19.3 ± 3.2Fish4 66.7 ± 16.8a 45.7 ± 4.7 33.7 ± 3.8 28.3 ± 2.9 26.7 ± 4.0 21.3 ± 2.1Fish5 72.0 ± 9.0 56.7 ± 7.8 42.7 ± 1.5 39.0 ± 2.0� 42.3 ± 4.7 44.0 ± 1.7Beef3 43.0 ± 6.4 29.7 ± 10.2 42.3 ± 4.9 32.7 ± 0.6 25.0 ± 4.6 22.7 ± 2.3Beef4 76.3 ± 8.7 42.3 ± 3.3 48.7 ± 7.6 42.0 ± 3.5 85.3 ± 4.9� 61.7 ± 10.7�

Sausage 49.7 ± 3.5 36.0 ± 7.0 32.7 ± 1.5 24.7 ± 5.5 28.3 ± 6.0 24.0 ± 2.6Pepper salami 75.3 ± 1.6 36.3 ± 5.5 67.0 ± 7.6 47.0 ± 8.7 42.3 ± 7.1 35.0 ± 2.0French fries 43.0 ± 3.0 34.0 ± 2.0 31.0 ± 9.0 28.7 ± 4.0 28.7 ± 5.8 19.3 ± 6.8Mashed potatoes 34.0 ± 1.9 29.0 ± 2.5 31.0 ± 1.0 17.3 ± 3.1 33.0 ± 2.0 25.3 ± 10.2Hamburger* 41.7 ± 5.7 31.0 ± 4.5 38.0 ± 3.6 24.3 ± 3.5 39.0 ± 2.0 32.3 ± 8.3Hamburger** 46.3 ± 9.0 31.3 ± 7.6 42.0 ± 3.6 30.0 ± 3.6 45.3 ± 2.9 38.7 ± 17.5

Key: 1: smoked; 2: honey-roasted; 3: grilled; 4: cold-smoked; 5: fried.* Chicken.** Beef.� Significantly different from respective controls (P < 0.05).a Significantly different from aggregate control (P < 0.05).

Table 3aNumber of revertants generated by the highest concentrations (10 per g of food sample) of extracts of ready-to-eat food snacks on Salmonella TA 100 (mean ± SD) in the standardplate incorporation assay.

Food product Revertants per gram

Batch 1 Batch 2 Batch 3

+S9 �S9 +S9 �S9 +S9 �S9

Cheese Burger 334.0 ± 19.5� 279.7 ± 24.5� 325.3 ± 25.5� 213.7 ± 27.0 255.7 ± 7.6 250.3 ± 13.3Chicken� 319.0 ± 12.0� 236.0 ± 34.0 292.3 ± 15.0� 254.3 ± 3.1� 312.0 ± 42.1� 204.3 ± 42.9French fries 200.7 ± 6.7 174.7 ± 23.6 150.0 ± 6.6 159.7 ± 12.1 183.3 ± 8.2 146.7 ± 6.8Hamburger* 253.7 ± 16.9 222.7 ± 22.2 158.7 ± 21.9 109.7 ± 10.4 211.0 ± 17.2 209.7 ± 16.8Hamburger** 330.7 ± 25.2� 269.3 ± 23.0� 244.3 ± 45.6a 202.3 ± 15.6 316.3 ± 14.6� 212.0 ± 37.0

Key.� Nugget.* Chicken.** Beef� Significantly different from respective controls (P < 0.05).a Significantly different from aggregate control (P < 0.05).

Table 3bNumber of revertants generated by the highest concentrations (1.0 per g of food sample) of extracts of ready-to-eat food snacks on Salmonella TA 98 (mean ± SD) in the standardplate incorporation assay.

Food product Revertants per gram

Batch 1 Batch 2 Batch 3

+S9 �S9 +S9 �S9 +S9 �S9

Cheese burger 53.7 ± 4.0 17.3 ± 3.2 52.7 ± 3.2 35.7 ± 4.5 45.7 ± 3.2 37.7 ± 3.1Chicken� 55.7 ± 3.2 35.0 ± 4.6 61.0 ± 13.1a 40.0 ± 4.6 56.3 ± 10.0 40.0 ± 9.6French fries 43.0 ± 2.7 19.3 ± 2.3 34.7 ± 1.5 20.3 ± 2.1 28.0 ± 4.6 31.3 ± 3.5Hamburger* 41.7 ± 6.4 23.3 ± 6.8 40.7 ± 7.6 26.7 ± 5.7 40.3 ± 7.6 29.3 ± 5.5Hamburger** 66.7 ± 06.0� 37.3 ± 5.1 53.3 ± 8.6 37.0 ± 4.0 54.3 ± 3.2 38.0 ± 11.5

Key.� Nugget.* Chicken.** Beef.� Significantly different from respective controls (P < 0.05).a Significantly different from aggregate control (P < 0.05).

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3.4. Modified Ames tests

In the modified Ames tests (Supplementary Tables 2–9), theoutcome proved to depend on bacterial strain, type of food, S9 sta-tus, and assay, albeit for most food samples the complementaryassays tended to reduce the number of revertants per gramobtained compared with the standard plate incorporation assay(some examples are shown in Figs. 1 and 2).

Extracts of industrially processed food (cold cuts of smokedchicken, honey-roasted chicken and cold-smoked beef) whichrequired metabolic activation for their mutagenicity in the stan-dard plate incorporation assay, were, unexpectedly, directly muta-genic in Salmonella TA 100 strain in the MC overlay assay(Supplementary Table 2). One of these samples (cold-smoked beef)produced the same outcome with the treat-and-wash assay (Sup-plementary Table 6). None of the ready-to-eat food samples exhib-ited any form of mutagenicity in either of these auxiliary assayswith Salmonella TA 100 (Supplementary Tables 4 and 8) in contrastto the outcome in the standard plate incorporation assay. Extract ofready-to-eat beef burger, however, exhibited indirect mutagenic

activity with the Salmonella TA 98 strain in the treat-and-washassay (Supplementary Table 9).

Although cold cuts of grilled turkey (batch 1) showed clearmutagenic activity in all three assays in the presence or absenceof S9 mix with the Salmonella TA 100 strain, both the MC overlayand treat-and-wash procedure produced fewer revertants com-pared with the standard assay (Fig. 1B). With the Salmonella TA98 strain, however, by far the most conspicuous mutagenicresponse was recorded in the treat-and-wash assay, in which itproduced revertants more than 13- and 5-fold control levels, withand without S9 mix, respectively (Supplementary Table 7 andFig. 2). The other batches of grilled turkey were also mutagenicwith the TA 98 strain in the presence or absence of S9 mix, butnot to the same extent (Supplementary Table 7).

3.5. Cytotoxicity assays

The cytotoxicity of the four concentrations of all food extractswas determined by both trypan blue exclusion and LDH secretionof HepG2 human hepatocellular carcinoma cells, as well as by

Fig. 1. Mutagenicity of the highest extract concentration (1.0 per g of food sample) of (A) smoked chicken; (B) grilled turkey; (C) cold-smoked beef on strain TA 100 in threeindependent assays. Data are mean ± standard deviation of triplicate samples. The asterisks depict statistically significant differences vs. the corresponding solvent controls atp < 0.05.

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the boar sperm motility assay. The percentage viability of HepG2cells in the trypan blue exclusion test exhibited an inverse correla-tion with the concentration of extracts from smoked chicken,grilled turkey and cold-smoked beef following 48 h exposure (Sup-plementary Fig. 2). However, the 50 ± 5% cytotoxicity limit(Nymark et al., 2012) was not reached at any concentration.Although there was a direct correlation between extract concentra-tion and LDH release, the maximum release never exceeded 25% ofthat induced by the positive control substance applied (lysis solu-tion; Supplementary Fig. 2A). Hence, the extracts were classifiednon-cytotoxic in this assay following exposure for 4, 24 or 48 h.Moreover, none of the food extracts tested affected boar spermmotility in a statistically significant manner. These samples werealso not cytotoxic in another independent cytotoxicity assay car-ried out on the yeast strain Saccharomyces cerevisiae BMA64/luc(Omoruyi, Kabiersch, & Pohjanvirta, 2013).

3.6. Comet Assay

As a follow-up of the results obtained in the conventional Amestest, samples showing mutagenic potential in at least one strain ofSalmonella were screened for single-strand breaks, DNA–DNA/DNA–protein cross-linking and alkali-labile sites by the CometAssay. None of the samples investigated tested positive when com-pared with both positive and negative controls. In the positive con-trols (treatment with hydrogen peroxide), the expected DNA

migration towards the anode was observed by microscopicexamination.

3.7. Recovery analysis

The percentage recovery for both benzo[a]pyrene and 2-amino-anthracene was approximately 73%.

4. Discussion

Industrially processed and packaged foodstuffs as well as ready-to-eat snacks are consumed in increasing quantities all over theworld. Therefore, it is important to ensure in addition to microbialsafety, that products do not contain chemicals that might pose atoxicological risk to consumer health. A conceivable potential riskin this regard is the formation of genotoxic compounds during theprocessing of industrial foodstuffs or cooking of snacks. Regularscreening studies are necessary to verify that the methods usedby food industry and for example, fast-food outlets are appropriatefrom this point of view. The present investigation aimed to explorethe current situation in Finland.

While the majority of the samples analyzed in our study provednegative, the mutagenic activity observed with extracts from somebatches of industrially processed foods (smoked chicken, honey-roasted chicken, grilled turkey, cold-smoked beef, fried fish, andpepper salami) and ready-to-eat snacks (cheese burger, hamburgercontaining beef, and chicken nuggets) indicates that exposure tomutagenic compounds arising from commercial food products inFinland cannot be completely excluded. Levels may be toxicologi-cally significant, because the magnitude of mutagenic activitieswas 2.5- to 13-fold those of negative controls. However, for someproducts there was a notable (up to over 10-fold) variation inmutagenic potency between batches, and among equivalent prod-ucts and different products processed in the same way. There wasalso variation in mutagenic potency among equivalent food prod-ucts with the three assays (standard plate incorporation, MC over-lay as well as treat-and-wash assays), in particular for ready-to-eatsnacks, casting doubt on the significance of some findings. Con-versely, the positive results obtained with the MC overlay andtreat-and-wash assays for some industrially processed food prod-ucts (smoked chicken, grilled turkey and cold-smoked beef) cou-pled with their mutagenic response in the standard plateincorporation assay imply the effect is genuine (i.e., not secondaryto histidine release from the food products) and thus of concern.

