STUDIES ON GENOTOXICITY AND MUTAGENICITY OF CERTAIN PESTICIDES

By

SANGEETA SAXENA

Date :

Approved :

Supervisor

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF PHILOSOPHY IN BIOTECHNOLOGY OF THE ALIGARH MUSLIM UNIVERSITY, ALIGARH

1994

DS2504

;^^''i'^i>m A ijfj^

I? 3 FEB W<>

C E R T I F I C A T E

I certify that the work presented in the following

pages has been carried out by Ms Sangeeta Saxena and that it

is suitable for the award of M.Phil, degree in Biotechnology

of the Aligarh Muslim University, Aligarh.

/ ^ ^ v ^ ^ ^ (SAAD TAWAB)

Senior Lecturer Interdisciplinary Biotechnology Unit

Aligarh Muslim University Aligarh 202002 (U.P) INDIA

ACKNOWLEDGEMENT

I wish to express my deep sense of gratitude to my

supervisor Dr. Saad Tayyab for his able guidance , keen

intrest, moral support and help extended to me in the

accomplishment of this task.

I am extremely grateful to Dr. Javed flussarrat for

his invaluable guidance, constructive and intelligent

criticism and encouragement , throughout the course of

this study.

I wish to thank Prof. A. Salahuddin, Coordinator,

Interdisciplinary Biotechnology Unit, A.M.U. for

providing necessary research facilties.

I thank Dr. Arshad Rahman for the help I received

from him.

I also wish to express my heartfelt gratitude to

Dr. P.V. Sane and Dr. K.li. Srivastava, NBRI , Lucknow

for their cooperation and genuine help in the

compilation of this dissertation.

I sincerely acknowledge the cooperation which I

received from my seniors, Aftab Bhai, Sadhna apa, Zoya

apa, Rizwan Bhai, Shama apa and Pasha Bhai.

I owe a lot to my friends liani ,Fariha, Ronana,

Nishat, Sunita, Sakhra, Sadia, Moin, Mozzafar, Jalal,

Fahim, Madhvi, Vipin and Rizwan for their genuine help

and love they showered on me.

I wish to express mv deep sense of appreciation to

my friend and colleague, Ashok for his great help,

moral support and always being there at my hour of

need.

ili

I sincerely acknowledge my parents ,SORADH ,

GAIJRAV and RA.TEEV for the encouragempn t , TIP 1 p and

cooperation I received in my academic endeavours.

I also extend my thanks to Mr. Fazlur Rahman Khan,

Mr. Lai Mohammad Khan and the other non-teaching staff

of the department.

1 am also thankful to Mr. Meraj Uddin, Design

Centre, Aligarh for painstaking, and timely typing of

this manuscript.

(SANGEETA SAXENA )

CONTENTS

PAGE NO

CERTIFICATE ii

ACKNOWLEDGEMENT iii

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS viii

ABSTRACT ix

I. REVIEW OF LITERATURE 1

II. GENERAL MATERIALS AND METHODS 3 7

III. MUTAGENICITY ASSAY 45

IV. GENOTOXICITY ASSAY 58

V. DISCUSSION 68

VI. REFERENCES 73

VII. BIOGRAPHY 88

LIST OF TABLES

PAGE

Table I Categories of different pesticides. 5

Table II High-risk cancers in mortality 16

surveys of farmers.

Table III Types of cancer associated with 18

specific pesticide exposure.

Table IV The Salmonella typhimurium and 37

Escherichia coli strains used in

this study .

Table V Relevant genetic and biochemical 56

markers of Ames Strains.

LIST OF FIGURES

PAGE NO.

Fig. 1. Dose response curves of captan (-S9)

with Ames tester strains. 47

Fig. 2. Dose response curves of captan (+S9)

with Ames tester strains. 49

Fig. 3. Dose response curves of foltaf (-S9)

with Ames tester strains. 50

Fig. 4. Dose response curves of phosphamidon

(-S7) with Ames tester strains. 52

Fig. 5. Dose response curves of furadan (-S9)

with Ames tester strains. 53

Fig. 6. Survival of SOS defective Ej;_ col i

K-12 strains exposed to captan. 61

Fig. 7. Survival of SOS defective E^. col i

K-12 strains exposed to foltaf. 63

Fig. 8. Survival of SOS defective E^ coli

K-12 strains exposed to phosphamidon. 64

Fig. 9. Survival of SOS defective Ej_ coli

K-12 strains exposed to furadan. 66

Iviii

LIST OF ABBREVIATION

Amp Ampicillin resistant

CFU Colony forming units

g Gram

Kg Kilogram

L Litre

ug Microgram

ml Millilitre

mg Milligram

N.B. Nutrient Broth

SCE Sister chromatid exchange

STT Short term test

Str Streptomycin resistant

UV Ultra violet

V.B. Salts Vogel Bonner Salts

(vi

ABSTRACT

Among the plethora of environmental mutagens,

pesticides belong to a noxious chemical class of compounds,

posing a major threat to global environment and human

health. Many of them possess mutagenic, carcinogenic and

teratogenic properties. However, very few of them have been

tested for their mutagenic and/or carcinogenic properties.

It is generally believed that a large proportion of human

cancers are caused mainly due to the genotoxic chemicals

present in the environment. It is therefore necessary to

identify the entire spectrum of chemical carcinogens by the

short-term mutagenicity and carcinogenicity assays in

combination with epidemiological investigations.

It is against this background that we have

evaluated the genotoxic and mutagenic potential of four

pesticides viz., captan, foltaf,phosphamidon and furadan.

Our studies have provided a complete profile of the

mutagenic potential and provided insights into their

mechanism of repair in bacteria. Of the four pesticides

studied, captan showed highest mutagenic response followed

by foltaf, phosphamidon and furadan. Captan was found to be

most mutagenic in the absence of metabolic activation (-S9).

However, in the presence of S9 mix its mutagenic activity

dropped significantly. About 1.6 to 13 times higher

ix

mutagenic response of captan was observed compared to the

reported values. Foltaf, phosphamidon and furadan have been

detected as comparatively weak mutagens.

A clear role for pol^ recA and lexA genes in the

repair of the pesticide induced DNA damage is suggested by

studies on the genotoxic effect of the pesticides on DNA

repair defective mutants of E^ coli. The wild-type strain

AB1157 showed a decline in survival in the presence of

captan, foltaf and furadan. The mutants polA~, recA~ and

lexA~ also showed a significant decrease in survival on

exposure to the pesticides. Based on our repair studies, it

is suggested that inducible error-prone repair pathways

involving the recA and lexA genes are in operation in the

enzymatic removal of pesticide induced lesions in DNA.

x

CHAPTER -1

REVIEW OF LITERATURE

REVIEW OF LITERATURE

The human environment is being polluted

continuously by the ever increasing presence of toxic

pollutants, introduction of synthetic chemicals, rapid

and unplanned industrialization, deforestation, burning

of domestic and industrial fuels and many such

activities. While there are a large number of

environmental agents to which people are exposed in an

occupational setting, carcinogenic substances are also

entering our system through food, air and water(Gupta,

1989 ) .

The realization that many chemicals have a

potential of damaging the genetic material has led to

considerable scientific effort to detect, in the tiuman

environment, the noxious substances that might induce

mutations culminating in tumorigenesis. In recent years

it has become more and more substantiated that

mutagenic chemicals pose danger to the present

generation because of the high correlation between

mutagenicity and carcinogenicity (Gocke et. al.» 1981).

Many heavy metals, several food mycotoxins, pesticides

which may contaminate food , synthetic hormones and

other food additives have been found to be carcinogenic

(Ray, 1986 ; Culliney gt. al . 1992).

It has long been known that cancer can arise as a

result of exposure to a variety of agents, and studies

on the mechanism of the induction process have revealed

that pure chemicals themselves are able to cause cancer

(Miller, 1978). Damage to DNA by environmental mutagens

may be the main cause of death and disability in

advanced societies (Cairns, 1975). It is believed that

this damage, accumulating during the lifetime initiates

most human cancer and genetic defects and is quite

likely a major contributor to aging (Burnet, 1974) and

heart disease as well (Pearson et. aX> 1975).

Pesticides - Uses and Consequences :

A pesticide is defined as "any substance or

mixture of substances for destroying, preventing,

repelling or mitigating any pest, including chemicals

intended for use as a plant regulator, defoliant or

dessicant" (CFR, 1986). The purpose of a pesticide is

to control insect and animal vectors in human disease

and to increase agricultural productivity which has led

to a 307. increase in food production worldwide (Jain,

1992). It has been projected that there will be a 117.

increase in the overall share of pesticide use in

developing countries from the current 24% to 35% by

the year 2000 (Jain, 1992).

