RESEARCH ARTICLE

Assessment of DNA damage in floriculturists in southern Brazil

Camila Mörschbächer Wilhelm & Adriani Kunz Calsing &

Luciano Basso da Silva

Received: 22 October 2014 /Accepted: 4 December 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The aim of this study was to assess possiblegenotoxic effects on floriculturists in a region of the state ofRio Grande do Sul, in the south of Brazil, using the micronu-cleus test (MN) and comet assay. Thirty-seven floriculturists and37 individuals not exposed to pesticides participated in the study.The micronucleus test was performed with epithelial cells of theoral mucosa. In the microscopic analysis, 2000 cells were eval-uated per subject, verifying the frequency of MN and thefrequency of other nuclear abnormalities (nuclear buds, binucle-ated cells, and karyorrhexis). For the comet assay in the periph-eral blood lymphocytes, 100 cells were classified in five classes,according to the migration of DNA fragments, thereby generat-ing the frequency of damaged cells and the damage index. Therewas no difference between the exposed and control groups in thefrequencies of MN and other nuclear abnormalities in the epi-thelial cells of the oral mucosa. However, the comet assayshowed that both the frequency of DNA damaged cells andthe damage index were significantly greater in the exposedgroup. The results therefore indicate that floriculturists are ex-posed to mixtures of pesticides with genotoxic potential.

Keywords Occupational exposure . Floriculturists .

Pesticides . Micronucleus test . Comet assay

Introduction

Pesticides are intended to prevent damage to plants caused byorganisms such as insects, fungi, and weeds. Ornamentalflowers are highly susceptible to pests, and their productionrequires the use of a wide variety of pesticides, belonging todifferent chemical classes (Bolognesi 2003; Bolognesi et al.2004). The action of a pesticide depends on its class, molec-ular structure, method of application, intensity of use, andappropriate agricultural practices. However, these substancesseldom present absolute selectivity and may become a risk tothe health of workers (Mostafalou and Abdollahi 2013). Flo-riculturists are therefore occupationally exposed to a complexmixture of potentially toxic chemicals (Bolognesi 2003). Ad-ditionally, the conditions of greenhouse work environments,involving confined spaces, high temperatures and humidity,the equipment used to apply pesticides, incorrect use of per-sonal protective equipment, the handling of plants, andreentering the greenhouse shortly after spraying, prolong andintensify floriculturists’ exposure to pesticides (Bolognesi2003; Bull et al. 2006; Ribeiro et al. 2012).

Prolonged exposure to pesticides has been linked to in-creased risk of chronic diseases such as cancer, Parkinson’s,Alzheimer’s, multiple sclerosis, diabetes, and cardiovascularand renal diseases. Given that DNA damage is one of theprimary mechanisms for the development of some of thesechronic diseases (Mostafalou and Abdollahi 2013),genotoxicity biomarkers have been suggested as predictorsof risk for the development of cancer, for example (Ellinger-Ziegelbauer et al. 2009; Valverde and Rojas 2009). Geneticbiomonitoring is also important since it enables the identifi-cation of risk factors at a time when control measures may stillbe implemented (Sailaja et al. 2006; Ergene et al. 2007).

The micronucleus test (MN) and comet assay are amongthe most widely used cytogenetic tests in the biomonitoring ofoccupational exposure to pesticides (Bolognesi 2003; Bull

Responsible editor: Philippe Garrigues

C. M. WilhelmCurso de Biomedicina, Universidade Feevale, Novo Hamburgo, RS,Brazil

A. K. CalsingCurso de Farmácia, Universidade Feevale, Novo Hamburgo, RS,Brazil

L. B. da Silva (*)Programa de Pós-Graduação em Qualidade Ambiental, Grupo dePesquisa em Saúde Humana e Ambiente, Universidade Feevale, RS239, 2755. CEP, 93352-000 Novo Hamburgo, RS, Brazile-mail: lucianosilva@feevale.br

Environ Sci Pollut ResDOI 10.1007/s11356-014-3959-4

et al. 2006). Genetic damage at the chromosomal levelresulting from chromosomal breakage or loss can be detectedby the MN test (Fenech et al. 1999). The version of the MNtest using epithelial cells of the oral mucosa presents advan-tages in that it is minimally invasive, does not require a cellculture and is considered more specific for detection of theeffects of exposure by inhalation or ingestion (Holland et al.2008; Bolognesi et al. 2013). It has been suggested that a highfrequency of MN in epithelial cells of the oral mucosa maypredict cancer risk for the upper aerodigestive tract and lungs(Holland et al. 2008). The MN test in oral mucosa can alsoassess other nuclear abnormalities, such as nuclear bud (alsoindicative of chromosomal instability or DNA damage), binu-cleated cells (indicative of cytokinesis failure and susceptibil-ity to aneuploidy), and karyorrhexis (abnormality associatedwith cell death), according to Bolognesi et al. (2013).