It is also noteworthy that extracts of certain food items (e.g.cold-smoked beef, Batch 3) exhibited mutagenic activity withoutmetabolic activation in all three assays, strongly suggesting directmutagenicity. Based on the classification of Abbas, Mirocha, andShier (1984), no cytotoxic effects were observed in the assays con-ducted with different concentrations of extracts from food samplesused in the mutagenicity studies. This is in keeping with a previousreport (Sharif et al., 2008), further strengthening the validity andrelevance of the mutagenicity findings and, concurrently, justifyingour requirement for dose–response across the entire range of con-centrations examined. On the other hand, none of the samples wasdiscernibly different from the negative control using the CometAssay. This has mechanistic implications, since the Comet Assaydetects mainly DNA strand breaks (He, Chen, Jin, & Jin, 2000),whereas the Salmonella strains TA 100 and TA 98 are sensitive tobase-pair substitutions and frame-shift mutations, respectively(Mortelmans & Zeiger, 2000).

Pepper salami has not previously been regarded as a potentialsource of mutagens. In our study, this product exhibited bothdirect and indirect mutagenicity in one batch, and indirect inanother when examined using TA 100 strain in the standard plate

Fig. 2. (A) Plate showing the number of revertants produced by strain TA 98 intreat-and-wash assay. (B) Mutagenicity of the highest extract concentration (1.0 perg of food sample) of grilled turkey on strain TA 98 in three independent assays. Dataare mean ± standard deviation of triplicate samples. The asterisks depict statisti-cally significant differences vs. the corresponding solvent controls at p < 0.05.

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incorporation assay. The number of revertants obtained with Sal-monella TA 100 ranged from 371.00 ± 04.58 to 433.00 ± 30.05 rev/g. All the values were at least twice their respective negative con-trols but the lack of dose–response in one of the batches resulted inits classification as non-mutagenic. Both positive samples ofpepper salami (batch 2 and 3) were also found to be mutagenicwith the MC overlay assay, in the presence of S9 mix, but not withthe treat-and-wash assay (209.3 ± 13.4 and 204.0 ± 0.00 rev/g).Thus, the true mutagenicity of these samples remains unclear.Further studies on this food item are warranted.

Three out of five products of ready-to-eat snacks examinedwere found to be mutagenic, in at least two batches, using theconventional Ames test. However, except for beef burger in thetreat-and-wash assay (TA 98, without S9), all these samples werenegative in the modified Ames tests suggesting their initial positiveresults stemmed from local or general histidine release. In thiscontext, it is noteworthy that in contrast to hamburgers purchasedfrom the fast-food outlet, hamburgers acquired from the super-market were all non-mutagenic as measured by the standard plateincorporation test. Overall, these results imply the industrialcooking methods currently used in Finland are carefully consideredalso from the toxicological point of view. Previous studies havereported mutagenic compounds in a hamburger extract (Stavricet al., 1995) and in the urine of subjects after a hamburger meal(Gabbani et al., 1998). However, in both of these studies, thesubjects prepared the hamburgers without monitoring the cookingtemperature and time, which could account for the high level ofmutagenicity observed. Homemade food products cooked at a hightemperature are reported sources of human exposure to genotoxiccompounds (Tikkanen, 1991). Also, the authors did not take intoconsideration the possible release of histidine from the burgerproducts, which apparently may have accounted for the highnumber of revertants observed (Khandoudi et al., 2009;Thompson et al., 2005).

The negative results obtained for French fries (both those pro-cessed industrially and those obtained from a hamburger restau-rant) as well as mashed potatoes with the standard Ames testand Comet Assays were not unexpected. Acrylamide is the princi-pal mutagen formed in both potato products, and studies haveshown that this compound is not mutagenic in the Ames test,either in the presence or absence of metabolic activation(Dearfield et al., 1995; Knaap et al., 1988). Furthermore, two inde-pendent studies have shown that acrylamide is not mutagenic inChinese hamster V79 cells (Baum et al., 2005; Tsuda et al., 1993).Acrylamide is formed when food high in carbohydrate is processedat a high temperature, usually between 120 and 180 �C. While wemay attribute the negative results obtained to such findings, it isalso worth mentioning that modified processing methods for pota-toes, at relatively low temperatures for shorter periods of time, cansignificantly reduce, or eliminate, the formation of genotoxic com-pounds (Peters et al., 2004). Moreover, the use of a potato varietylow in carbohydrate can have the same effect (Sowokinos, 1990).

The localized release of proteins, peptides or histidine from foodsamples has been reported as a source of false positive results inthe Ames test (Khandoudi et al., 2009; Thompson et al., 2005). Toprevent this potential misinterpretation and exaggeration of osten-sible mutagenicity, treat-and-wash as well as MC overlay assayswere performed for all samples eliciting a positive outcome inthe conventional Ames test. The results from these complementaryassays for some samples (cold cuts of smoked chicken, grilled tur-key and cold-smoked beef) were consistent, further reinforcing ourinitial findings with the Ames test. Among these, extracts of grilledturkey stood out for their ability to induce revertants in the pres-ence and absence of metabolic activation system in both bacterialstrains. Grilled turkey also yielded the highest number of coloniesin the MC overlay assay in both strains (with S9 mix). Interestingly,

one of the lots of grilled turkey (batch 1), substantially enhancedthe generation of revertants, with colonies more than 13- and5-fold their controls, produced in the presence and absence of S9mix, respectively, as determined using the treat-and-wash assay.Possible reasons for this finding are the long pre-incubation periodin the presence of a nutrient broth, increased viability of the bacte-ria, and removal of substances increasing or inhibiting reversemutation. It is notable that two other batches of grilled turkey alsoproduced significant effects in the treat-and-wash assay in TA 98strain (in TA 100, only batch 1 was examined), both in the presenceand absence of S9 mix.

Although it is challenging to compare results for processed foodproducts worldwide, the data obtained in our studies are similar tothose reported elsewhere (Peters et al., 2004; Stavric et al., 1995)but somewhat at odds with those of a previous study in Finland,where the majority of ready-made industrial food samples weremutagenic and, for example, grilled chicken generated up to1400 revertants in the TA 98 strain (Tikkanen, 1991). Moreover,Tikkanen (1991) did not control for possible histidine release. Itshould also be noted that most of the earlier studies in Finlandand elsewhere have employed Salmonella TA 98 alone (and some-times only in the presence of S9). Based on our findings, TA 98strain may be more or less sensitive to food mutagens than TA100, depending on sample type and assay conditions. On the otherhand, the observed generally low levels of mutagenic activity inindustrially processed food products in Finland today are in agree-ment with the more recent data of Jägerstad and Skog (2005). Theyreported the daily intake of nitrosodimethylamine (a known muta-gen formed as a result of processing especially at high tempera-ture) to be significantly lower in Finland (0.08 lg/day) whencompared with other European countries (0.12–0.38 lg/day). Thistogether with our data, suggest the food industry in Finland hasrecognized its responsibilities and taken appropriate steps towardsproducing products that do not pose genotoxic hazards to consum-ers. However, the process has clearly not been brought to comple-tion yet.

In conclusion, our study demonstrated that, while in most cases,the risk of genotoxicity associated with consumption of industri-ally processed and ready-to-eat foodstuffs is low in Finland, thereare still products the production of which should be refined furtherto reduce the potential risk for consumer. Our data may have tox-icological implications and further studies are, therefore, needed.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by Grants from the ResearchFoundation of the University of Helsinki and Walter EhrströmFoundation. Benson Idahosa University, Benin City, Nigeria is alsoacknowledged for a fellowship given to Iyekhoetin MatthewOmoruyi.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2014.04.055.

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214 I.M. Omoruyi, R. Pohjanvirta / Food Chemistry 162 (2014) 206–214

III

Int. J. Environ. Res. Public Health 2014, 11, 8347-8367; doi:10.3390/ijerph110808347

International Journal of Environmental Research and

Public Health ISSN 1660-4601

www.mdpi.com/journal/ijerph Article

Dietary Exposure of Nigerians to Mutagens and Estrogen-Like Chemicals

Iyekhoetin Matthew Omoruyi 1,2,*, Derek Ahamioje 2 and Raimo Pohjanvirta 1

1 Food and Environmental Toxicology Unit, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, P.O.Box 66, 00014 Helsinki, Finland; E-Mail: raimo.pohjanvirta@helsinki.fi

2 Department of Basic Sciences, Faculty of Basic and Applied Sciences, Benson Idahosa University, P.M.B. 1100, Benin City, Edo State, Nigeria; E-Mail: dahamioje@biu.edu.ng

* Author to whom correspondence should be addressed; E-Mail: matthew.omoruyi@helsinki.fi; Tel.: +358-9191-5709.

Received: 3 June 2014; in revised form 30 July 2014 / Accepted: 7 August 2014 / Published: 15 August 2014

Abstract: Food and drinking water are poorly delineated sources of human exposure to chemical food mutagens and endocrine-disrupting chemicals. In this study, we investigated the presence of mutagens and chemicals exhibiting estrogenic activity in the daily diet of Nigerians, using in vitro assays. Commercially processed foods or snacks and various brands of pure water sachets were extracted by solid-phase extraction and liquid-liquid extraction, respectively. Mutagenicity was determined by the conventional Ames test and two complementary assays on two strains of Salmonella (TA 100 and TA 98), while the estrogenic activity was assessed by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). A third of the food varieties investigated (chin-chin, hamburger, suya and bean cake) were mutagenic in all three assays, either in the presence or absence of S9 mix. Of the packed water samples, five out of the sixteen investigated (31%), were found to be estrogenic, with estradiol and bisphenol A equivalents ranging from 0.79 to 44.0 ng/L and 124.2 to 1,000.8 ng/L, respectively. Hence, although the current situation in Nigeria does not appear to be substantially worse than, e.g., in Europe, regular monitoring is warranted in the future.