The benefits of pesticide use should also be

weighed against their harmful effects on human health,

biological interactions with non-target species,

pesticide resistance, and alterations to and /or

accumulation of pesticide in the environment. The

increased and continued use of pesticide is associated

with human health risks, adverse effect on non target

organisms and contamination of air, soil and water.

Less than 0.17. of the applied pesticides are estimated

to actually reach the target pests and therefore large

amounts are entering the environment and contaminating

soil and water resources (Young, 1987). In order to

minimize the risks associated with the use of

pesticides, Young (1987) advocated a better

understanding of pesticide pharmacology and toxicology,

pests and pest management, and potential hazards

associated with pesticide use.

All pesticides are considered to be toxic to

humans and other organisms. The degree of harmfulness

to humans and other living organisms depends on

pesticide characteristics, the amount or dosage of the

pesticide, and the duration of exposure or contact

time. Studies on mammalian toxicity including acute,

subchronic, chronic, mutagenicity and special tests

provided a data base for evaluating the hazards and

risk assessment associated with pesticides (Cardona,

1987).

Substantial amount of literature is available

indicating that the pesticide / insecticide load in the

food chain is increasing (Anon , 1973; Manske and

Johnson, 1977 ),due to their intensive, widespread and

indiscriminate use. Even the mother's milk has been

reported to contain certain level of pesticides.

Various fruits, vegetables and cereals also accumulate

biologically significant quantities of pesticides. (

Kannan et. al . 1992).

For this reason, the genotoxic and mutagenic

activities of a number of pesticides have been the

subject of extensive research. Such studies have a

predictive value for the potential of the pesticide to

cause cancer ( Innes et. ai.5l969; Ames et, al_»i*?75; Mahr

and Miltenburger, 1976; Simmon, 1978; Waters et. al ,

1980; Moriya et. al_, 1983; Rosenkranz et. al.. 1984),

Classification of Pesticides :

The term "pesticide" is a generic name, which

includes not only insecticides but also herbicides,

fungicides, rodenticides, fumigants , other growth

regulators and repellents. A list of some

representative pesticides is given in table I.

Table-I: Categories of different pesticides

(1) Insecticides

(a) Chlorinated hydrocarbons DDT, Aldrin, Dieldrin, Heptachlor, (organochlorines) Toxaphene, Chlordane, Methoxychlor

Endosulfan, BHC, Endrin.

(b) Organophosphates Malathion, Parathion, Diazinon, Guthion, Phosphamidon, Dimethoate, Monocrotophos, Dichlorvos.

(c) Carbamates Sevin, Zectran, Baygon, Temik, Furadan, Carbaryl, Isolan.

(d) Pyrethrins Allethrin and Cyclethrin

(2) Fungicides

(a) Dithiocarbamates

(b) Nitrogen containing compounds

Ferbam, Ziram and Maneb.

PhenyImercuric acid acetate, Triazines, Quinones, Heterocyclics, and some heavy metals.

(c) Organochlorine Hexachlorobenzene, Captan, Foltaf.

(3) Herbicides

(a) Phenoxy acids

(b) Aquatic herbicides

(4) Rodenticides :

(a) Inorganic

(b) Organic

2 , 4 - D and 2 , 4 , 5 - T .

E n d o t h a l and D i q u a t .

Barium carbonate.

Sodium F l u o r o a c e t a t e , W a r f a r i n ,

(5 ) Fuffligants ;

(a) Organic Methyl Bromide, CS2•

(6) Others :

(a) Plant growth regulators 1-Naphthylacetic acid.

(b) Repellents Deet, Dimethyl phthalate.

(von Rumker e^ al., 1975)

Pesticides as Carcinogens :

There are numerous reports on the mutagenicity of

captafol (Difolatan) and captan (Seller , 1973; Kada et.

al . 1974). Kada et. ai. (1974) have reported captafol to

be a weak base change mutagen. Moriya ejt al_ (1983) have

also reported the same for captafol by E.coli WP2 her

strain. Captafol, an alkylating agent, has produced

genotoxic effects in, in. vitro systems (IPCS, 1990a).

Captan, a broad spectrum fungicide is also an

alkylating agent which has demonstrated genotoxic

properties iri. vitro (IPCS, 1990b). Captan has also

been reported to be a weak base change mutagen (Siebert

et aX, 1970; Clarke, 1971; Brideges et. al_ , 1972).

Moriya et. al_ (1983) have also reported captan to be a

base-change mutagen in a test on WP2 her, TA1535,TA100,

TA1537, TA1538 and TA 98. Moriya et. al (1978) have

reported that mutagenicity of captan decreased by S9 as

also by blood and cysteine. Captan has been shown to

increase in mutagencity in TA98 by the plant extract

activation (S14), while in TA100 it decreased

(Rasquinha et. al., 1988). Captan induced mutation has

been observed with the recA assay of B. subtilis (Kada

et al. 1974) and the involvement of the error-prone

recombination - dependent repair system and the

recombination- dependent repair mechanisms in both the

£.. coli K-12 as well as E.coli WP2 repair systems

(Rashid and Ralph, 1986). A Microscreen phage-induction

assay performed by Houk and DeMarini (1787) showed that

captan was genotoxic and induced prophage. Reuber

(1989) has reviewed the carcinogenicity of captan in

mice and rats.

Carbofuran or Furadan, is a broad spectrum

carbamate and used as an insecticide and nematicide.

This insecticide has been shown to be a frame-shift

mutagen. Carbofuran has been tested for mutagenicity

and shown to be positive in frame shift type strains

(Moriya et. al_) 1983). Carbofuran and its metabolite 3-

ketocarbofuran have been reported to be acutely toxic

in the Microtox method of toxicity assessment (Kross et.

aJL, 1992).

Mutagenicity of organo-phosphorous pesticides ,

especially phosphamidon has been reported by Vishwanath

and Jamil (1986). This was found to be a weak mutagen

and caused base change mutations in the Ames Salmonella

assay. This pesticide has also been reported to be

mutagenic • in the E. coli WP2 Try-systems and

clastigenic in the human leucocyte tests system

(Patankar and Vaidya, 1980). Phosphamidon has been

shown to have mutagenic activity in. vitro and iji vivo

in mammalian systems (Georgian, 1975; Usha Rani et. al.

1980) .

It has been reported that mutagenic activation or

inactivation of the ingested chemicals can occur

through various metabolic processes in the animal body

8

(Lu et. aX» 1^72; Prins, 1978). In such transformation

of the chemicals, liver microsomal enzymes and

intestinal microflora play major roles (Prins, 1978).

Pednekar et. al_ (1987) have evaluated the mutagenic

activities of commonly used insecticides - endosulfan

(organochlorine), phosalone and malathion

(organophosphorus) and permethrin(pyrethroid) before

and after activation with fecal microbial extracts or

with liver post mitrochondrial fraction (39 fraction)

or rat in Ames test with Salmonella tvphimurium tester

strains TA97a, TA98 and TA100.

The microtitration SOS chromotest to study

genotoxicity, showed that nine pesticides were capable

of inducing SOS response in E.. col i. The SOS inducing

potency was least for captafol and captan (Xu and

Schurr, 1990). Wong et. aj. (1989) have reported that

diazinon, dimethoate, fenitrothion, malathion,

phenothoate and quinolphos were non-mutagenic in the

absence of S9, diazinon, dimethoate and quinolphos were

mutagenic in the absence of S9, while in the presence

of S9, diazinon, dimethoate and quinolphos were

mutagenic. Several new chemical mutagens in

pesticides have been detected by the rec-assay

procedures. The mutagenic specificities of Captafol;

DAPA (Dexon) and NBT (Nirit) were determined using gj.

coli WP2 and Ames Salmonella system by Kada et. al.

(1974).

Genotoxic and Non-genotoxic Carcinogens.

The classification of carcinogens into two

categories, genotoxic and non-genotoxic has been

proposed to give a logical foundation on which cancer

risk assessment is reasonably based. The term

"genotoxic carcinogen' indicates a chemical capable of

producing cancer by directly altering the genetic

material of target cells, while "non-genotoxic

carcinogen" represents a chemical capable of producing

cancer by some secondary mechanism not related to

direct gene damage. Most classical carcinogens such as

polycyclic aromatic hydrocarbons, nitroso-compounds,

aromatic amines and azo dyes belong to the genotoxic

category. However, sex hormones like estrogen and

progesterone can be raised as typical examples of non-

genotoxic carcinogns (Hayashi, 1972). While this

classification can not be applied, at present to all

instances due to insufficiencies in necessary

information or limited current knowledge on

carcinogenesis, the genotoxic versus non-genotoxic

carcinogen concept can still contribute to a basic

understanding of carcinogenic mechanisms and

establishment of strategies for cancer risk evaluation

from exposure to the environmental chemicals.