The alkaline version of the comet assay (pH>13) is capableof detecting single-strand breaks, double-strand breaks, alkali-labile sites (apurinic/apyrimidinic sites), cross-linking, andincomplete DNA repair sites (Valverde and Rojas 2009). Itis a quick and sensitive method for the detection of DNAdamage in single cells, induced by a variety of genotoxicagents (Tice et al. 2000).

Studies on DNA damage in workers exposed to pesticidespresent both positive as well as negative results (Bolognesi2003; Bull et al. 2006). The inconsistent responses betweenstudies may reflect different exposure conditions, such as theintensity of exposure, the use of personal protective equip-ment, and the specific genotoxic potential of the pesticidesused (Ergene et al. 2007). Studies specifically considering theuse of theMN test in epithelial cells of the oral mucosa and thecomet assay to assess genotoxicity in floriculturists are scarceas well as contradictory (Bull et al. 2006). Accordingly, theaim of this study was to evaluate the rates of DNA damage infloriculturists in the south of Brazil.

Material and methods

Subjects

The exposed group consisted of 37 floriculturists from theeastern region of the state of Rio Grande do Sul, in the south ofBrazil. The control group consisted of 37 individuals,employed in the fields of administration, commerce, andeducation, from the same region. Individuals of both sexes,over 18 years of age, participated in the study. All participantssigned an informed consent document and completed a ques-tionnaire in which they informed their age, gender, occupa-tion, contact with pesticides, and smoking and alcohol habits.In addition to this information, floriculturists also provideddetails regarding their work, such as length of service (used toestimate exposure time to pesticides), the activities performed

in floriculture, the herbicides, fungicides and insecticidesused, the frequency with which they come into contact withpesticides, the use and types of personal protective equipment(PPE), and the method of applying pesticides. Samples werecollected at the end of the working week, during the periodfrom February to April 2013. The study protocol was ap-proved by the Feevale University Research Ethics Committee.

Cytogenetic assays

Buccal cell samples were obtained by gently rubbing theinside of the cheeks with a cytobrush. The cells were storedon ice in a microtube containing a saline solution until pro-cessing. The samples were washed twice with saline, thenfixed in ethanol/acetic acid (3:1), and stored under refrigera-tion. For staining, 130 μL of the cell suspension in the fixingsolution was dropped onto clean, cold slides. The slides wereallowed to dry overnight, and the DNA-specific Feulgen-Fastgreen staining was then performed, in accordance with Tolbertet al. (1992). For each subject, 2000 cells on coded slides wereanalyzed under light microscopy, by the same observer, toverify the frequencies of MN, nuclear buds, binucleated cells,and karyorrhexis (according to Bolognesi et al. 2013). Thecriteria used for inclusion of the cell under analysis were thosesuggested by Tolbert et al. (1992): intact cytoplasm and rela-tively flat cell position on the slide, little or no overlap withadjacent cells, little or no debris, and nucleus normal andintact, and nuclear perimeter smooth and distinct.

For the comet assay, performed in accordance with Ticeet al. (2000), a single drop of whole blood was collected bymeans of a capillary puncture with an automatic, retractable,and single-use lancet. The drop of blood was stored in amicrotube containing heparin and protected from light. Toprepare the slide, 5 μL of the sample was mixed with 95 μLof low-melting agarose (0.7 % in a PBS solution). This mix-ture was dispersed on a slide pre-coated with 1 % normalagarose. A cover slip was placed over the mixture, and theslides were placed in an incubator for 7 to 10 min at 4 °C.Afterward, the cover slip was gently removed, and the slideswere placed in a lysis solution (2.5 M NaCl, 100 mM EDTA,10 mM Tris, 10 % DMSO and 1 % Triton X-100) at 4 °C, inthe dark, for 24 to 48 h. After this time had elapsed, the slideswere incubated in an alkaline electrophoretic buffer (300 mMNaOH, 1 mM EDTA, pH>13) for 25 min, and this wasfollowed by electrophoresis (1.4 V/cm across the chamberplatform 300 mA, 25 min). The slides were then placed underthe action of a neutralization buffer (0.4 M Tris and HCl toadjust pH to 7.5), allowed to dry at room temperature, andthen fixed (15% trichloroacetic acid, 5% zinc sulfate, and 5%glycerol). Afterward, the slides were again allowed to dry atroom temperature and then stained with silver nitrate, inaccordance with Nadin et al. (2001). For each subject, 100cells on coded slides were analyzed under light microscopy,