OPEN ACCESS

Int. J. Environ. Res. Public Health 2014, 11 8348

Keywords: mutagenicity; endocrine-disrupting chemicals; estrogenic activity; processed food; pure water sachet

1. Introduction

Food and drinking water are major sources of human exposure to both mutagens and endocrine-disrupting chemicals (EDCs) globally [1–7]. This is alarming in view of the fact that food and water are prerequisites of human life.

The sources of chemical mutagens in food vary remarkably, depending on the foodstuff and processing methodology. However, emphasis has traditionally been placed on reducing the levels of possible mutagenic residues in meat, grain, vegetables etc. prior to processing, neglecting the possibility of a less clear-cut risk: the formation of these mutagenic compounds in food as a result of processing. Yet, processed food items are reported to contain chemical substances known to have mutagenic, genotoxic and carcinogenic effects, and thus acting as a key global contributor to human cancer risk [8–11]. Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines have been reported in processed food (mainly meat and fish products) at various concentrations all over the world [12–15]. The formation of these chemical mutagens during food processing has been demonstrated to depend on a number of factors such as cooking time, method of cooking and type of heat source [3,8,12]. For example, the Ames test shows a correlation between meat-processing temperature and the number of revertants generated per gram of meat [3,4]. Chung et al. [12] also reported that charcoal-grilled pork contained higher levels of PAHs (10.2 μg/kg) compared with other methods of processing. Likewise, high concentrations of PAHs have been found in smoked-cured fish in Ghana [13]

The contamination by mutagenic PAHs of thermally treated high-protein foods such as charcoal-grilled meat products is mainly due to the direct pyrolysis of food fats and the deposition of PAHs from smoke produced through incomplete combustion of the thermal agents [16]. Unfortunately, this method of food processing is the method of choice in most developing nations, including Nigeria. Although knowledge of proper processing techniques would help reduce the risk of generating mutagenic compounds in food, a recent study showed that only 4.76% of 63 subjects involved in food processing in Nigeria had a formal training in a food safety/hygiene-related discipline [17]. Similar percentages have also been reported in Kenya and Ghana [18,19].

Regarding EDCs, the bulk of information available is on compounds possessing estrogen-like activity. Phytoestrogens and food contact materials are the main sources of human exposure to xenoestrogens in food [20–22]. While the health effects of phytoestrogens remain controversial, synthetic xenoestrogens have been associated with certain cancer types, reproductive disorders, developmental abnormalities and other adverse physiological effects in both humans and wildlife [23–25]. In this light, it is quite worrisome that drinking water sources as well as bottled mineral and flavored waters have been reported to contain estrogenic substances [5–7,26]. The estrogenic activity in bottled mineral and still water is mainly attributed to the prevailing use of several phthalates and other plasticizers including bisphenol A in packaging materials [5,6]. These chemicals are increasingly

Int. J. Environ. Res. Public Health 2014, 11 8349

raising concern, because they may leach into consumer products in normal use [27–29]. There are over 50 chemical compounds authorized for use in food contact materials which are known to have endocrine-disrupting potential [30]. Interestingly, when food contact materials are assessed for their health risk, they are not routinely tested for their endocrine-disrupting potential [31]. However, the Endocrine Society has expressed its concern about the widespread exposure of humans to these chemicals, as they are capable of affecting multiple endpoints within a living system [32].

Chemical mutagens and EDCs in food and water samples have usually been determined by various methods of analytical chemistry. However, these methods suffer from a number of limitations in their ability to elucidate the entire range of chemical mutagens and EDCs in a single experiment, including an unknown number of yet-to-be identified compounds. In vitro assays offer the advantage of detecting all substances that contribute to the functional property (mutagenicity or estrogenic activity) being assessed in food, water and environmental samples. Therefore, in the present study, we sought to determine the genotoxic and estrogenic properties of food and water samples by in vitro assays. We focused on Nigerian products, because the customary food processing methods there are potentially risky in this regard (see above) and because, to the best of our knowledge, such information does not yet exist in the body of scientific literature.

2. Materials and Methods

2.1. Materials

All chemicals used in this study were of analytical grade. The NADP and glucose-6-phosphate used were obtained from Roche Biochem (Stockholm, Sweden). Aroclor-induced S9 from rat liver was purchased from Trinova Biochem (Giessen, Germany). Histidine, potassium chloride, magnesium sulfate, potassium phosphate dibasic anhydrous and sodium ammonium phosphate were purchased from Merck AG (Darmstadt, Germany). Magnesium chloride hexahydrate and citric acid monohydrate were acquired from VWR international (Leuven, Belgium). Biotin, tryptophan, methylcellulose (MC), dimethyl sulfoxide (DMSO), benzo[a]pyrene, 2-aminoanthracene, sodium azide, estradiol, bisphenol A, progesterone and testosterone were purchased from Sigma-Aldrich (Steinheim, Germany). D-Luciferin was obtained from Biotherma (Handen, Sweden). Yeast nitrogen base medium without amino acids was obtained from Becton Dickinson (Franklin Lakes, NJ, USA).

2.2. Microorganisms

The bacteria, Salmonella enterica sv. typhimurium strains TA 100 and TA 98, were obtained from Pasteur’s Institute (Paris Cedex, France). Two recombinant yeast strains Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc [33] were used in this study. In the yeast bioluminescent assay, BMAEREluc/ERα served as a reporter strain, in which the ERα is expressed. Upon ligand binding, the dimerized receptor binds the estrogen response elements in the promoter region of the luc reporter gene. In S. cerevisiae BMA64/luc, luciferase is expressed constitutively, and this strain was used for determination of cytotoxicity of the test samples. Both yeast strains are kind gift donations by Dr. Johanna Rajasärkkä of the Department of Food and Environmental Sciences, Faculty of Agriculture

Int. J. Environ. Res. Public Health 2014, 11 8350

and Forestry, University of Helsinki, Finland. Yeasts were grown on Difco Yeast Nitrogen Base medium without amino acids, supplemented with 40% glucose and their respective amino acids.

2.3. Cell Line

Human hepatocellular carcinoma-derived cell line (HepG2) was obtained from American Type Culture Collection through LGC standards (Boras, Sweden) and cultured in Eagle’s Minimum Essential Medium (LGC standards) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, Steinheim, Germany). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air atmosphere incubator (NuAire Inc., Plymouth, MA, USA).

2.4. Sampling and Sample Preparation

A total of 36 samples (3 lots of 12 varieties) representing commonly consumed, commercially processed food items in Nigeria were evaluated for their mutagenic potential. All varieties were obtained from different vendors since no quality control is carried out in the production of these food items. Moreover, equivalent food products obtained from the same manufacturer have been previously reported to vary in their mutagenic potential [2,4]. Since the major source of xenoestrogens in processed food items are phytoestrogens and food contact materials, and all our food samples were informed to be free of soy (a highly significant source of phytoestrogens) and mostly unpackaged, we targeted water samples as possible sources of exposure to EDCs. Sixteen sachet pure water samples sold in Benin City metropolis, Edo State, Nigeria were acquired for the purpose of this study. Food samples were extracted by solid phase extraction method [4], while possible estrogenic compounds were extracted from the water samples (1000 mL each) by liquid–liquid extraction as described by [34]. The final extracts were concentrated to approximately 2 mL using a rotary evaporator, and the concentrates were shipped on ice to the Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland. Upon arrival, samples were further concentrated to dryness under nitrogen. Food samples were reconstituted in DMSO, while water samples were reconstituted in 5% ethanol for in vitro analyses. Food packaging materials were extracted for possible estrogenic activity as described previously [1].

2.5. Cytotoxicity Assays

The cytotoxic effect of the concentrations of food extracts used in this study was investigated by two independent assays measuring trypan blue exclusion and lactate dehydrogenase (LDH) activity as previously described [2]. Briefly, HepG2 cells were grown in 24-well plates (VWR, Finland) for 48 h, and further exposed to different concentrations of food extracts for 4, 24 or 48 h. After exposure, the cells were trypsinized and centrifuged for 5 min at 2500 rpm. Pellets were then resuspended in PBS, after which 10 μL of the cells were mixed with 5 μL (0.8 mM) trypan blue dye for microscopic observation. LDH activity was performed according to the instructions provided in the Cytotoxicity Detection KitPLUS (LDH), version 6 (Roche Biochem, Stockholm, Sweden).

Int. J. Environ. Res. Public Health 2014, 11 8351

2.6. Mutagenicity Assay

The mutagenic potential of food extracts was initially determined by the standard plate incorporation assay. Samples showing mutagenic potential in this assay were subsequently subjected to “treat-and-wash” as well as methylcellulose overlay assays to ascertain to what degree a localized release of proteins, peptides or histidine from the samples contributed to the outcome.

2.6.1. Standard Plate Incorporation Assay

The standard plate incorporation assay was performed as described by Maron and Ames [35] using Salmonella strains TA 100 and TA 98 with and without metabolic activation (S9 mix). The amount of S9 used in the S9 mix was 10%. Water and DMSO were used as negative controls for both strains while sodium azide (0.04 mg/mL) and 2-aminoanthracene (0.02 mg/mL) served as positive controls for TA 100 and TA 98, respectively. Benzo[a]pyrene (0.1 mg/mL) was also used as a positive control for both strains. The volume of controls used was 50 μL/plate in triplicate plates. Sodium azide is a known direct mutagen in Salmonella TA 100 [36], whereas 2-aminoantracene is metabolically activated by mono-oxygenases of the CYP1A family in rat liver [37]. Likewise, benzo[a]pyrene requires metabolic activation for mutagenicity [38].