Carcinogenesis is a multi-step evolutionary

process occuring both in. vivo and in cell culture

(Newbold,1983; Boone et. art, 1992). The concept of

multistage carcinogenesis was initially developed

10

decades ago in rodent skin models (Rous and Kidd, 1941;

Mottram, 1744) and has since been shown to apply more

generally to cancers of many species and cell types. It

is now beleived that experimental carcinogenesis

proceeds through at least 3 distinct stages viz.

initiation, promotion and progression. The first stage

of carcinogenesis, initiation, results from

irreversible genetic alterations, most likely one or

more simple mutations, transversions, transitions

and/or small deletions in DNA. The reversible stage of

promotion does not involve changes in the structure of

DNA but rather in the expression of the genome mediated

through promoter—receptor interactions. The final

irreversible stage of progression is characterized by

karyotypic instability and malignant growth (Pitot,

1993). Oncogene research has provided new insight into

the cellular signal pathways, as well as genetic

alterations leading to neoplastic growth. The current

understanding of the processes regulating tumor

initiation and progression is based on the observation

that genes found in tumorigenic retroviruses, genes

from spontaneus human tumors that could transform

recipient murine or rat cells, and genes located at

sites of chromosomal translocations in human cancers

have similar origins (Bishop, 1991; Boyle, 1992;

Rozengurt, 1992) . These genes commonly called as

oncogenes (Garrett, 1986) are mutated forms of normal

11

cellular genes, designated protooncagenes (Temin, 1974)

which are components of the pathways regulating normal

cellular proliferation and differentiation. (Schmandt

and Mills, 1993). There are principally two sets of

oncogenes in the cell- those acting in the nucleus, and

those acting in the cytoplasm. The most thoroughly

studied cytoplasmic oncogene is the family of ras,

which is probably involved in over 307. of human cancer

(Ramel, 1991). The product of ras (p21 ) functions as

a GTPase in combination with other proteins, such as

GTPase-activating proteins, GAP. This process, implies

an amplification of the trans-membrane signals that

control cell proliferation and differentiation. Mutant

ras interrupts the GTP-GDP cycle, causing a

constitutive signal output (McCormick, 1989).

Activating mutations in ras are provided bv base

substitutions in DNA, located mainly in codons 12, 13

and 61 which coincide with regions for binding between

p21 and GAP (Khosravi-Far and Der, 1994). Nuclear

proto-oncogenes on the other hand are activated by

genetic alterations affecting the expression of the

genes. A continuous expression of the oncogene mvc has

been demonstrated in Burkitt's lymphoma as a result of

translocations of mvc to the vicinity of the promoter

of immunoglobulin genes (Ramel, 1991). Also, nuclear

oncogenes are often activated by gene amplification

causing increased output of the gene product.

12

Another critical molecular target during the

stages of carcinogenesis are tumor suppressor genes

(Marshall, 1991). In this instance, the mutational

alteration of the gene leads to a complete loss of

function or to a change in function such that its

activity in suppressing the neoplastic phenotype is

drastically altered. In the human, the best

documentation that a tissue -specific neoplasm arises

as a result of deletion or mutation of both copies of

the tumor supressor gene is the retinoblastoma

(Knudsen, 1971). Mutations in p53 tumor suppressor gene

have now turned out to be the most common genetic

alterations yet identified in human cancers (Ryberg et.

al , 1994; Deng et. al., 1994). The genetic alteration of

a single copy of a tumor suppressor gene may be a

critical event for the stage of initiation, with

alteration of the other allele occurring as the

determinant in the transition from the stage of

promotion to that of progression.

Although other potential critical molecular

targets exist, such as those involved in the cell cycle

(Nishimoto et. aX ,1992; Kaufmann and Kaufman,

1993),whose alterations result in neoplasia. What is

not clear, with few exceptions, are the ultimate number

and characteristics of critical molecuar alterations

that absolutely define the malignancy of a cell. Hence,

the molecular nature of neoplasia and its development

stand as a continuing challenge to the human intellect.

13

Carcinogenic Effects of Pesticides

In recent years there has been increasing concern

that exposure to pesticides may pose a potentially

serious cancer risk to the general population. The

currently available data are insufficient to estimate a

pesticide-related cancer rate for the general

population. However, a variety of pesticides are known

to cause cancer in laboratory animals (Blair et. al ,

1990; Hooever and Blair, 1991). In a recent survey of

47 pesticides evaluated for carcinogenecity, it was

found that six pesticides (13'/.) were positive in both

sexes in mice and rats, ten pesticides (217.) were

positive in both sexes of one species and six

pesticides (13"/.) were positive in one sex of atleast

one species (Hooever and Blair, 1991). Of 16 chemicals

that were positive in both sexes of atleast one

species, included organochlorine and organophosphate

insecticides, herbicides, fungicides and fumigants,

suggesting that no chemical class of pesticides can be

considered problem free. Similarly, the International

Agency for Research on Cancer recently reported that

43'/. of a wide variety of pesticides were carcinogenic

in humans (Blair et. al., 1990). With pesticides of all

the variant types showing some evidence of

carcinogenicity, the concern about human exposure seems

to be well founded. With regard to mechanisms of

carcinogenesis, a survey of the genotoxic activities of

65 pesticides by Garrett et. al_ (1987) revealed that 33

14

of the pesticides (54'/.) tested positive, including

organophosphates, thiocarbamate insecticides,

pyrethroid insecticides, and a variety of herbicides

and fungicides. However, some non-genotoxic pesticides

also may be involved in carcinogenesis via their

epigenetic properties, such as tumor promotion,

inhibition of intercellular communication, or induction

of peroxisome proliferations.

Substantial amount of epidemiologic data is

available on the carcinogenicity of pesticides based on

the persons employed in agricultural or related

occupation (Weisenburger, 1793). Although farmers

typically perform many tasks resulting in a variety of

exposures it is the pesticides that have received the

most attention. Table II shows the cancers that have

been associated with occupational exposures to specific

pesticides or class of pesticides (Blair and Zahm,

1991). The epidemiologic data linking specific

pesticide exposures to hematopeitic cancer (i.e., non-

Hodgkin's lymphoma, leukemia and multiple myeloma) are

the strongest compared to the data on soft tissue

sarcoma and Hodgkin's disease. Very little is known

about specific pesticides and cancer of the brain,

although ovarian carcinoma has been shown to be

associated with exposure to triazine herbicides (Donna

et al. 19B9) Neverthless, the leukemia and brain cancer

in children have also been reported to be associated

15

Table II: High-risk cancers in mortality surveys of farmers

Type of Cancer No. of Studies

No. of studies with RR> 1.0

Range of RRs

Lip

Melanoma

Leukemia

Non- Hodgkin's lymphoma

Multiple myeloma

Hodgkin's disease

Soft tissue sarcoma

Brain

Stomach

Prostate

9

12

21

21

16

13

9

20

23

24

9

8

12

11

12

10

6

15

12

17

1.3 - 3.1

0.5 - 6.3

0.3 - 2.0

0.6 - 2.6

0.4 - 3.1

0.6 - 1.5

0.9 - 1.5

0.7 - 6.5

0.5 - 1.7

0.9 - 2.0

Abbreviation : RR, risk ratio

(Blair and Zahm, 1991 ; Hoover and Blair, 1991

16

with parental employment in agriculture and exposure to

pesticides (Lowengart et. aj., 1987; WilkinS and sinks,

(1990). Table III shows a list of different types of

cancer associated with specific pesticide exposures.

17

Table -III Types of cancer associated with specific pesticide exposures.

Cancer Pesticides

Soft tissue sarcoma and lymphoma

Non-Hodgkin's lymphoma

Soft tissue sarcoma

Leukemia

Lung Carcinoma

Phenoxyacetic acid herbicides (2,4,D, 2,4,5-T), DDT

Organophosphate and Organochlorine insecticides, fumigants

Chlorophenols, animal insecticides

Dichlorvos, crotoxyphos, famphur, methoxychlor, pyrethrins, DDT, chlordane, heptachlor, lindane, ethylene oxide

Organochlorine insecticide (DDT), arsenicals

Ovarian Carcinoma Triazines

18

Immunologic Effects of Pesticides:

Although there is little evidence to suggest that

pesticides exposure compromise human health through

interference with immune system, however, Thomas et. ai.

(1990) reported that pesticides or pesticide

contaminants can modulate the immune response in

experimental animals. Sharma and Kour (1990) suggested

that contact dermatitis is a common disease caused by

pesticides and also the asthma-type reactions can be

triggered by pesticide exposures. Although altered

immunoglobulin and competent levels and changes in T-

cell populations have been reported in human exposed to

pesticides (Thomas et. aX> 1990), no adverse health

effects have been noted. However, impaired neutrophil

chemotaxis accompanied by an increase in respiratory

tract infections was correlated with the length of

occupational exposure to organophosphate pesticides

(Hermanowicz and Kossman, 1984). Further studies on the

immunological effects of pesticides in humans are

needed since depressed immunity is known to predispose

humans to a variety of cancers, including non-Hodgkin' s

lymphoma.