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classifying them visually, in accordance with Anderson et al.(1994) into five classes (0, I, II, III, and IV), according to thesize of the tail of the comet formed in this technique. In thissystem, a cell with no damage is categorized as class 0, while acell with the greatest damage falls under class IV. Based onthis classification, the frequency of damaged cells (I to IV)was determined together with the damage index, calculatedaccording to Pitarque et al. (1999) by the sum of the number ofnuclei multiplied by the value of the respective class, gener-ating an index that ranges from 0 to 400.

Statistical analysis

The studied cytogenetic variables departed significantly fromnormality, and therefore, the nonparametric Mann–WhitneyUtest was applied to the data. The associations between the twovariables were analyzed by means of the Spearman’s correla-tion. The chi-square test was used to compare frequenciesbetween groups. The level of significance was taken as p≤0.05. All analyses were conducted using the statistical pack-age for social sciences (SPSS) for Windows, version 18.0.

Results

Table 1 presents the characteristics, obtained from the ques-tionnaire, of floriculturists and the control group. The groupsdid not differ in terms of age, sex ratio, and smoking habits.However, the control group presented a higher frequency ofindividuals who consumed alcohol. In the group of floricul-turists, the average exposure time to pesticides was 9.7 years,with a median of 7 years.

With regard to activities required for flower production,51.4 % of workers performed pesticide application, 32.4 %prepared the mixture of pesticides, 51.4 % moved the hoses,and 48.8 % handled plants after application. The fungicides,herbicides, and insecticides most frequently used by the floricul-turists are shown in Table 2. Twenty-six different active ingredi-ents were cited, some of which are classified as moderatelyhazardous according to the toxicological classification of theWorld Health Organization (WHO 2010), such as copper sulfateand paraquat, while others are classified as possibly carcinogenicto humans (according to the U.S. Environmental ProtectionAgency (EPA)), including fosetyl and, once again, paraquat.

The frequency of pesticide application was once a week for86.5 % of workers, once every 15 days for 8.1 %, and twice aweek for 5.4 %. The methods of application cited were withthe aid of a tractor (40.5 %) and/or manually (73 %). The useof full personal protection, including overalls, gloves, boots,mask, goggles, and head protection, was reported by 45.9 %of workers, but only at the time of pesticide application. Withregard to the most frequently used PPE, 64.9 % reported

wearing gloves, 56.8 % wore a mask, 32.4 % used goggles,62.2 % wore boots, and 24.3 % reported using flip-flopsduring the workday.

Table 1 Characteristics of the floriculture worker (exposed) and con-trol groups

Characteristics Exposedgroup

Controlgroup

p value

No. of subjects 37 37

Age (mean in years±SD) 35.6±11.3 37.1±12.7 0.497

Gender

Female (%) 40.5 51.4

Male (%) 59.5 48.6 0.350

Smoking

Smoker (%) 5.4 5.4

Nonsmoker (%) 94.6 94.6 1000

Consumption of alcoholic beverages

Yes (%) 35.1 64.9

No (%) 64.9 35.1 0.011

Exposure time (mean in years±SD) 9.7±10.3 –

Table 2 Pesticides most frequently used by floriculturists and theirclassification and active ingredient

Classification Active ingredient Use Class(WHO)a

Class(EPA)b

Fungicides Mancozeb 67.6 % U c

Procymidone 67.6 % U NL

Iprodione 48.7 % III c

Thiophanate-methyl 46.0 % U c

Fosetyl 37.8 % U C

Copper sulfate 32.4 % II NL

Captan 27.0 % U c

Difenoconazole 24.3 % II c

Fenamidone 24.3 % c c

Pyraclostrobin 24.3 % c c

Others 37.8 %

Herbicides Glyphosate 83.8 % III D

Paraquat and diuron 27.0 % II and III C and c

Others 13.5 %

Insecticides Abamectin 51.4 % c c

Thiamethoxam 37.8 % c c

Others 13.5 %

NL not listed, IImoderately hazardous, III slightly hazardous, U unlikelyto present acute hazard, C possible human carcinogen, D not classifiableas to human carcinogenicitya Toxicological classification according to the World Health Organization(WHO)b Classification according to the U.S. Environmental Protection Agency(EPA)c Information not available

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The results of the cytogenetic tests are shown in Table 3.No differences were observed between the groups in terms ofthe frequencies of MN, nuclear buds, binucleated cells, andkaryorrhexis. In the comet assay, significantly higher valuesfor both the frequency of damaged cells as well as the damageindex were observed in the group of floriculturists.