For all samples, four different concentrations of the food extracts (25, 50, 100 and 200 mg/mL) were tested in triplicate plates (50 μL/plate). The highest concentration (200 mg/mL) was equivalent to 1 g of the food sample. The plates were incubated at 37 °C for 48 h.

The results of the mutagenic activities are presented as the number of revertant colonies per gram of food sample. Only the mean and standard deviation of the highest concentration for all food extracts are shown.

2.6.2. Treat-and-Wash Assay

The treat-and-wash assay was conducted according to the method described by Thompson et al. [39]. The protocol applied was as per the standard plate incorporation assay with the exception that the S9 mix, bacteria and sample extract were incubated for 90 min prior to the addition of molten top agar. Briefly, a 500 μL aliquot of S9 mix/phosphate buffer (0.2 M, pH 7.4) was combined with 100 μL each of late-log bacterial culture and sample extract solution in a sterile 15 mL tube. The mixture was incubated for 90 min in a mechanical shaker (180 rpm) at 37 °C. The extended duration of bacterial exposure compensated for the absence of bacterial exposure on plates, as the test sample was washed away prior to plating. After a 90-min preincubation, 10 mL of wash solution (Oxoid No. 2 nutrient broth in phosphate-buffered saline (1:7 v/v)) was added, and the washed bacteria were collected by centrifugation at 2,000 g for 30 min. All but approximately 700 μL of the supernatant was removed and discarded, and the bacteria were resuspended in the residual supernatant prior to plating via top agar.

2.6.3. Methylcellulose Overlay Assay

Methylcellulose overlay assay was performed as previously described [39]. Briefly, a 500 μL aliquot of S9 mix/phosphate buffer (0.2 M, pH 7.4) was combined with 100 μL of late-log bacterial culture in a sterile 15 mL tube. A 2 mL aliquot of the MC overlay suspension was added to the tube,

Int. J. Environ. Res. Public Health 2014, 11 8352

and a 100 μL aliquot of the sample extract solution was added immediately afterward. The mixture was overlaid on a pre-warmed (37 °C) minimal glucose plate. Plates were held at 4 °C for 1 h after plating to ensure gelling of the MC overlay, and subsequently incubated (not inverted) at 37 °C for 48–72 h. The MC overlay was prepared on the day of the test, and the mixture was stirred at 50–60 °C throughout use.

2.7. Yeast Bioluminescent Assay

The yeast bioluminescent assay was performed as previously described [1]. Estradiol and bisphenol A were used as positive controls, while progesterone and testosterone served as negative controls.

2.8. Statistical Analysis/Interpretation of Data

The mutagenic potency of each food sample was determined from the slope of the linear portion of the dose-response curve by linear regression analysis using the software program Prisma 4.0 (GraphPad software Inc., San Diego, CA, USA). In addition to the requirement of a statistically significant (p < 0.05) dose-response effect, only those samples were considered mutagenic whose highest test concentration generated at least twice as many revertants as the negative control (DMSO). For proper interpretation and clarity, the number of revertants obtained was compared with both their experiment-specific controls and aggregate controls across all experiments. The p values of these comparisons in the tables are derived from the regression analyses. In the estrogenic activity assays, the fold induction, fold induction corrected (FIC) and limit of detection (LOD) were calculated as described previously [33]. The sigmoidal dose-response curves for increasing concentrations of estradiol and bisphenol A were obtained using Prisma 4.0. The estradiol and bisphenol A equivalents of food samples showing estrogenic activity were calculated from probit transformation of the curves.

3. Results

3.1. Plate Incorporation Assay: Control Substances

The results obtained with the control substances on both strains of Salmonella are presented in Table 1. In the test system, the number of revertants generated by sodium azide was 4–5 fold the negative control (DMSO) in TA 100 strain, both in the presence and absence of metabolic activation (S9 mix). This was an expected outcome, because sodium azide is the recommended direct chemical mutagen for TA 100 strain [36]. Meanwhile, the number of revertants generated by benzo[a]pyrene was 3–4 and 2–3 fold that of DMSO with and without metabolic activation, respectively. In Salmonella TA 98 strain, 2-Aminoanthracene behaved as expected, with the number of revertant colonies being 17–23-fold higher than that of the control substance, in the presence of S9 mix. On the other hand, only a 2–3-fold increment was observed in the absence of S9 mix. No mutagenic effect/potency was observed with benzo[a]pyrene in the absence of metabolic activation; co-incubation with S9 resulted in an approximately three-fold increment in colony formation.

Int. J. Environ. Res. Public Health 2014, 11 8353

Table 1. Ranges for revertant colonies obtained with control substances in the standard plate incorporation assay.

Controls Number of Revertant Colonies

Respective Controls * Aggregate Controls ** +S9 −S9 +S9 −S9

Salmonella TA 100 Water 134.3–177.7 112.7–134.3 161.8 ± 22.4 125.2 ± 11.0 DMSO 138.7–166.3 111.0–128.3 155.3 ± 17.9 120.2 ± 11.3

Sodium azide 568.7–651.0 432.7–516.3 621.6 ± 78.8 470.1 ± 42.7 Benzo[a]pyrene 438.0–517.3 248.0–270.0 471.6 ± 48.5 260.7 ± 75.7

Salmonella TA 98 Water 32.3–42.7 19.0–34.0 37.4 ± 3.1 25.0 ± 4.8 DMSO 35.7–36.7 19.0–24.0 36.2 ± 2.8 21.5 ± 3.6

2-Aminoanthracene 624.3–837.7 55.0–69.3 754.2 ± 68.2 61.4 ± 11.2 Benzo[a]pyrene 102.0–122.0 20.3–29.7 116.8 ± 10.4 24.0 ± 5.1

Notes: * Range; ** Mean ± SD.

3.2. Plate Incorporation Assay: Test Substances/Food Samples

The mutagenic activity of commercially processed food items, obtained by the standard plate incorporation assay, is presented in Tables 2 and 3. The majority of samples investigated (75%) exhibited fairly high mutagenic activity (the maximal responses being comparable to those elicited by benzo[a]pyrene), mainly in Salmonella TA100 strain. However, there was notable lot-to-lot variation.

In TA 100 strain, chin-chin, hamburger, suya and bean cake were the most mutagenic food samples investigated. The number of revertants generated by these samples was over twice that of DMSO in all the batches analyzed and mostly independent of the S9 mix. A somewhat surprising result was found with the potato products (french fries and potato chips), as at least one of the lots of both products proved directly mutagenic. Roasted maize, plantain chips and coconut-candy did not show any evidence of mutagenic potency in this strain (Table 2).

In Salmonella TA 98, only three food or snack varieties (potato chips, peanut and suya) exhibited mutagenic potency in at least one of the batches investigated (Table 3). Suya displayed the most coherent outcome with all its three batches being mutagenic in the presence of S9 mix. In support of the result with TA 100, the same batch of potato chips (number 3) exhibited direct mutagenicity also in TA 98.

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193.

3 ±

07.0

18

8.7

± 06

.5

256.

7 ±

11.4

†,ф

Plan

tain

chi

ps

124.

7 ±

11.8

10

9.3

± 01

.5

159.

3 ±

27.3

19

2.7

± 11

.1

185.

7 ±

11.1

15

4.7

± 06

.5

Pean

ut

293.

3 ±

30.6

† 24

2.7

± 04

.6 †,ф

339.

0 ±

62.6

†,ф

272.

7 ±

61.6

†,ф

215.

0 ±

26.2

17

5.3

± 28

.4

Roa

sted

mai

ze

252.

3 ±

23.0

18

7.3

± 04

.7

176.

7 ±

29.9

15

4.0

± 07

.0

215.

7 ±

20.0

20

2.7

± 18

.5

Suya

38

3.0

± 20

.7 †,ф

240.

7 ±

10.0

†,ф

401.

7 ±

12.1

†,ф

296.

0 ±

06.0

†,ф

308.

7 ±

14.6

26

3.3

± 05

.8 †,ф

Frie

d ch

icke

n 13

8.7

± 06

.0

133.

0 ±

08.5

39

4.7

± 14

.7 †,ф

204.

3 ±

06.0

38

1.0

± 35

.5 †,ф

156.

0 ±

23.5

B

ean

cake

31

2.0

± 25

.4 †,ф

241.

7 ±

36.8

†,ф

365.

0 ±

22.6

†,ф

159.

0 ±

25.9

29

4.7

± 12

.3

278.

7 ±

09.1

†,ф

Not

es: †

: Sig

nific

antly

diff

eren

t fro

m re

spec

tive

cont

rols

(p <

0.0

5); ф

: Sig

nific

antly

diff

eren

t fro

m a

ggre

gate

con

trol (

p <

0.05

).

Int.

J. E

nvir

on. R

es. P

ublic

Hea

lth 2

014,

11

8355

Tab

le 3

. Num

ber

of r

ever

tant

s ge

nera

ted

by th

e hi

ghes

t con

cent

ratio

ns (

1.0

per

g of

foo

d sa

mpl

e) o

f fo

od e

xtra

cts

on S

alm

onel

la T

A 9

8

(mea

n ±

SD) i

n th

e st

anda

rd p

late

inco

rpor

atio

n as

say.

Food

Pro

duct

s R

ever

tant

s per

Gra

m

Bat

ch 1

B

atch

2

Bat

ch 3

+S

9 −S

9 +S

9 −S

9 +S

9 −S

9 D

ough

nut

40.3

± 8

.4

22.0

± 8

.2

30.7

± 1

1.6

19.0

± 4

.6

28.0

± 3

.0

19.0

± 6

.1

Chi

n-ch

in

34.0

± 1

.7

23.3

± 2

.1

31.0

± 7

.9

20.0

± 5

.3

37.7

± 6

.5

27.3

± 4

.2

Ham

burg

er

46.0

± 1

4.0

26.3

± 3

.1

31.7

± 4

.5

20.7

± 3

.5

43.0

± 7

.0

20.7

± 2

.1

Coc

onut

-can

dy

38.7

± 4

.9

24.3

± 1

.6

40.7

± 6

.1

25.3

± 5

.1

34.7

± 9

.3

21.3

± 1

.5

Fren

ch fr

ies

32.3

± 1

.5

36.0

± 6

.6

30.3

± 3

.5

17.0

± 2

.0

31.3

± 4

.0

18.7

± 6

.4

Pota

to c

hips

36

.3 ±

5.9

26

.7 ±

3.8

33

.0 ±

4.6

26

.3 ±

4.0

31

.7 ±

2.1

72

.7 ±

18.