Carcinogenicity Testing System :

It has become increasingly apparent that the

traditional methods for identifying carcinogens by

using long term studies in rodents are unable to meet

the demands for a quick, sure and inexpensive

19

identification of environmental carcinogens. This led

to an intensive search for appropriate test systems and

over the last few decades more than one hundred short-

term assays for mutagenicity have been developed (

Ramel , 1991).

Despite the fact that long-term tests are

expensive and time consuming they provide considerable

evidence in estimating the potency of a carcinogen

(Farmer, 1982). However, there is a problem of

predicting the response of humans to the carcinogens

because of the perplexing differential responses

exhibited by the animal model systems. Therefore, the

extrapolation of the risk factor from rodents to humans

could be quite contentious (Ames et_ aX 1987). Short-

term tests (STTs) •'or genotoxicity testing have

therefore been developed to study the mechanism of

chemically induced DNA damage and to assess the

potential genetic hazard of chemicals to humans. The

most commonly employed short-term tests are chromosome

aberrations, sister chromatid exchange (SCE),

mutagenicity assay using mouse lymphoma cells and the

Ames-microsome test. The chromosomal abberations are

the manifestation of the deleterious effects that occur

anywhere in the genome. Because of the difference in

size between a genome and a gene, the cytogenetic

effects manifested as gross chromosome aberration occur

at a much greater frequency than do mutations observed

at a single locus. This test has provided a very

20

sensitive method for determining, whether or not

environmental agents can interact with the genetic

material (Wolff, 1984). SCE method has been widely used

to detect whether or not a chemical is potentially

dangerous and which of the metabolites of a

premutagenic and precarcinogenic agent could be a

potential candidate to interact with DNA resulting in

adduction (Wolff, 1984).

Ashby and Tennant (1988) reported that no

combination of the STTs is any better than the

Salmonella test, on its own for flagging probable

carcinogens. The significance of STTs has increased

tremendously because of observations that many rodent

carcinogens have been found to be genotoxic in in, vitro

STTs (Ames, 1979). The in. vitro STTs have the advantage

that they can be conducted relatively quickly and

inexpensively compared to long- term carcinogenicity

assays with rodents. These short-term tests have

significantly contributed to the identification of many

natural mutagens and carcinogens in the human diet

(Kapadia, 1982). There is a range of applications in

which STTs have been successfully used for the

identification of mutagenic fractions in complex

mixtures such as cooked meat (Hatch et. al_f 1984;

Sugimura, 1985) or air pollutants (Schuetzle and

Lewtas, 1986). Nevertheless, there are two impediments

to a thorough evaluation of the ability of these tests

21

to predict carcinogenicity . For most STTs there is a

dearth of positive results for documented

noncarcinogens (Shelby and Stasiewiez, 1784; Kier et.

aJL, 1786; Ray et. aj., 1787) and statistically

insignificant number of chemicals had been tested in

multiple STTs to provide meaningful comparisons of the

ability of different STTs and STT combinations to

predict carcinogens. Even with a battery of assays, not

all rodent carcinogens have been found to be in. vitro

mutagens nor are all in. vitro mutagens proven as

rodent carcinogens. Even then STTs do, contribute to

offer an economical, rapid and dependable means to

detect genotoxic chemicals (Tennant et. al_, 1787).

Ames Testing System :

The Salmonel la mutagenicity test (Ames et. al ,

1775), has been extensively used to survey a variety of

substances in the environment for their mutagenic

activities (Ames, 1784). The assay measures back

mutation in several specially constructed mutants of

Salmonella typhimurium strains. The method has been

adapted for use in detecting chemicals which are

potential human carcinogens or mutagens supplementing

homogenates of rat liver (S7 fraction) directly to the

petri-plates, as an approximation of mammalian

metabolism (Ames et. al_> 1773). The compounds are tested

on petri-plates with several specially constructed

mutants of Salmonella typhimurium LT2 type selected

22

for sensitivity and specificity in being reverted from

a histidine requirement back to prototrophy by a wide

variety of mutagens (Ames et. al., 1973). These histidine

requiring mutants have GC base pairs at the critical

site for reversion e.g. -C-C-C in the base pair

substitution strain, TA100 (Barnes et. aj^, 1982), -C-C-

C-C-C-C in the frameshift tester strain, TA97a (Levin

et al, 1982b) and -C-B-C-G-C-G-C-G in the frame shift

tester strain, TA98 (Isono and Yourno, 1974). However,

TA102 and TA104 have AT base pairs at the critical site

for reversion. These strains detect a variety of

oxidants and other agents as mutagens. (Levin ez_ al .

1984).

A new mutagenicity testing system to test a

chemical with more than one strain simultaneously (the

"SIMULTEST') has been develped . It involves the jse of

different Salmonella typhimurium tester strains with

and without R-plasmid, in combination on the same

plate. This approach may be useful in reducing the

workload associated with mutagenicity testing with

Salmonel la (Nestman et. al., 1987).

A great deal of controversy is over the popular

view that synthetic carcinogens are posing a higher

risk than the natural ones. According to Ames e^ al_

(1987) the carcinogenic hazards from current levels of

pesticide residues or water pollution are likely to be

of minimal concern relative to the background level of

natural substances.

23

Animal models on the other hand provided valuable

information related to the mechanism of carcinogenesis

(Harris, 1985; Hay, 1988). Extrapolation of this

information from experimental animals to humans however

remains a problematic endeavour. Most scientific

workers consider the qualitative extrapolation to be

accurate, i.e., a chemical , carcinogenic in

experimental animals is likely to be carcinogenic in

humans. Although there is a positive association

between adduct levels and tumor initiating potency in

many but not all studies using animal models (Wogan and

Gorlick, 1985), it is not known whether such

association exists in human carcingenesis (Harris,

1985). Lifetime carcinogenicity studies will still be

needed for those non- genotoxic carcinogens that are

tissue, sex, species specific in contrast to the

genotoxic carcinogens that cause tumors in different

species at multiple sites. The latter will need only a

limited bioassay (Hay, 1988). A study on mutagenic

and non-mutagenic rodent carcinogens shows that the

tested carcinogens in rats are potentially mutagenic

in S_i_ typhimurium compared to their non-mutagenic

counterparts (Rosenkranz, and Ennever, 1990).

DNA Repair in Prokaryotes

DNA is the primary carrier of genetic information

and the structural integrity of DNA is a prerequisite

for gene expression (Modak, 1972). It is a known fact

24

that the primary structure of DNA is dynamic and

subject to a constant change (Friedberg, 1785).

Ionizing and UV-radiations as well as a multitude of

other chemical agents upset the genetic and metabolic

machinery of the living system. That would perhaps

render our planet barren were it not subjected to the

constant cellular monitoring and repair. Moreover, the

contemporary global environment also posed a continual

threat to the hereditary material. The living systems

have therefore evolved repair processes to maintain

structural and functional fidelity of DNA against a

large range of insult (Friedberg, 1985). The molecular

mechanisms involved in repair of damaged portions of

DNA and their restriction into functionally intact

informational units is fundamental to the maintenance

of the genomic integrity (Modak, 1972). Defective DNA

repair could cause an accumulation of lesions or

mutations which might be either lethal, lead to an

altered phenotype, or neoplastic transformation. Repair

at the cellular and macromolecular level is multiple in

its form and varies as a function of the cell cycle

(Hart et. al, 1979) .

A great deal of research has been directed towards

gaining new insights into the mechanistic regulation of

repair machinery in Escherichia coli. The physiological

studies on the recovery from DNA damage have

established the existence of DNA repair pathways.

Moveover, the genetic studies have identified a large

25

number of genes participating in the repair of damaged

DNA (Walker, 1985).

The earliest suggestion on recovery of bacteia

after exposure to ultraviolet (UV) light was made by

Hollaender and Curtis in 1935. Later, Kelner (1949)

observed that exposure of UV-irradiated bacteria to

visible light reversed the killing and mutagenic effect

of UV light and coined the term, 'photo-reactivation'.

The isolation of an E.coli by Hill (1958) provided the

first evidence on genetic control of radiation

sensitivity . Setlow and Carrier (1964) and Boyce and

Howard-Flanders (1964) independently demonstrated that

UV-induced thymine dimers in bacterial DNA were not

excised in a UV-sensitive strain but were excised in

the wild type strain. This suggested that excision of

thymine dimers from bacterial DNA may be important for

cell survival and that it is genetically controlled.