Considering both the total sample and the exposed andcontrol groups separately, no significant differences wereobserved in relation to gender, alcohol consumption, orsmoking, for the parameters analyzed in the MN test as wellas those in the comet assay (data for the control group areshown in Table 4 and for the exposed group in Table 5).

When the exposed group was subdivided according to themedian of exposure time to pesticides, cytogenetic damage infloriculturists with less than 7 years of exposure was notdifferent to those exposed for 7 years or more (Table 6).Table 6 also shows that the floriculturists responsible forperforming pesticide application showed no difference inrelation to workers with other duties in flower production.Similarly, the workers who reported using full PPE did notshow different rates of DNA damage compared to those whodid not use PPE or used incomplete PPE (Table 6).

No significant correlations were seen with cytogeneticdamage or age, both in the exposed group as well as in thecontrol group (with the exception of the expected correlationbetween frequency of damaged cells and damage index in thecomet assay), or with exposure time in the exposed group(with the exception of the expected correlation between ageand exposure time; Table 7).

Discussion

The exposure to pesticides has become a concern to humanhealth due to the association between direct or indirect contactwith pesticides and various diseases, particularly cancer. Cy-togenetic studies with different biomarkers have shown in-creased DNA damage in farmers (Bolognesi 2003; Bull et al.2006). More specifically, floriculturists suffer the action ofpesticides on the body more intensely compared to farmers onopen ground, given that the characteristics of the greenhouse

environment can favor exposure (Bolognesi 2003). Severalcytogenetic studies applied to floriculturists have shown pos-itive results for MN test (Bolognesi et al. 1995; Falck et al.1999; Gómez-Arroyo et al. 2000; Bolognesi et al. 2002), sisterchromatid exchange (Shaham et al. 2001), chromosomal ab-errations (Dulout et al. 1985; De Ferrari et al. 1991; Landeret al. 2000), and comet assay (Castillo-Cadena et al. 2006).However, negative results have also been observed (Scarpatoet al. 1996; Lucero et al. 2000; Piperakis et al. 2003;Bolognesi et al. 2004).

The few studies that have applied theMN test with epithelialcells of the oral mucosa in floriculturists have also reportedcontradictory results. Gómez-Arroyo et al. (2000) found asignificant increase in the frequency of MN, while the resultsof this study and Lucero et al. (2000) showed no difference.The mean exposure time in these three studies is similar (ap-proximately 10 years of exposure); however, in the studyconducted by Gómez-Arroyo et al. (2000), it was reported thatthe floriculturists did not use any type of personal protectiveequipment, unlike our study and the study conducted by Luceroet al. (2000), in which the majority used at least one type ofPPE. Similarly, there are few studies in which the comet assayhas been specifically applied to floriculturists. Piperakis et al.(2003) found no significant difference between floriculturistsand control subjects, unlike Castillo-Cadena et al. (2006) andthis study, which show an increased rate of DNA damage.

There is evidence of DNA damage for some of the mostfrequently used pesticides in this study, according to informa-tion presented by Bolognesi et al. (2004) in their genotoxicitydatabase. In this sense, negative results for the MN test andpositive results for the comet assay were also observed in onestudy of farmers exposed to pesticides also reported in thisstudy, such as diuron, glyphosate, mancozeb, and paraquat(Remor et al. 2009). However, positive results for both tests infarmers have also been reported (Da Silva et al. 2012;Benedetti et al. 2013), with one study including pesticides incommon with this study, such as captan, glyphosate,mancozeb, and paraquat, but with greater exposure time forone of these (Da Silva et al. 2012). In the other study, 80 % ofworkers did not use PPE during the preparation and applica-tion of pesticides (Benedetti et al. 2013).