0 †,ф

Plan

tain

chi

ps

29.0

± 5

.6

29.0

± 2

.6

32.3

± 2

.9

18.7

± 5

.5

38.3

± 6

.1

21.0

± 4

.6

Pean

ut

69.7

± 5

.6

34.7

± 4

.0

78.0

± 1

2.5 †,ф

37.0

± 1

4.9

35.0

± 2

.0

27.3

± 5

.0

Roa

sted

mai

ze

24.3

± 2

.1

20.0

± 2

.0

31.7

± 4

.9

28.0

± 5

.3

27.3

± 5

.0

23.7

± 7

.4

Suya

86

.7 ±

5.8

†,ф

27.7

± 2

.5

83.0

± 3

.5 †,ф

19.0

± 4

.6

97.3

± 7

.6 †,ф

20.7

± 3

.5

Frie

d ch

icke

n 32

.0 ±

5.0

28

.3 ±

5.1

24

.0 ±

4.0

19

.7 ±

3.8

28

.3 ±

3.2

20

.7 ±

2.5

B

ean

cake

32

.0 ±

5.3

11

.0 ±

1.7

28

.7 ±

8.5

22

.3 ±

5.8

32

.3 ±

3.1

21

.0 ±

2.0

N

otes

: †: S

igni

fican

tly d

iffer

ent f

rom

resp

ectiv

e co

ntro

ls (p

< 0

.05)

; ф: S

igni

fican

tly d

iffer

ent f

rom

agg

rega

te c

ontro

l (p

< 0.

05).

Int. J. Environ. Res. Public Health 2014, 11 8356

3.3. Modified Ames Tests

To ascertain to which degree a localized release of proteins, peptides or histidine contributed to the mutagenicity test results obtained with the standard plate incorporation assay, “treat-and-wash” as well as MC overlay assays were performed (Tables 4 and 5). The outcome proved to depend on bacterial strain, type of food, S9 status, and assay. For some food extracts initially found to be mutagenic in the standard plate incorporation assay, the number of revertants decreased below the two-fold limit level in comparison with the negative control. Hence, the original Ames test result was in these cases interpreted to be of secondary nature and not due to genuine mutations. However, in a large number of cases, the food extracts were mutagenic in all three assays both in the presence and absence of S9 mix. For some food items (hamburger, suya and bean cake), a single lot was mutagenic in all three assays but only in the presence of S9 mix. In the absence of S9, the outcome with these three products varied. In contrast to this pattern, a single batch of hamburger (batch 2) was mutagenic in all three assays, both in the presence and absence of S9 mix. A couple of surprises also emerged in these complementary assays. Extracts of fried chicken (batch 2) and bean cake (batch 2) that required metabolic activation for their mutagenicity in the standard plate incorporation assay, were, unexpectedly, directly mutagenic in the treat-and-wash assay in Salmonella TA 100 strain (Table 4). One of these samples (bean cake, batch 2) behaved the same way also in the MC overlay assay (Table 4). An identical shift from indirect to direct mutagen was recorded in Salmonella TA 98 strain for extracts of peanut (batch 2) and suya (batch 2) (Table 5).

3.4. Cytotoxicity Assays

The cytotoxicity of the four concentrations of all food extracts was determined by both trypan blue exclusion and LDH secretion assays in HepG2 human hepatocellular carcinoma cells. The non-survival percentage of HepG2 cells in the trypan blue exclusion test did not exceed 50%. Also, there was a significant difference between the positive control (lysis solution) and the test samples in the amount of LDH released. Hence, the extracts were classified non-cytotoxic in these assays following exposure for 4, 24 or 48 h.

3.5. Estrogenic Activity Assay: Control Substances

The positive and negative control compounds used in this study behaved as expected with the S. cerevisiae BMAEREluc/ERα yeast strain. Both positive controls (estradiol and bisphenol A) produced a sigmoidal dose-response curve (Figure 1), while the negative controls (progesterone and testosterone) did not elicit any luciferase activity in the test system. This is in keeping with previously published data [29,40]. The limit of detection (LOD) in the yeast bioluminescent assay was 2.4-fold induction corrected (FIC), corresponding to 76 fM and 1.2 nM of estradiol and bisphenol A, respectively.

Int. J. Environ. Res. Public Health 2014, 11 8357

Table 4. Number of revertants in the treat-and-wash as well as methylcellulose overlay assays generated by the highest concentrations (1.0 per g of food sample) of food extracts showing mutagenic potential on Salmonella TA 100 (mean ± SD) in the standard plate incorporation assay.

Food Product Batch Revertants per Gram

Treat-and-Wash Assay Methylcellulose Overlay Assay +S9 −S9 +S9 −S9

Doughnut 1 115.0 ± 4.2 84.3 ± 8.7 215.0 ± 34.6 191.3 ± 10.6 Chin-chin 1 192.7 ± 17.5 58.7 ± 7.8 128.3 ± 9.8 95.0 ± 11.4

Hamburger 1 633.0 ± 23.3 * 124.7 ± 10.6 330.0 ± 10.4 * 166.7 ± 28.3 French fries 1 121.3 ± 11.7 103.0 ± 1.4 84.3 ± 22.6 86.0 ± 4.9

Peanut 1 108.0 ± 7.1 123.5 ± 13.4 182.3 ± 26.2 93.0 ± 15.6 Suya 1 366.0 ± 22.6 * 113.0 ± 1.4 382.0 ± 17.2 * 271.7 ± 9.4 *

Bean cake 1 736.3 ± 85.1 * 156.0 ± 13.9 * 401.0 ± 28.4 * 238.2 ± 12.1 Chin-chin 2 618.7 ± 58.7 * 32.3 ± 12.5 126.0 ± 8.5 125.7 ± 10.6

Hamburger 2 397.7 ± 21.2 * 408.0 ± 32.5 * 304.7 ± 19.4 * 263.3 ± 9.3 * Peanut 2 141.3 ± 23.3 42.0 ± 11.3 344.3 ± 31.8 * 201.3 ± 2.1 Suya 2 181.0 ± 16.9 240.0 ± 42.4 * 165.0 ± 10.6 76.0 ± 5.0

Fried chicken 2 174.3 ± 12.4 150.3 ± 12.8 * 192.3 ± 13.9 138.3 ± 12.4 Bean cake 2 470.0 ± 16.3 * 260.7 ± 33.2 * 324.7 ± 28.6 * 271.3 ± 19.4 * Chin-chin 3 126.3 ± 12.0 131.7 ± 10.6 183.3 ± 9.9 164.0 ± 16.3

Hamburger 3 135.0 ± 18.4 126.3 ± 9.2 166.7 ± 0.7 162.0 ± 22.6 French fries 3 139.7 ± 6.8 97.0 ± 8.5 110.0 ± 3.5 95.3 ± 2.1 Potato chips 3 194.7 ± 11.6 108.3 ± 9.4 90.7 ± 10.0 106.0 ± 18.2

Suya 3 179.0 ± 9.9 209.0 ± 19.7 * 194.3 ± 7.8 271.0 ± 14.4 * Bean cake 3 371.3 ± 12.1 * 228.0 ± 23.3 * 267.7 ± 14.3 290.0 ± 21.9 *

Fried chicken 3 117.7 ± 12.3 108.3 ± 11.4 158.0 ± 12.8 124.3 ± 11.4 Note: *: Significantly different from control (p < 0.05).

Table 5. Number of revertants in the treat-and-wash as well as methylcellulose overlay assays generated by the highest concentrations (1.0 per g of food sample) of food extracts showing mutagenic potential on Salmonella TA 98 (mean ± SD) in the standard plate incorporation assay.

Food Product Batch Revertants per Gram

Treat-and-Wash Assay Methylcellulose Overlay Assay +S9 −S9 +S9 −S9

Suya 1 78.0 ± 8.5 * 17.0 ± 1.4 48.0 ± 4.8 24.3 ± 2.7 Peanut 2 30.0 ± 8.5 158.0 ± 51.0 * 31.3 ± 5.0 84.3 ± 9.6 * Suya 2 33.3 ± 5.0 154.0 ± 0.0 * 29.3 ± 1.9 78.7 ± 6.6 *

Potato chips 3 48.3 ± 7.2 21.7 ± 4.1 33.0 ± 2.4 19.3 ± 2.1 Suya 3 42.0 ± 9.9 18.0 ± 3.1 39.7 ± 5.2 26.7 ± 3.8

Note: *: Significantly different from control (p < 0.05).

Int. J. Environ. Res. Public Health 2014, 11 8358

Figure 1. Dose-response curves of increasing concentrations of estradiol and bisphenol A.

3.6. Estrogenic Activity of Pure Water Sachets

The estrogenic activities of the 16 pure water samples investigated ranged from below LOD to 44.0 ng/L (median: 23.0 ng/L) estradiol equivalent (the amount of estradiol needed to bring about the same effect as the sample analyzed in an assay specific for estrogens) concentrations (EEQs). Five out of the 16 sachets produced luciferase activities greater than the LOD. The positive water samples were coded W1 to W5 (Table 6). W1 had the lowest value of 0.79 ng/L or 124.2 ng/L estradiol vs. bisphenol A equivalent concentrations, respectively. Concurrently, the highest values found (for sample W2) extended to 44.0 ng/L (estradiol equivalent) or 1000.8 ng/L (bisphenol A equivalent).

Table 6. Estradiol (EEQ) and bisphenol A (BPAEQ) equivalent concentrations of sachet water samples.