Further, the same repair system has been shown to

operate on the damage induced in DNA by carcinogens,

mutagens and other hazardous chemicals ( Seeberg, 1981;

Friedberg, 1985). The foregoing developments paved the

way for the systematic study of DNA repair mechanism in

prokaryotes and thus led to the discovery of additional

repair systems. The following repair systems have been

shown to be existing in bacteria.

26

Photoreactivation :

The simplest class of repair pathway is

photoreactivation that directly rectifies the cyclo-

butane type pyrimidine dimers in UV-irradiated DNA

without the formation of new phosphodiester bonds

(Walker ejt al_, 1985). This type of recovery process

requires the exposure of cell to visible light. It was

the first system to be observed iri. vitro (Rupert et. al.

1958) and was the first to be characterized with regard

to mechanism (Rupert, 1962).

Photoreactivation is a universal phenomenon

because it is known to occur in E.. col i , yeast and

possibly in higher animals and plants. (Schild et_ al,

1984).

Excision Repair :

An important mechanism for cell survival after UV-

irradiation depends upon the release or excision of

pyrimidine dimers from the DNA by excision enzymes, and

the subsequent reconstruction of the double helix by

repair enzymes that trjake use of the intact opposite

strand as template. Excision repair appears to be a

significant source of DNA repair virtually in all

organisms (Walker et. al_, 1985). UV-induced damage in

cellular DNA is also repaired in the dark by excision

repair system. Mechanism of repair of DNA by excision

has been studied extensively using mutants of E. coli

sensitive to ultraviolet radiations (Setlow and

27

Carrier, 1964; Boyce and Howard-Flanders, 1964). Two

basic types of excision repair have been described in

bacteria (van Houten, 1990). One system, base excision

repair, removes smaller types of base damage, such

lesions caused by ionizing radiation or monofunctional

alkylating agents. Another system, nucleotide excision

repair, removes bulky DNA adducts of which the UV-

induced pyrimidine dimer is the prototype (Bohr, 1991).

The basic steps involved in excision repair includes

(i) pre-incision recognition of DNA damage, (ii)

incision of the damaged DNA strand near the defect,

(iii) excision of the defective site with concomitant

degradation of the excised DNA strand (iv) replication

of a repair patch to replace the excised strand with

the opposite intact strand being used as a template and

(v) ligation of the repair patch at its 3" end. The

resulting patch in nucleotide excision repair is much

larger (20-100 bases) than in base excision repair (1-5

bases) (Bohr, 1991).

Although some of the enzymes involved in

prokaryotic DNA repair have been characterized, their

mammalian counterparts are less well described. Two

prokaryotic enzymes, T4 endonuclease V and uvrABC

excinuc lease, play roles in the excision pathways of E..

coli and have proven particularly useful in the study

of gene-specific repair. The T4 endonuclease V, which

recognizes cyclobutane pyrimidine dimers, has both DNA

glycosylase and Ap endonuclease activities resulting in

28

a single-stranded nick in DNA at the site of a

pyrimidine dimer (Bohr et. al., 1985). The E_; coli ABC

excinuclease, encoded by the uvrA, uvrB and uvrC genes,

recognizes a wide range of bulky DNA adducts. These

proteins act in concert to accomplish the first three

steps of excision repair i.e. recognition, incision and

excision. This complex enzyme has also been used in

gene specific repair studies because the damage formed

by several environmental and chemotherapeutic agents,

including cisplatin, polycylic aromatic hydrocarbons,

mitomycin C and psoralen, can be recognized by the

enzyme (Thomas et. al^, 1988).

Post Replication Recombinational Repair :

The DNA lesions, especially UV-induced pyrimidine

dimers that are neither split photoenzymatically nor

removed from DNA by excision repair, block the

continuous progress of the DNA replication fork.

However, they do not prevent the re-initiation of DNA

synthesis at a point beyond the dimer (Rupp and Howard-

Flanders, 1968). As a result, gaps are produced in the

daughter strand opposite the lesions. The continuity of

daughter strand is interrupted by gaps of about 1000

nucleotides (Howard-Flanders, 1968; Iyer and Rupp,

1971; Benbow et. al_, 1974). This type of enzymatic DNA

repair by which the molecular weight of the newly

synthesized strand increases is called post replication

repair. This was first demonstrated in E. coli by Rupp

29

and Howard-Flanders (1768). In excision deficient uvr

mutants that are otherwise normal, the major mechanism

of post replication repair is recombinational ,

requiring the activity of rec genes.( Howard-Flanders

et al, 1781). The most fascinating property of the RecA

protein of Escherichia coli is its ability to form

filaments with nucleic acid (Flory and Radding, 1782;

Dunn et. aX, 1782). These filaments interact with linear

DNA molecules and exhibit end-joining activity

(Register and Griffith, 1786). The RecA protein also

plays an important role in SOS induced targeted

mutagenesis in addition to its recombinations and

regulatory roles (Walker, 1784).

Recently, Lu ejt aJL. (1786) have proposed a model

for targeted mutagenesis. According to this model (i)

RecA protein should bind more effectively to UV-

irradiated double stranded DNA than to un-irradiated

DNA (ii) RecA should inhibit the 3'-5' activity of the

isolated £ subunit of DNA polymerase III.

Inducible Error Prone (SOS) Repair :

The term 'SOS' (international distress signal)

implied to an error prone repair, is induced under

enormously stressed condition of growth as a last

resort to the survival of cells. The existence of

'SOS'network was clearly postulated by Defais et. ai.

(1771) and was further developed and amplified by

Radman (1774, 1775) and Witkin (1776). The exposure of

30

E. coli to the agents that damage DNA or interfere with

DNA replication results in the induction of a diverse

set of physiological responses called "SOS' response.

+ +

It requires recA and lexA genotypes of host (Muira

and Tomizawa, 1968; Defais et. ai., 1971).'SOS' repair is

a highly integrated and sophisticated regulatory

network that requires de. novo protein synthesis for

expression (Kovel, 1986). It is an inducible repair

process and is believed to be responsible for common

mutagenic pathways (Radman, 1974, Witkin, 1976; Walker,

1985). In response to DNA damage calling for the SOS

repair, DNA repair systems in Ej_ coli are activated,

cell division is altered, integrated viruses ars

induced and respiration is blocked (Witkin, 1976;

Little and Mount, 1982).

Several bacterial genes have been identified which

coordinately function in 'SOS' repair. These are uvrA

and uvrB (DNA repair), umuC (mutagenesis), sfiA

(filamentation), himA (site specific recombination) and

several din genes with known functions in additon to

the recA and lexA genes (Witkin, 1976;Kenyon, 1983).

Role of recBC and recN genes has also been suggested in

'SOS' induction (Chaudhury and Smith, 1985; Finch et.

al, 1985).

The recA and lexA genes are the regulators which

control "SOS" response. Under normal conditons, LexA

protein represses the subordinate genes of the system

31

and RecA protein derepresses these loci in response to

DNA damage (Kenyon, 1983). LexA protein is a self

repressor and also binds to similar operator sequences

in each gene (Little et. BI_, 1981; Sancar et. aj., 1982;

Little, 1984). During 'SOS' induction, the LexA

repressor is cleaved between ala-gly bond by the second

regulator, the RecA protein (Little 1984). RecA protein

if acquires a specific protease activty to form RecA when

it interacts with intracellular molecule that results

from certain type of DNA damage and therefore, RecA

protein is considered to play a key role in the

induction of 'SOS' response (Radman, 1975; Takahashi et_

al , 1986) .

The "SOS' response is transient and thus following

DNA repair and removal of inducing stimulus (i) RecA

protein loses its protease activity, (ii) level of LexA

repressor rises and (iii) repression of the SOS genes

resume (Brent and Ptashna, 1981; Little et, al., 1981;

Sancar et. aX, 1982).

The accumulation of certain deoxynucleotide

monophosphates and a high level induction of recA gene

expression have been suggested to stimulate the "SOS'

repair (Gottesman, 1981). Moreover, Salles et. al_ (1983)

reported the full amplification of RecA protein without

any amplification of other single strand binding

proteins under 'SOS' inducing conditions. Bebenek and

Janion (1985) proposed the possible role of mismatch

repair also in the induction of 'SOS'.