Table 3 Values obtained in the MN test and comet assay for the exposed and control groups

Group MN testa Comet assay

MN Nuclear bud Binucleated cells Karyorrhexis Damaged cells (%) Damage index

Control 0.11±0.21 1.02±1.39 0.41±0.51 0.84±1.02 1.51±2.55 1.95±3.88

Exposed 0.16±0.43 1.14±1.74 0.27±0.55 0.66±0.92 4.22±3.89 4.73±4.27

p 0.73 0.96 0.12 0.33 <0.001 <0.001

Values expressed as mean±standard deviationa In 1000 cells

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The review of Bull et al. (2006) on the genotoxicity ofpesticides showed that in studies where the majority(over 60 %) of workers use PPE, the results are nega-tive, and significant increases in cytogenetic damage aregenerally seen in studies where workers use little or noprotection. The use of epithelial cells of the oral mucosahas been indicated for biomonitoring of certain occupa-tional groups, given that this type of tissue is one of thefirst barriers in cases of exposure to inhaled or ingestedsubstances (Bolognesi 2003; Bolognesi et al. 2013). Inthis study, the use of at least one type of PPE wasreported by 70 % of floriculturists (and 56.8 % used amask). This suggests some degree of effectiveness ofPPE in preventing the mutagenic effects of pesticidesdetected by the MN test on cells of the oral mucosa.However, the floriculturists appear to be unprotected

from the genotoxic effects detected by the comet assayin peripheral blood. Additional studies should investigate thispossibility.

In this study, the effect of PPE use on cytogenetic damagewas not clearly determined since no differences were foundbetween subjects who reported using PPE and those who didnot use this type of protection. A factor that may explain thisresult is the lack of information on whether reported use wasthe same as actual use. In this sense, many floriculturistsreported using full PPE mainly during application butneglected to use this during other activities. Another possiblefactor is that the small size of the exposed group (37 subjects)may have reduced the statistical power of this study.

It was shown that DNA damagewas not correlated with theduration of pesticide exposure. Furthermore, when comparingfloriculturists with less than 7 years of exposure to those

Table 4 Values (expressed asmean±standard deviation)obtained in the MN test andcomet assay in control groupaccording to gender, alcoholconsumption, and smoking

Group MN test (in 1000 cells) Comet assay

MN Nuclear bud Binucleatedcell

Karyorrhexis Damagedcells (%)

Damageindex

Gender

Female 0.13±0.23 1.25±1.83 0.37±0.52 0.73±1.03 1.79±3.12 2.37±5.00

Male 0.09±0.21 0.77±0.64 0.46±0.57 0.96±1.02 1.22±1.80 1.50±2.23

p 0.58 0.80 0.58 0.36 0.71 0.83

Smoking

Smoker 0.00±0.00 0.50±0.71 0.25±0.35 1.00±0.71 0.50±0.71 0.50±0.71

Nonsmoker 0.12±0.22 1.05±1.42 0.42±055 0.83±1.04 1.57±2.60 2.03±3.97

p 0.49 0.68 0.85 0.49 0.67 0.64

Alcohol consumption

Yes 0.06±0.17 0.72±0.70 0.47±0.59 0.97±1.12 1.46±2.59 2.00±4.31

No 0.20±0.26 1.57±2.08 0.31±0.43 0.61±0.79 1.62±2.57 1.85±3.08

p 0.07 0.56 0.50 0.35 0.99 0.92

Table 5 Values (expressed asmean±standard deviation)obtained in the MN test andcomet assay in exposed groupaccording to gender, alcoholconsumption, and smoking

Group MN test (in 1000 cells) Comet assay

MN Nuclear bud Binucleatedcell

Karyorrhexis Damagedcells (%)

Damageindex

Gender

Female 0.07±0.18 0.55±0.61 0.26±0.49 0.50±0.80 5.07±4.54 5.47±5.11

Male 0.23±0.53 1.55±2.12 0.27±0.59 0.77±1.00 3.64±3.36 4.23±3.64

p 0.59 0.26 0.87 0.39 0.34 0.54

Smoking

Smoker 0.00±0.00 0.75±1.06 0.00±0.00 0.75±7.06 3.50±0.71 3.50±0.71

Nonsmoker 0.17±0.44 1.16±1.78 0.28±0.56 0.65±0.93 4.26±4.00 4.80±4.38

p 0.58 0.94 0.46 0.89 0.99 0.81

Alcohol consumption

Yes 0.29±0.61 1.85±2.52 0.42±0.67 0.62±0.82 4.31±3.15 4.76±3.72

No 0.09±0.27 0.75±0.99 0.19±0.46 0.68±0.99 4.16±4.30 4.71±4.62

p 0.38 0.33 0.18 0.90 0.51 0.71

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exposed for 7 years or more, no significant difference wasobserved. The median value adopted as the cutoff (7 years) isrelatively low compared to other studies. Bolognesi et al.(2002) found significant differences for the MN test in lym-phocytes of floriculturists but used a cutoff of 20-year expo-sure, while Shaham et al. (2001) found significant differencesfor sister chromatid exchange, also in floriculturists, between agroup exposed for less than 21 years and those exposed formore than 21 years.