Sample Code Water Samples Sachet/Packaging Material

EEQs (ng/L) BPAEQs (ng/L) EEQs (pg/L) BPAEQ (pg/L) W1 0.79 124.2 14.5 224.0 W2 44.0 1000.8 <LOD <LOD W3 28.0 597.8 10.2 186.1 W4 23.0 442.8 <LOD <LOD W5 15.0 269.7 <LOD <LOD

Median 23.0 443.0 12.4 205.0 Average 7.0 152.0 2.0 26.0

As an attempt to further trace the origins of the estrogenic activities observed, the sachets themselves were analyzed for possible leaching of estrogenic substances into the water. The packaging material of three out of the five positive samples did not generate any positive signal in the yeast-based assay. However, a feeble response was obtained from the packaging material of the two other samples (Table 6).

Int. J. Environ. Res. Public Health 2014, 11 8359

4. Discussion and Conclusions

Processed food items and bottled water are consumed in increasing quantities all over the world. Therefore, it is of utmost importance to ensure that in addition to their microbial safety, the products do not contain chemicals which might pose a toxicological risk to consumer health. A conceivable potential risk in this regard is the formation of genotoxic compounds during the processing of foodstuffs and leaching of food contact materials into food and water. Regular screening studies are necessary to verify that the methods used by food vendors are appropriate and sound also from this point of view. The present investigation aimed at exploring the current situation in Nigeria.

To the best of our knowledge, this is the first study on mutagenicity of food products from Africa. To compare our data with those of previous studies is challenging because of the paucity of published data on mixture effects combined with the wide variation in food types in different parts of the world. However, it is possible to compare foodstuffs based on the number of revertants their extracts generate in the Ames test and its derivatives, and we will utilize this approach.

Food processing methods as well as the sales of processed food items in Nigeria are poorly—if ever—regulated. Furthermore, it has been reported that the majority of Nigerians involved in food processing do not have formal training on food safety issues or related techniques [17,41]. This may bear on the present finding that the majority of food items (75%) investigated were mutagenic in the standard plate incorporation assay for at least one of the three batches when studied in Salmonella TA 100 strain. On the other hand, in Salmonella TA 98 strain only 25% of food extracts were found to yield a mutagenic response, possibly due to a weaker sensitivity of this strain compared with TA 100 or to the type of mutagens present.

The conventional Ames test outcome cannot, however, be taken at its face value in the case of food extracts as these may be sources of localized release of proteins, peptides or histidine itself onto the bacterial plates [2]. To prevent this potential misinterpretation of ostensible mutagenicity, “treat-and-wash” as well as methylcellulose overlay assays were performed for all samples eliciting a positive outcome in the conventional Ames test. The results of these complementary assays were consistent for some samples (bean cake, suya, hamburger, fried chicken and chin-chin), further reinforcing our initial findings with the Ames test. Extracts of bean cake and suya stood out from among the positive samples. All batches of bean cake exhibited mutagenic activity in the treat-and-wash assay with the Salmonella TA 100 strain, both in the presence and absence of S9 mix. One of these lots (batch 1) generated a conspicuously high number of revertants, almost five-fold its control (DMSO), with metabolic activation. A similar situation was observed with the MC overlay assay, as all bean cake samples were mutagenic in Salmonella TA 100 strain in the presence of S9 mix. Similarly, all batches of suya were consistently mutagenic in the treat-and-wash assay, with the Salmonella TA 100 strain, either in the presence or absence of S9 mix.

Bean cake is commonly consumed in different parts of Nigeria, irrespective of ethnicity, religion or social status. A probable explanation for the mutagenicity test results observed with extracts of bean cake is in the method of its processing. Bean cake is processed by deep-frying for several minutes. Deep-frying has previously been reported to result in the formation of mutagenic and genotoxic compounds in the final product [42]. Food vendors in Nigeria are also known to repeatedly reuse their frying oil, which is often already of questionable quality, for several days or weeks. This may have

Int. J. Environ. Res. Public Health 2014, 11 8360

contributed to the high number of revertants obtained with extracts of bean cake and fried chicken. Double heat-treatment of cooking oil has been shown to cause an increase in the genotoxic activity of food products [43,44]. During frying, cooking oil undergoes deterioration through various chemical and physical processes such as oxidation, polymerization, hydrolysis and cyclization, leading to the formation of both volatile and non-volatile undesirable by-products [43]. These derivatives are partially absorbed by the fried food, which thus becomes carcinogenic [45]. For example, the PAH compounds benzo[a]pyrene and benzo[a]anthracene are all well-known human carcinogens which have been detected in different types of cooking oil [45].

The positive mutagenicity test results obtained with suya were not unexpected. Suya is 100% beef, and it is a special type of delicacy, mainly consumed in Nigeria, irrespective of social status. All suya products are processed the same way: by charcoal-grilling. After processing, the products are left to be heated on the charcoal for several hours, until they are purchased. This processing method typically explains the reason for the mutagenicity test results obtained with extracts of suya in our study. The contamination of thermally treated high-protein foods, such as charcoal-grilled meat products, by PAHs and heterocyclic aromatic amines is well established [12,46–48]. The building up of PAHs in this case is due to their generation by direct pyrolysis of food fats and the direct deposition of PAHs from smoke produced through incomplete combustion of the thermal agent [16]. Heterocyclic aromatic amines, in turn, are formed through the condensation of creatine/creatinine and the strecker degradation radicals (pyridines and pyrazines) generated from the reaction of sugars and amino acids during the Maillard reaction [49]. The present findings are worrisome, because meat-cooking habits have been linked with several forms of cancer [50–53]. In Argentina, for example, cooking meat at a high temperature and close to the cooking source has been linked with increased incidence of colorectal cancer [54]. This is also the case in Hawaii and the Netherlands [55,56]. More recently, a number of PAHs have been reported in different types of smoked meat in Serbia, Latvia and Sweden [14,57,58]. However, no nexus has been established in relation to increased incidence of cancer in these countries. In Nigeria, there is a paucity of data on the incidence of different cancer types, but the two major forms, breast and prostate cancers, may be increasing [59]. Both of them have been associated with meat-cooking habits [60].

Hamburger products have previously been reported as a major source of chemical food mutagens to consumers [38,61]. The results obtained in our study further reinforce this view, as two different lots of hamburgers examined were found to be mutagenic in all three assays in Salmonella TA 100 strain, with one of the lots (batch 2) being both directly and indirectly mutagenic in all three assays. Stavric et al. [61] previously reported hamburger products purchased in Ontario, Canada, to be mutagenic in a similar assay, but only with Salmonella TA 98 strain. The number of revertants generated in that study ranged from 63 to 1042 rev/g (average: 199 rev/g). This is in contrast to our study, in which hamburger products were only mutagenic with the Salmonella TA 100 strain, and not TA 98. In a recent study in Finland [2], the number of revertants generated with extracts of hamburger products were slightly lower than those obtained in this study, both with Salmonella TA 100 and 98 strains. Although these findings might seem to implicate the current cooking methods of hamburgers in Nigeria, the present outcome may not be entirely attributable to the processing methods. This is due to the fact that high levels of potassium bromate, a well-known mutagen and human carcinogen, have been detected in bread in different parts of Nigeria [62–64]. In one of these cases, Alli et al. [62]

Int. J. Environ. Res. Public Health 2014, 11 8361

found that even the lowest level of potassium bromate in their bread samples was over 150 times higher than the maximal permissible limit.

Overall, the mutagenicity test outcome of our study is in keeping with previously published data on food mutagenicity elsewhere [3,4,61], but somewhat at odds with a recent study published in Finland, where only 40% of the processed food items investigated showed mutagenic properties in the conventional Ames test [2]. In further contrast with the current findings, for most food varieties in the study by Omoruyi and Pohjanvirta [2], only a single batch proved positive. This may reflect more refined food processing techniques in Finland as compared with Nigeria.

In Nigeria, it is estimated that about 25% and 53% of people living in urban and rural areas, respectively, lack access to pure, portable water [65]. This is related to recent outbreaks of several water-borne diseases in major states of the country, specifically cholera [66,67]. It has prompted entrepreneurs to continuously establish water plants, in which pure water samples are mainly packaged in plastic sachets.

Our study demonstrates that pure water sachets may contain estrogen-like chemicals. Five of the 16 samples investigated were discovered to be estrogenic in our in vitro test system, with EEQs ranging from 0.79–44.0 ng/L. Both the frequency of positive samples and their concentrations were actually lower than we feared, considering that the proprietors of pure water sachet factories in Nigeria are principally entrepreneurs with little or no knowledge of water quality (microbiological, physicochemical or toxicological). There are two recent studies carried out in Europe in which estrogenic activity of water samples was assessed by a comparable in vitro yeast assay to that of ours. Pinto and Reali [68] analyzed mineral waters packed in polyethylene terephthalate (PET) bottles in Italy. The levels they detected varied from 0.03 through 23.1 ng/L (mode 9.5 ng/L) EEQs. Somewhat surprisingly, tap water made of either surface water or ground water contained approximately 15 ng/L EEQs. In another study, Wagner and Oehlmann [6] determined estrogenic activities in 20 major brands of bottled water in Germany. Twelve of these samples proved positive with the levels ranging from 2.64 to 75.2 ng EEQ/L (average 18.0 ng/L). Interestingly, in their material, the highest estrogenic activities were recorded for waters packaged in either non-reusable PET or reusable glass bottles, and even water packed in Tetra Pak™ bricks contained levels that were similar to those found in our study (14–44 ng/L). Thus, substances exhibiting estrogen-like activity are common in water samples in both industrialized and developing countries.

It is widely believed that the decline in male reproductive functions, increased incidence of different cancer types amongst young men and women and neurobehavioural diseases observed in the population of different countries may, at least partly, be attributable to exposure to estrogenic compounds, particularly during the intrauterine phase or during critical periods of postnatal development [23–25,69]. Studies in recent years have shown, for example, that the commonly used plasticizer, di(2-ethylexyl)phthalate (DEHP), alters gene expression in rats and that, at appropriate concentrations, it alters the development of the central nervous system in the fetus [70]. Similarly, certain compounds, such as benzophenone used as food contact material, are reported to almost completely block the 17β-hydroxysteroid dehydrogenase type 3 enzymes that are required for testosterone synthesis [71].