32

Mutagenesis resulting from'SOS' processing of

damaged DNA template is targeted and is not due to the

induction of some random mutator activity (niller,

1783; Walker, 1984). Radman (1974, 1975) suggested that

the appearance of mutation during "SOS' processing may

be due to the inducible infidelity of DNA replication,

because pyrimidine dimers were found to block the chain

elongation by purified DNA polymerase I and III on UV-

irradiated DNA template. Subsequent studies revealed

the involvement of inducible inhibition of proof

reading (3'-5' exonuclease) activity of DNA polymerases

.(Radman et. ai, 1977; Boiteux et. aj^, 1978; Villani et.

al. 1978). Moreover, the lexA dependent conversion

involving the £ subunit of DNA polymerase III

holoenzyme to 'SOS' polymerase has also been postulated

(Scheuermann et. aX, 1963; Piechocki et. aJL, 1986). Lu eX.

al (1986) infact, have demonstrated the recA mediated

inhibition of 3'-5' nuclease activity of isolated £

subunit of DNA polymerase III and was suggested to be

responsible for targeted mutagenesis.

Gene Specific DNA Repair:

Recent studies demonstrated that in several

biological systems, the DNA repair processes are

heterogeneous within the genome (Bohr, 1991). It has

been reported that active genes are preferentially

repaired in hamster (Bchr et. aJL ,1985) and human cells

(Mellon et. al_. 1986) after exposure to UV radiation.

33

This heterogeneity is further extended to a strand bias

of DNA repair after UV exposure and has been

experimentally proved that the transcribed strand is

repaired much more efficiently than the nontranscribed

strand (Mellon et. al, 1987; Stevnsner and Bohr, 1993).

The first study of gene specific repair in heritable

disorder Xeroderma pigmentosum (XP) group 6 cells

revealed 10-307. repair in the dihydrofolate reductase

(DHFR) gene in XP4R0 strain (Bohr et. aJL, 19B6a).

Recently, Venema et. al_ (1990) reported that adenosine

deaminase and DHFR genes were preferentially repaired

in XP-C fibroblasts from individuals XPITE and XP

21R0. The strand bias of the repair in these genes in

XP-C (XPITE) cells has also been analyzed (Venema et.

al, 1991).

Preferential DNA repair is a general feature in

many biological systems. It has been observed in

various mammalian cell lines, in fish, yeast (Terlleth

et al , 1989) and in E^. coli (Mellon and Hanawalt,

1989). Its occurrence in bacteria suggests that the

preferential DNA repair of active genes is not

dependent upon chromatin structure. Studies on the fine

structure of DNA repair of UV damage in the hamster

DHFR gene suggest that the region of the preferential

repair could be larger than the size of the gene.

Characterization of a DNA repair domain containing the

DHFR gene in CHO cells indicated the size of the domain

in the range of 60 -80 Kb (Bohr et. JLL. I' BA.b) . Since

34

this is the size of the higher -order structures in

chromatin, it is possible that DNA repair is regulated

within such chromatin loops. One parameter to consider

is the degree of genomic demethylation. There are very

few studies on the relation of DNA repair to genomic

methylation patterns, at the gene level showing that

genomic demethylation could affect the pattern repair

in the hamster DHFR domain .

Several studies have demonstrated that the

efficiency of repair in a gene also correlates to its

transcriptional activity, as in the case of the hamster

and human metallothionein genes and can be blocked by

the transcription inhibitors . Moreover, with respect

to repair, different genomic regions have been

categorized from the lowest to the highest repair

efficiency as follows (i) the inactive genomic DNA ,

(ii) the inactive genes that are turned off early in

development, (iii) the structural genes that are

capable of transcription at certain times in

metabolism, (iv) the essential genes that are always

active but at a low level of transcription and finally

(v) an active gene transcribed at a high rate. This

categorization is obviously speculative and needs to be

explored (Bohr, 1991).

In view of the present literature we have

undertaken this study ta determine the mutagenic and

genotoxic potential of four pesticides viz captan,

35

foltaf, phosphamidon and furadan.

The major objectives were as follows:-

(1) Screening of the pesticides for mutagenicity by

means of Ames testing system. (Plate incorporation

assay) .

(2) The effect of the pesticides on the survival of

SOS repair defective strains of E.. col i K-12.

36

CHAPTER - II

GENERAL MATERIALS AND METHODS

Table IV: The Salmonella typhimurium and Escherichia coli strains

used in this study have been tabulated as under :

Strain designation

Relevant genetic markers Source

Ames Strains TA97a

TA98

TA100

TA102

TA104

uvrB. hisD6610, bio, rfa. R-factor plasmid-pkMlBi

urvB. hi5D5052. bio, rfa. R-factor plasmid-pkH101

uvrB. hi5G46. bio, rfa. R-factor plasmid-pkM101

uvrB. hi5G428. bio, rfa R-factor plasmid-pkM101 multicopy plasmid-pAQl

uvrB, hi5G42B. rfa. R-factor plasmid-pkM101

Ames, B.N.

Ames, B.N.

Ames, B.N.

Ames, B.N.

Ames, B.N.

E. coli K-i2 strains

AB1157

AB2463

AB2494

AB2470

KL400

KL 403

MM159

thi-1. arqE3. thr-1. 1euB6. ProA2. hisG4. lacYl. F-, Str' . A'^

recA13. thi-1. argE3» thr—1. leuBd). proA2. hisG4. F-, StrPr~^

lexA. thi-1. thi-1. leuB6. proA2. hisG4, metB. lacYl. F-, Str-, A"'

thr-1. leuB6. lacYl. ^, bisG4. recB21. arqE—. thi-1.

thi-1. leuB6. proC32. hi5FB60. thvA54. lacZ36. 5trA109. A .

leuB6. lacZ36. DrDC32. ^ , hisF860. thvA54. polAl. thi-i

qalK2./f-. raaL200. ijvrAi57. IN (rrnD-rrnE)l

Howard-Flanders,P,

Howard-F1anders,P.

Howard-F1anders,P.

Howard-Flanders,P.

K.B.Law laboratory,

K.B.Low laboratory

Meselson, M.

37

METHODS

Maintenance and growth of bacteria : Each strain of

Salmonella typhimurium was streaked over master plate.

A single colony was picked up, grown in minimal medium

and repurified by streaking over fresh master plate.

Likewise each strain of E.coli was streaked over

nutrient agar plates. A single colony was picked up and

repurified by streaking over agar plates. The culture

was tested on the basis of associated genetic markers

raising it from a single colony from the master plate.

Having satisfied with the test clone the culture was

raised and streaked over minimal and nutrient agar

slants. It was then allowed to grow over night at Zl'C

and stored at 4°C. Every month, cultures were

transferred over fresh slants with TA102 strain as an

exception. It was transferred after every 15 days.

Stabs were prepared for longer storage.

Overnight culture of S_ typhimurium strains were

used as such for experiments. Overnight culture of

E.coli strains were raised in nutrient broth at 37'C

The culture was diluted fifty times in fresh broth

followed by shaking at 37'C till the cell density

8 ~1 reached to about 2 x 10 viable counts ml . Such

exponential cultures were used in all the experiments.

38

Media

Media far Ames Strains :

Medium for master plates and slants : The composition of the

medium for Ames tester strains to prepare master plates and

slants is as under:

Sterile 50X VB Salts 20 ml

Sterile agar 15 g per 910 ml

Sterile 407. glucose 50 ml

Sterile histidine HCI.H2O 10 ml

(2 g per 400 ml H2O)

Sterile 0.5mM biotin 6 ml

Sterile ampicillin solution 3.15 ml

(a mg/ml 0.02 N NaOH)

Sterile tetracycline solution 0.25 ml

(8 mg/ml 0.02 N HCl)

The above components were mixed with the molten agar to

prepare the plates. Tetracycline was added only for use with

TA102 which is tetracycline resistant.

Stock solution of VB salts (IX) was prepared using the

following ingredients :

MgSD4.7H20 0.2 g/1

Citric acid monohydrate 0.2 g/1

K2HPO4 (anhydrous) 10.0 g/1

NaHNH4Pa4.4H20 3.4 g/1

39

The salts were added in the order indicated to warm

distilled water and each salt was allowed to dissolve

completely before adding the next. The solution was then

autoclaved for 20 min. at 121"C.

Minimal glucose plates for mutagenicity assay :

Sterile VB salts 20 ml

Sterile 407, glucose 50 ml

Sterile agar 15 g/930 ml distilled water

The above components were mixed with the molten agar

and poured atleast 30 ml in each plate.

Top agar for mutagenicity assay : The top agar contained

0.67. Difco agar and 0.57. NaCl. At the time of experiment

i0ml of sterile solution of 0.5 mM histidine.HCl and 0.5 mM

biotin was added to the molten agar and mixed thoroughly by

swirling.

0.5 mM histine/biotin solution for mutagenicity assay :

D-Biotin 30.9 mg

L-Histidine HCl 24.0 mg

Distilled water 250 ml

Dissolved biotin by heating the water to the boiling

point and mixed histidine to it. Autoclaved for 20 minutes

at 121'C.

40

Media far E.coli K-12 strains :

Nutrient broth (13g/l): Nutrient broth had the following

composition.