It is known that various factors extrinsic to occupationalexposure can affect the frequency of cytogenetic damage,including age, gender, and smoking (Holland et al. 2008).

These variables were, therefore, also assessed in this study.A higher number of subjects in the control group ingestedalcohol, consuming once a week. However, this variable, aswas the case with age and smoking, did not significantlyinfluence the rate of DNA damage, in line with previousstudies (Castillo-Cadena et al. 2006; Singh et al. 2011; DaSilva et al. 2012).

It was not possible to detect the effect of clastogenic oraneugenic substances with the MN test on epithelial cells ofthe oral mucosa. However, the results of the comet assaysuggest that floriculturists are exposed to genotoxic agents.The exposed group evaluated in this study used 26 different

Table 6 Values (expressed asmean±standard deviation)obtained in the MN test andcomet assay in floriculturists,grouped according to exposuretime, activity, and use of PPE

Group MN test (in 1000 cells) Comet assay

MN Nuclear bud Binucleatedcell

Karyorrhexis Damagedcells (%)

Damageindex

Exposure time

<7 years 0.24±0.54 1.18±1.85 0.40±0.63 0.34±0.57 4.63±4.16 5.44±5.02

≥7 years 0.13±0.37 1.16±1.73 0.21±0.50 1.01±1.11 3.65±3.97 4.00±3.87

p 0.42 0.66 0.25 0.06 0.35 0.46

Activity

Applicators 0.17±0.39 1.51±2.21 0.38±0.65 0.63±1.04 3.44±3.15 4.06±3.57

Nonapplicators 0.16±0.47 0.79±1.07 0.16±0.41 0.68±0.82 4.95±4.44 5.37±4.86

p 0.91 0.52 0.23 0.60 0.39 0.59

Full PPE

Yes 0.15±0.35 1.98±2.57 0.38±0.68 0.66±0.86 4.00±3.38 4.75±3.93

No 0.17±0.47 0.74±0.99 0.22±0.47 0.66±0.97 4.32±4.17 4.72±4.50

p 0.98 0.26 0.41 0.73 0.81 0.98

Table 7 Spearman’s rank correlation coefficients (rS) between characteristics in the floriculture worker group (upper diagonal) and in the controlgroup (lower diagonal)

MN Nuclear bud Binucleated cell Karyorrhexis Damaged cells (%) Damage index Age Exposure time

MN 0.16 0.10 −011 0.00 −0.02 −0.07 −0.06p 0.33 0.56 0.53 1.00 0.90 0.67 0.75

Nuclear bud 0.16 0.16 0.03 0.20 0.26 −0.15 −0.14p 0.35 0.13 0.86 0.23 0.13 0.38 0.44

Binucleated cell 0.20 0.17 −0.06 0.20 0.27 −0.03 −0.24p 0.23 0.31 0.72 0.24 0.11 0.87 0.19

Karyorrhexis −0.09 −0.09 0.11 0.04 0.06 −0.22 0.33

p 0.61 0.58 0.21 0.83 0.73 0.20 0.08

Damaged cells (%) 0.01 −0.03 0.16 0.06 0.97 −0.19 −0.12p 0.96 0.85 0.34 0.71 <0.001 0.27 0.50

Damage index −0.02 −0.04 0.17 0.03 0.99 −0.19 −0.10p 0.92 0.81 0.32 0.87 <0.001 0.26 0.57

Age −0.19 −0.02 −0.26 −0.08 −0.14 −0.12 0.35

p 0.26 0.91 0.09 0.65 0.39 0.47 0.047

Exposure time – – – – – – –

p – – – – – – –

Statistically significant correlation coefficients are marked in bold

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chemical pesticides, many of which have no toxicologicalclassification (as shown in Table 2). However, it is worthnoting that the estimated rates of genetic damage are relatedto exposure to a mixture of pesticides and it is not possible torelate the results to a single active ingredient or specificchemical group (Bull et al. 2006). The results also indicatethe need to investigate more fully the effect of continuous useor nonuse of PPE on cytogenetic damage in workers exposedto pesticides.

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