The presence in or leaching into water samples of endocrine-disrupting chemicals is influenced by a number of factors such as storage conditions, exposure to sunlight and ambient temperature [72,73]. Unfortunately, the environmental conditions in Nigeria (abundant sunlight and high temperature) tend

Int. J. Environ. Res. Public Health 2014, 11 8362

to favor the migration of endocrine-disrupting chemicals from the packaging materials into water, as, for example, during transport of water containers. Therefore, sachets of water stored or transported in less appropriate conditions than our samples would be at risk of containing higher concentrations of estrogenic substances.

The bulk of dietary xenoestrogen exposure for adults has been proposed to emanate from dairy products, and total daily intake of estrogens has been estimated to be 80–100 ng [74]. Assuming an average daily water consumption of 3 L at Nigerian latitude [75], in the worst-case scenario based on our sample material, the intake from pure water sachets would double the estimated exposure. Hence, every effort should be taken to reduce the estrogen levels in these waters in the future.

In conclusion, the results obtained in our study show that both commercially processed food items and sachet-packed pure water sold in Nigeria, are sources of mutagen and estrogen-like chemicals, respectively. Although their concentrations are not alarming in the light of food and water analyses from other countries, measures should be taken to reduce them further and monitor their levels regularly. Since the number of samples examined here was relatively low, further survey studies are also warranted.

Acknowledgments

This research was supported by grants from the Research Foundation of the University of Helsinki, Finland, Walter Ehrström Foundation, Finland, and Benson Idahosa University, Benin City, Nigeria.

Author Contributions

Iyekhoetin Matthew Omoruyi was involved in planning the study, conducting the research and writing the manuscript; Derek Ahamioje did the extraction of food and water samples; Raimo Pohjanvirta was involved in planning the study, supervising the research and writing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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IV

© 2015 the Nordic Societies of Public HealthDOI: 10.1177/1403494815591686

Scandinavian Journal of Public Health, 770–775

Introduction

In recent times, there has been an increased aware-ness of human exposure to exogenous estrogenic chemicals and an intensified debate on the effects of such exposure on humans over time. Examples of known xenoestrogens include bisphenol A, polyvinyl-chloride (PVC), di(2-ethylhexyl)phthalate (DEHP), polychlorinated biphenyls (PCBs), dioxin, and dioxin-like chemicals, etc. These chemicals have been reported in most of the daily products used by man,

including food, food cans, plastic bottles, toys, liners of metals, and some pesticides [1,2].

Influent and effluent waters from wastewater treat-ment plants (WWTPs) have also been shown to be major potential sources of estrogenic substances to both terrestrial and aquatic environments. Table I gives a summary of specific endocrine-disrupting com-pounds (EDCs) reported in influent and effluent waters from WWTPs in different parts of the world [3–12].

Estrogenic activity of wastewater, bottled waters and tap water in

Finland as assessed by a yeast bio-reporter assay

IYEKHOETIN MATTHEW OMORUYI & RAIMO POHJANVIRTA

Department of Food Hygiene and Environmental Health (Food and Environmental Toxicology Unit), Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland

Abstract

Aims: Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled drinking water are potential, but yet poorly characterized, sources of human exposure to endocrine disrupting chemicals globally. Here, we investigated the current situation in the most densely populated region in Finland. Methods: Influent and effluent bi-monthly samples from a major wastewater treatment plant in Helsinki were obtained over a preceding 2-year period at two time-points (in 2011 and 2014). Equivalent samples from a household water purification plant (located in the same region) were also analyzed, together with various brands of bottled still and mineral water as well as tap water from residential buildings. Samples were obtained in one liter sterile containers, extracted by solid-phase extraction method, and their estrogenic potential determined by a yeast bioluminescent assay. Results: The estrogenic activities of influent samples from the wastewater treatment plant in Helsinki were generally low (from less than limit of detection to 0.7 ng/L estrogen equivalent quantities (EEQ)), except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/L EEQ, respectively) were obtained. Meanwhile, no estrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, influent and effluent samples from the drinking water plant, as well as tap water, bottled still, and mineral waters. Conclusions: These findings indicate that the purification method applied in Helsinki

wastewater treatment plant, activated sludge with mechanical, chemical and biological purification steps, is

effective in reducing estrogenic activity, and that tap or bottled waters are not a significant source of these

compounds to the population in this region.

Key Words: Endocrine-disrupting chemicals, wastewater, estrogenic activity, drinking water, tap water, mineral water, still water, bioassays

Correspondence: Iyekhoetin Matthew Omoruyi, Department of Food Hygiene and Environmental Health (Food and Environmental Toxicology Unit), Faculty of Veterinary Medicine, Agnes Sjöbergin Katu 2, University of Helsinki, P.O. Box 66, 00014, Helsinki, Finland. E-mail: matthew.omoruyi@helsinki.fi

(Accepted 24 May 2015)

591686SJP0010.1177/1403494815591686IM Omoruyi et al.Estrogenic activity and waterresearch-article2015

ORIGINAL ARTICLE

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Estrogenic activity of water samples 771

The concentrations of EDCs in the environment are poorly documented globally, and are, indeed, a difficult task to undertake. However, the overall pres-ence of these compounds in the environment is wor-risome due to an increase in hormone-related disorders, especially in industrialized countries, and their shown linkages with EDC exposure [13]. From the point of view of risk assessment, EDCs present a particular challenge because they may have non-monotonous dose–response curves not adequately covered by conventional toxicological experimenta-tion, and because of their capability of causing unto-ward impacts at environmentally prevailing low concentrations [14]. Although limited scientific information is available on the potential adverse

human health effects of EDC exposure, concern arises because EDCs present in the environment at very low concentrations have been shown to have adverse effects in wildlife species as well as in labora-tory animals [15–17]. The difficulty of assessing pub-lic health effects is magnified by the fact that people are typically exposed to multiple endocrine disrup-tors simultaneously.

In fish, the impacts of EDCs include altered sex-ual development, appearance of intersex individuals, and changed mating behavior. High incidence of intersexuality has been reported in roach and wall-eye populations in rivers receiving effluents from municipal WWTPs that contain estrogenic hor-mones [15]. Chronic exposure of fathead minnows

Table I. Summary of specific EDCs reported in different types of influent and effluent samples from WWTPs in different parts of the world.

Country Matrix Estrogenic compound/range Detection method/Assay Reference

Australia Effluent (WWTP) Estrone: 0.35–29.0 ng/L ELISA 3 Estradiol: 0.27–0.35 ng/L Estuary Estrone: 2.25–23.16 ng/L ELISA Estradiol: < 0.025–0.18 ng/L Fresh water Estradiol: 2.95–7.26 ng/L ELISA Estradiol: 0.05–0.33 ng/L Republic of Korea Sewage effluent Estrone: 4.32–5.99 ng/L Yeast two-hybrid 4 Estradiol: nd Bisphenol A: nd–91.29 ng/L Industrial effluent Estrone: nd–5.30 ng/L Estradiol: nd Bisphenol A: nd–1501 ng/L Livestock effluent Estrone: nd–21.9 ng/L Estradiol: nd–18.5 ng/L Bisphenol A: nd–198 ng/L China Influent (WWTP) Estrone: 11.6–110 ng/L GC-MS 5 Estradiol: 3.7–140 ng/L Estriol: nd–760 ng/L Effluent (WWTP) Estrone: 0.2–74.2 ng/L Estradiol: nd–3.9 ng/L Estriol: nd–5.1 ng/L China Influent (WWTP) 1136 ± 269 ng/L EEQ YES 6 Primary effluent 1417 ± 320 ng/L EEQ Anaerobic effluent 959 ± 69 ng/L EEQ Czech Republic Effluent (WWTP) nd–4.8 ng/L EEQ YBA 7Italy Textile effluent 0.72–4.18 ng/L EEQ E-Screen, MELN RGA 8South Korea Surface water Estrone: 3.6–69.1 ng/L E-Screen 9 Waste water Estradiol: 1.2–10.7 ng/L Bisphenol A: 7.5–335 ng/L France Influent (WWTP) nd–25.0 ng/L EEQ SRB 10Spain Influent (WWTP) Estrone: < 2.5–115 ng/L LC/MS 11 Estradiol: 11.0–30.4 ng/L Effluent (WWTP) Estrone: < 2.5–8.1 ng/L Estradiol: < 5.0 ng/L Germany Influent (WWTP) Estrone: 54.9–76.6 ng/L GC/MS 12 Estradiol: 12.2–19.5 ng/L Effluent (WWTP) Estrone: < 1 ng/L Estradiol: < 1 ng/L

nd: not detected; EEQ: estradiol equivalent concentration; ELISA: enzyme linked immunosorbent assay; YES: yeast estrogen screen; YBA: yeast bioluminescent assay; SRB: steroid hormone receptor/reporter gene bioassay; RGA: gene reporter assay; WWTP: wastewater treatment plant.

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to environmentally relevant concentrations of EDCs in an experimental lake area resulted in the femini-zation of males with induced vitellogenin produc-tion; this led to a near wiping out of the species in the lake. Also, early exposure to ethinyl estradiol at a concentration of 9.86 ng/L resulted in diminished courting behavior of female zebrafish and reduced female reproductive success [16]. More recently, Marmuqi et al. [17] reported that long-term expo-sure of mice to bisphenol A during adulthood leads to hyperglycemia and hypercholesterolemia. On the other hand, in humans the evidence is less solid. Studies have reported lowered sperm count, declin-ing male reproductive health and elevated incidence of breast cancer as an aftermath of increased expo-sure to EDCs [18]. In Finland, the incidence of tes-ticular germ cell cancer (TGCC) is on the increase and is attributed to environmental factors such as EDCs [19]. Also, the use of postmenopausal hor-mone therapy drugs in Finland between 1995 and 2007 has been associated with an increased risk of primary fallopian tube carcinoma [20].