Peptone 5 g/1

NaCl 5 g/1

Beef extract 1.5 g/1

Yeast extract 1.5 g/1

pH (approx.) 7.4 + 0.2

Nutrient agar (Hard agar) :

Nutrient broth 13 g/1

(Hi media)

Agar powder 15 g/1

(Hi media)

Buffers

MgS04.7H20 solution (B.BIM) : For all dilution, 0.31M MgS04

solution was used.

B.2 M sodium phosphate buffer, pH 7.4 :

NaH2P04.H20 13.8 g Na2HP04 14.2 g Distilled water 500 ml

Adjusted the pH to 7.4 and then sterilized it at 121'C

for 20 minutes.

41

CHEMICALS

The fallowing chemicals were used :

Chemicals

Agar Powder

Ampicil1 in

Biotin

Chloramphenicol

Dimethyl sulphoxide

Dipotassium hydrogen phosphate

Disodium hydrogen phosphate

D-glucose

Histidine monohydrochloride

Hydrochloric acid

Magnesium sulphate

Nutrient agar

Sodium ammonium phosphate

Sodium chloride

Sodium hydoxide

Tetracycline

Source

Hi-Media, India

Ranbaxy, India

Hi-Media, India

Biochem Pharmaceutical Industries, India

s.d. fine chemicals, India

Qualigens, India

Sarabhai Chemicals, India

s.d. fine chemicals, India

CDH, India

s.d. fine chemicals, India

s.d. fine chemicals, India

Hi-Media, India

E. Merck, Germany

s.d. fine chemicals, India

Qualigens, India

IDPL, India

42

structure and chemical composition of the pesticides used:

Pesticides Source

Captan 3a, 4, 7, 7a Tetra-

hydra-2- C(trichloromethyl) thio]- 1 H - isoindole -1-3 (2H) - dione !I N - (tri­chloromethyl thio)-4 cyclo-hexene-1, 2-dicarboximide.

Rallis Agrochemicals Research Station, Banglore

—S —CCi

(2) Foltaf : 3a , 4, 7, 7a -Tetrahydro-2-C(1,1,2,2 Tetrachloroethy1) thiol] -1 H -isoindole - 1, 3 (2H) -dione, ;! N - (1, 1, 2, 2 -tetrachloroethyl thio) - 4 -cyclohexane - 1, 2 dicarbo -ximide; ;; Difolaton.

Rallis Agrochemical Research Station, Banglore.

CHCk:

43

3. Furadan : 2, 3 - Dihydro-2,

2 - dimethyl - 7 methylcarbamate; carbamic acid 2, 2, 2 - dimethyl -furanyl ester.

benzofuranol :! methyl 3 - dihydro • 7 - benzo

Rallis India Ltd. Agrochemical Division (Maharashtra)

O.CO-NHCH

Phosphamidon : Dimecron ; ; 2 - chloro - N, N diethyl 3 hydroxycrotonamide dimethyl phosphate.

Hindustan Ciba Geigy

o

CHjO II

CHjO /

0

CH3 I

I a

o 11 c N

CzHs /

44

CHAPTER - lil

MUTAGENICITY ASSAY

MUTAGENICITY ASSAY

INTRODUCTION

There is considerable evidence that a large

proportion of human cancer may be caused by exposure to

toxic chemicals in the environment, very few of which

have been tested for carcinogenicity or mutagenicity. A

program of cancer prevention aimed at identifying and

eliminating human exposure to hazardous chemicals

requires the development of rapid, inexpensive, long-

term animal tests, to pinpoint dangerous chemicals

among the thousands to which humans are exposed.

The Salmonella/microsome mutagenicity test,

relating mutagenicity to the number of revertant

colonies, has been sufficiently developed and validated

to be seriously considered for widespread use in this

way (Ames, 1971; Ames et. al, 1973; McCann et al_, 1975;

van der Hoeven e . ajL., 1990). The evidence accumulated

so far indicates that with a few exceptions,

carcinogens are mutagens (Ames et. a_l.j 1973; Flessel et.

al. 1987; Zeiger, 1987). It thus supports the

desirability of using this type of rapid and economical

test system as a screening technique (Ames, 1971; Ames

et al. 1973). The work embodied in this chapter was

therefore, designed to screen the available pesticides

for their potential mutagenic and carcinogenic

behaviour employing the Ames testing system.

45

Methods

Bacteria, pesticides and media are listed in

Chapter II. All the media were freshly prepared. Plate

incorporation assay was routinely performed for testing

the mutagenicity of the pesticides.

Plate incorporation assay : This test was

performed by combining the pesticide, the bacterial

tester strain and 0.2M phosphate buffer (pH 7.4) in

soft agar which was poured onto a minimal agar plate.

Positive and negative controls were also included in

each assay. After incubation at ZVC for 48 hours,

revertant colonies were counted and the mutation

frequency calculated by subtracting the spontaneous

reversions from the induced reversions.

Routine examination of the bacterial background

lawn resulting from the trace amount of histidine added

to the top agar aided in determining the toxicity of

the test chemicals and in the interpretation of

results.

RESULTS

Assessment of the mutagenic potential of certain

pesticides

Mutagenicity of captan:

Mutagenicity of four different pesticides viz

captan, foltaf, phosphamidon and furadan have been

evaluated employing Ames salmonella tester strains.

Fig. 1 shows a dose dependent increase in the mutagenic

46

400

d) •U rt

1-1

a tn 4-1 C rt 4-1

(1) > OJ

u 14-1 O

o 2

'^00 \ j \ j \ j

200

100

0 0 0.25 0.50 1.00 1.50

Concentration (;jg)

Fig. 1. Dose response curves of captan (-S9) with Ames tester strains.

TA 97 a ( ); TA 98 ( - j — ); TA 100 (-- ) ; TA 102 (-X-); TA 104 (-Q") .

47

response of captan with five different histidine

mutants. At concentration range of 0- 1.5^g/plate, a

considerable increase in the number of his revertants

was observed with a certain degree of variability

depending upon the sensitivity of the strain. The total

number of induced revertants as shown in fig. 1 have

been obtained after subtracting the spontaneous

revertants. The results indicate that the tester strain

TA104, TA97a and TA100 are maximally sensitive compared

to TA102 and TA78 at a concentration of 1.0;jg/plate. A

higher dose of captan i.e. 1.5_^g/plate was invariably

found to be toxic to all the tested strains. At this

concentration, a sharp decline in the dose response

curve of TA104, corresponding to 987. decrease in the

number of revertants, was clearly observed. The maximum

mutagenic response was seen at a plate concentration of

l.B^g/plate captan, in the following order of

sensitivity: TA104> TA77a> TA100 > TA102 and TA98.

Interestingly, a 2-4 fold decrease in the mutagenic

response of his mutants was noticed in the similar

concentration range in presence of S9 mix (Fig. 2).

Mutagenicity of foltaf:

The mutagenic response of foltaf, evaluated in

concentration range of 0-2.5 g/ plate, is shown in

Fig. 3. The results show that the maximum number of

revertants were produced with TA102 and TA77a at a dose

of 1.0jjg/ plate. Any further increase in the dose of

48

150

Q) 4J

a CO

c u

>

o 2;

100

1.50

Concentration ( g)

Fig. 2. Dose response curves of captan (+S9) with Ames tester strains.

TA 97 a(-*-); TA 98 (-H); TA 100 {^); TA 102 (-D-) ; TA.104 (-H- ).

49

400

Concentration (>Jg)

Fig. 3. Dose response curves of foltaf (~S9) with Ames tester strains.

TA 97 a(-»-); TA 100 (-1—); TA 102 (-O-) ; TA 104 (- -] .

TA 98 (Not mutagenic).

50

foltaf resulted in decreased survival of the bacteria

except strain TA104 which has exhibited a concentration

dependent increase in the number of revertants upto the

level of i. 5)jg/plate. There after, a sharp decline in

the mutagenicity curve was observed, most probably due

to the toxic effect of the pesticide. No significant

induction in the mutagenic response in TA78 was

noticed. The maximum number of revertants, induced by

foltaf at a concentration of Ijjg/plate in different

tester strains were in the fallowing sequence: TA102 >

TA97a> TA104> TA10IZ) and TA78.

Mutagenicity of phosphamidon

Fig.4 shows the mutagenicity profile of

phosphamidon in a concentration range of 0-i0^g/plate.

A linear dose dependent mutagenic response was observed

upto a concentration of 7.5 ^pg/plate in the following

order of sensitivity;TA100> TA104 > TA97a > TA102 and

TA98. Further increase in dosage to 10^g/plate resulted

in reduced survival of the bacterial strains. The

strain TA9a was found to be least sensitive and

produced the lowest number of revertants even at the

optimum plate concentration of 7.5^g which is causing

maximum induction of mutagenicity in other tester

strains.