With compelling or emerging evidence of the impact of EDCs in wildlife and humans, respectively, it is of utmost importance to continuously evaluate the possible sources of human exposure to these chemicals. The current study was aimed at determin-ing the current situation in the most densely popu-lated region in Finland with respect to potential exposure to xenoestrogens.

Materials and methods

Sampling and description of study site

Treated and untreated water samples, taken bi-monthly, were obtained from the WWTPs in Viikinmäki, Helsinki, Finland, over preceding 20-month periods, on two occasions, in 2011 and 2014. Viikinmäki is the largest WWTP plant in Finland, serving over one million inhabitants, and processing both household (85%) and industrial (15%) wastewater from five different districts (Helsinki, Kerava, Tuusula, Järvenpää, Sipoo, and from the central and eastern districts of Vantaa) in Finland. It receives approximately 270,000 m3 of wastewater per day, and an average 100 million m3 each year. The influent samples were taken before any treatment of the wastewater and the effluent samples at the end of the purification process, right before discharge into the tunnel that leads to the Baltic Sea. At each time-point, both represent 24-hour stream-weighted aggregate samples of the same day.

Equivalent samples from a household water puri-fication plant (located in the same region) were also

obtained in March, April, and June of 2014. Crude water for the household water plant in Viikinmäki, Helsinki, comes from Lake Päijänne (located in central Finland) through a 120-km long tunnel (Figure 1).

Tap water (hot and cold) samples were collected twice a month both from the premises of the University of Helsinki, Viikki campus, Helsinki, and a residential building in Vantaa, Finland, over a 3-month (March–May) period in 2014. Ten different brands each of bottled still and mineral waters (Supplementary Table I) were purchased from a local grocery store (Prisma, Viikki, Helsinki).

Chemicals and medium

Estradiol, progesterone, and testosterone were pur-chased from Sigma-Aldrich (Steinheim, Germany). D-Luciferin was obtained from Biotherma (Handen, Sweden). Yeast nitrogen base medium without amino acids was obtained from Becton Dickinson (New Jersey, USA).

Microorganisms

Two recombinant yeast strains Saccharomyces cerevi-siae BMAEREluc/ER and S. cerevisiae BMA64/luc [21] were used in this study. BMAEREluc/ER served as a reporter strain, in which the ER is expressed. Upon ligand binding, the dimerized recep-tor binds to the estrogen response elements in the promoter region of the luc reporter gene. In S. cerevi-siae BMA64/luc, luciferase is expressed constitutively, and this strain was used for determination of cytotox-icity of the test samples. Both yeast strains were gifted by Dr Johanna Rajasärkkä of the Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Finland. Yeasts were grown on Difco Yeast Nitrogen Base medium without amino acids, supplemented with 40% glu-cose and their respective amino acids.

Sample preparation

Five-hundred milliliters each of all samples were extracted by solid phase extraction method as described by Kopperi et al. [22] using Phenomenex strata-X 33u polymeric reversed phase 500 mg/5 mL (Phenomenex, Aschaffenburg, Germany).

Bioassay

The yeast bioluminescent assay was performed as previously described [23]. Estradiol served as the positive control, while progesterone and testosterone

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Estrogenic activity of water samples 773

served as negative controls. The yeast bio-reporter assay offers a number of advantages in its robustness and ability to determine receptor activation without interference by endogenous receptors. This assay type has been compared and reported to be as effec-tive as liquid-chromatography tandem mass spec-trometry (LC-MS) in its ability to detect anabolic steroids in food supplements [24]. However, it fails to detect compounds that require metabolism to gain estrogenic activity, and exhibits only a modest response to estrogen antagonists.

Data analysis

The fold induction, fold induction corrected (FIC), and limit of detection (LOD) were calculated as described previously [21]. The sigmoidal dose–response curves for increasing concentrations of estradiol were obtained using the software program Prisma 4.0 (GraphPad software Inc. San Diego, CA). The estradiol equivalents of food samples showing

estrogenic activity were calculated from probit trans-formation of the curves.

Results and discussion

Environmental pollutants from WWTPs and plasti-cizers leaching from, e.g., plastic water bottles are subjects of continued debate on human exposure to EDCs, foremost xenoestrogens, globally. This is so because of recent evidence of a decline in male sperm count, an increased incidence of different cancer types amongst young men and women, and neurobe-havioral diseases observed in the populations of dif-ferent countries. These phenomena have epidemiologically been associated with EDC expo-sure, particularly during the intrauterine phase or during critical periods of postnatal development [13]. Thus, our study was aimed at evaluating the current situation in the most densely populated region in Finland with respect to existence of sub-stances possessing estrogenic activity in both influent

Figure 1. A diagram illustrating the main features of water management in Helsinki.

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774 I.M. Omoruyi and R. Pohjanvirta

and effluent samples from water treatment plants as well as in tap and bottled water.

The mean values of the two influent wastewater samples with detectable estrogenic activities are shown month-wise for both years (2011 and 2014) in Table II. The estrogenic activity level remained low, at approximately 0.5 ng/L EEQ, over this period, except for two months in 2011, March and August, when it peaked to 14 and 7.8 ng/L, respectively. The reason(s) for these peaks are currently unknown, but the latter might be linked to exceptionally high rain-fall in August 2011 [25]. However, in all treated (effluent) wastewater samples discharged into the Baltic Sea the estrogenic activity was below the detec-tion limit of our assay (data not shown).

In light of the data presented in the literature (Table I), the concentration rates found in the cur-rent study appear to be lower than those reported in other parts of the world, albeit one should be cau-tious in comparing data generated by different meth-ods. Also, the majority of these studies only screened effluent water samples for possible estrogenic activ-ity. Using a similar in vitro bio-reporter assay to that of ours in studying influent wastewater samples, Bellet et al. [10] reported estrogenic activity ranging from below detection limit to 25 ng/L EEQ in France, while a recent report revealed strikingly high levels (1136 ± 269 ng/L EEQ) in China [6]. Interestingly, in the latter study the value increased (1417 ± 320 ng/L EEQ) after primary treatment. In Spain, Germany, and China, the concentrations measured by mass spectrometry were also far higher than those determined in our study (Table I). All the cited stud-ies also reported varying levels of EDCs in effluent samples in contrast to the case here. Recently, 75 effluent samples from 16 European countries, includ-ing Finland, were screened for possible estrogenic activity. In accordance with our data, the effluent water from the Viikinmäki WWTP contained estro-genic activity below 0.5 ng/L EEQ [26]. Moreover, this was also the case for effluent waters from all other Finnish WWTPs (five) tested.

The overall outcome of the present study implies that the treatment method (activated sludge with mechanical, chemical and biological purification) currently employed in Helsinki, Finland is effective in removing estrogenic compounds from wastewater during treatment. Activated sludge and/or upflow anaerobic sludge blanket reactor followed by chlo-rination steps have also previously been found to be effective in removing EDCs from wastewater [27].

Tap and bottled water have been reported to rep-resent potential sources of human exposure to EDCs. There are two recent studies carried out in Europe in which estrogenic activity in this type of water samples was assessed by a comparable in vitro yeast assay to

that of ours. Pinto and Reali [1] analyzed mineral waters packed in polyethylene terephthalate (PET) bottles in Italy. The levels they detected varied from 0.03 through 23.1 ng/L (mode 9.5 ng/L) EEQs. Somewhat surprisingly, tap water made of either sur-face water or ground water contained approximately 15 ng/L EEQs. In another study, Wagner and Oehlmann [28] determined estrogenic activities in 20 major brands of bottled water in Germany. Twelve of these samples proved positive with the levels rang-ing from 2.64 to 75.2 ng EEQ/L (average 18.0 ng/L). Interestingly, in their material the highest estrogenic activities were recorded for waters packaged in either non-reusable poly(ethylene terephthalate) or reusa-ble glass bottles, and even water packed in Tetra Pak™ bricks contained high levels (14–44 ng/L). More recently, five sachet-packed drinking water samples (a third of the samples analyzed) from Nigeria were reported to be estrogenic, with estradiol equivalents ranging from 0.79 to 44.0 ng/L [29], fur-ther reinforcing the contention that exposure to EDCs via drinking water can be of concern in some parts of the world. Interestingly, in the present study estrogenic activity was neither found in any brand of bottled still or mineral water, nor in tap water, testify-ing to a high chemical quality of these products in Finland.

The negative estrogenic activity obtained in this study stems from legislation, good administration, extensive research, and follow-up approaches aimed at sustainable use of clean water resources in Finland. Efficient measures for water protection in Finland date back to early 1970s, and are based on long-term goals and proactive strategies [30]. The outcome of such concerted effort is the provision of high-quality drinking water and protection of the environment from wastewater discharges.

In conclusion, the treatment methods employed in Finland appear to be effective in reducing estrogenic activity during wastewater treatment. Drinking water, whether bottled or tap, does not pose a problem with respect to its xenoestrogen contamination.

Table II. Influent samples from Viikinmäki WWTP harboring estrogenic potential in 2011 and 2014.

2011 2014

Montha EEQ (ng/L) EEQ (ng/L)

January 0.7 0.4February 0.6 0.5March 14 0.5June 0.6 0.7August 7.8 0.6September 0.5 0.7

aThe values represent means of two samples taken two weeks apart. Only those months in which measurable estrogenic activity was detected are listed.

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Estrogenic activity of water samples 775

Acknowledgements

The authors are grateful to Dr Leena Maunula of the Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland for providing the wastewater sam-ples in 2011. Dr Tuula Laakso, Mr Kari Murtonen and Mr Reijo Nurmi of Viikinmäki Wastewater Treatment Plant, Helsinki, Finland, are also acknowl-edged for providing the wastewater samples in 2014. We are further obliged to Ms Mari Heinonen, Process Manager at Helsinki Region Environmental Services Authority, for useful information on water manage-ment in Helsinki.

Conflict of interest

None declared.

Funding

This research was supported by grants from the Research Foundation of the University of Helsinki, Helsinki, Finland; Walter Ehrström Foundation, Helsinki, Finland; and Benson Idahosa University, Benin City, Nigeria.

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