Mutagenicity of furadan

Fig. 5 shows the mutagenic response exhibited by

furadan at a concentration range of 0-30pg/plate. A

51

400

10.0

Concentration ( g)

Fig. 4. Dose response curves of phosphamidon (-S9) with Ames tester strains.

TA 97 a ( — ) ; TA 98 (-X-) ; TA 100 {-\-) ; TA 102 (-O-): TA 104 (^^) .

52

500

400

cd

a

^ 300 CO

> CD

O

O 2;

200

100

30.0

Concentration ( g)

Fig. 5. Dose response curves of furadan (-S9) with Ames tester strains.

TA 97 a (-»-); TA 98 (-+-); TA 100 (- ) ; TA 102 ( — > ^ ); TA 104 (—a—).

53

significantly high level of mutagenic response was seen

in strain TA1(Z)4 producing the highest number, 422

revertants/plate at concentration of 5;jg/plate followed

by TA102 (85 revertants/plate).Other strains including

TA97a, TA100 and TA98 did not exhibit any significant

increase in the reversion frequency. The results also

indicate that an increase in amount of furadan beyond

5ijg resulted in a remarkable decrease in the viability

of the bacterial strains.

Discussion

The Salmonella test was first validated in a study

of 300 chemicals most of which were known carcinogens

(McCann ejt aJL. 1975; licCann and Ames, 1976; McCann and

Ames 1977). It was subsequently employed in other

studies by the Imperial Chemical Industries (Purchase

et al. 1976), and the International Agency for Research

on Cancer (Bartsch et. ai., 1980). Nearly 90"/. of the

carcinogens tested were found to be mutagenic in these

studies, but there was considerable overlapping of

chemicals tested. It is customary to define the

compound as negative if none of the tester strains

responds with and without metabolic activation upto the

limit of toxicity not exceeding to 5ng/plate

concentration (deSerres and Ashby, 1981; Dyrby and

Ingvardsen, 1983). An increase in the number of

colonies (over the number of spontaneous background

revertants) indicates that the chemical is mutagenic

while the number of revertant colonies provides an

index of the mutagenic activity of the sample (deSerres

and Ashby, 1981; Levin et. aj., 1984; Flessel e^ al,

1987). Our results indicated an invariable increase in

number of revertant colonies with all the pesticides

viz. captan, furadan, foltaf and phosphamidon. Moreover

all the pesticides exhibited a remarkable degree of

mutagenicity with TA97a, TA1(Z)0, TA104 and TA102.

Table V, shows the relevant genetic and

biochemical markers of the Ames tester strains. All the

strains carry the pKM101 plasmid which is believed to

enhance the error—prone repair process (Shanabruch and

Walker, 1980; Levin et. aJL. 1982a). TA97a strain has

been recommended for detecting frameshift mutagens

(Levin et. al, 1982a), whereas TA102 is known to detect

the oxidative mutagens (Levin et. al, 1984). TA97a, TA98

and TA100 tester strains contain G-C base pairs at the

critical site for reversion (Isono and Yourno, 1974;

Barnes et. al., 1982; Levin et, al_, 1982a). With regard to

the individual difference among the G-C specific

mutants, TA97a, TA98 and TA100, the first two strains

are the frameshift type while TA100 is the base-pair

substitution mutant (Isono and Yourno, 1974, Barnes et.

al. 1982). The tester strain, TA102, is proficient in

the excision repair machinery but carries a mutation in

his G428 gene on the multicopy plasmid, pAQl. On the

contrary, TA104 carries the same mutation on the

55

Table V: Relevant genetic and biochemical markers of Ames Strains.

S.NO Markers TA97a TA98 TA100 TA102 TA104

1. Type of mutation Frame-shift

Frame-shift

Base pair substi tution

Transi­tion

Transi­tion

2. Location of mutation in histidine gene

his012A2

3. Excision-repair Auvrb

hi5D3052 hi5G46

^uvrb Auvrb

hisG^28 Ochre mutation on a multicopy plasmid pAQl

uvrb Proficient

hisG428 on the chroao-some

extra Auvrb

4 . Loss of p o l y ­sacchar ide layer ( r f a )

r f a r f a r f a r f a r f a

5. R- fac tor p lasmid pkri l01 pkni01 pkr i l01 pkn iB l pkI1101

Base-pai r a t the s i t e o f r eve rs i on

GC GC GC AT AT

7. Antibiotic resistence

Amp Amp Amp Amp Tet''

Amp

Spontaneous reversion frequency

90-180 30-50 120-200 240-320 350-^75

Replacement of the strains

TA1537 TA1538 TA1535

Symbols : A - deletion r - resistent

56

chromosome but carries a defective excision repair

gene, uvrB which obviously diverts the repair towards

the errorprone side and thus changes the mutagenic

activity of the compound (Levin e^ al_» 1982b). In view

of our findings, it is noteworthy that the pesticides

act on the A-T base pair mutants TAi04 and TA102 in

addition to TA97a and TA100 having G-C base pairs at

the site of mutation. However, the pesticide, captan

seems to revert the frameshift tester strain, TA97a,

more efficiently as compared to base-pair and

transition mutants.

We have then, concentrated our studies on four

pesticides. Quantitative Ames testing revealed that the

tester strains, TA97a, TAi00, TA102, TA104 were

responsive (Fig. 1,2,3,4 and 5). TA104 happened to be

the most responsive. In view of the present findings it

is clear that the pesticide treatment brought about a

transition mutation owing to the error prone repair of

the damaged cell. This idea gains further support by

the relatively strong mutagenic response of the test

compound on the uvrB defective TA 104 strain.

57

CHAPTER - IV

GEiNOTOXICITY ASSAY

Genotoxocity Assay

Introduction

Various physical and chemical environmental agents

have been shown to damage cellular DNA in. vivo. The

capacity of a system to repair such damage is basic to

the ability of an organism to withstand the biological

consequences of environmental stress. Correctly

repaired, genetic damage has little effect on the

biological function of a system. Studies on

microorganisms, mammmalian and plant cells have shown

that DNA damage results in change in the physiological

processes such as growth, division, transcription of

DNA, cell death, mutation and induction of

transformation (Elkind and Whitmore, 1967; Price and

Makinodon, 1973; Lohman and Bootsma, 1974).

E.coli responds to DNA damage with the expression

of a set of functions usually termed as SOS response.

This includes the induction of a transitory mutagenic

DNA repair system, the activation of inducible prophage

and of several other functions involved in cell

division and DNA metabolism (Radman, 1974; Witkin,

1976).

The work embodied in this chapter was therefore

planned to estimate the extent of injury produced, to

understand the mutagenic behaviour and the mode of

interaction of pesticides with DNA.

58

Our contention was that SOS defective strains of

E.coli K-12 might serve as a convenient model for this

purpose because even a slight change in environment

could be reflected in its survival pattern.

Methods

Bacteria, media, pesticides and buffers are listed

in Chapter II. Relevant genetic markers associated with

the strains is given in (Table IV ). Survival patterns

of E•coli strains have been studied in the presence of

pesticides.

The SOS defective E.coli strains were treated

with that concentration of pesticides at which the

highest peak was observed in the Ames test . Except

otherwise stated^ the E.coli cells were treated for 6

hours with pesticide. All the media were freshly

prepared.

Pesticide treatment to bacteria :

The SOS defective recA. recB. lexA. polA and uvrA

mutants of E.coli K-12 as well as the isogenic wild

type strain were harvested by centrifugation from g

exponentially growing cultures (1-3 x 10 viable counts

ml ). The pellets so obtained were suspended directly

in 0.01M MgS04 solution and treated with the pesticides

at various concentrations. Samples were withdrawn after

the treatment period of 6 hours, suitably diluted and

plated to assay the colony forming ability. Plates were

incubated over night at 37*C. Treated controls were

59

also run simultaneously.

RESULTS

Evaluation of the qenotoxic effect of the pesticides

Genotoxicity of captan

The genotoxicity of captan has been asssessed by

determining the colony forming ability of the bacterial

strains treated with varying doses of captan. The

relationship between the percent survival and the dose

of captan is shown in Fig. 6.The trend of the survival

curves of DNA repair defective mutants, recA , recB ,

lexA , and pol campared to their isogenic wild types

ABii57 and pol A clearly indicates the DNA damaging

activity of the captan. The mutants particularly pol ,

recA , and lexA exhibited a significant decrease in

the colony farming ability within a concentration range

of 2 to IB^g^itJesticide. Among all mutants, pol was

found to be the most sensitive and its survival reached

to the baseline, indicating 99.3X loss in viability

campared to the pol strain. The mutants recA and lexA

have also shown decrease in the colony forming

ability compared to their wild-type counterparts. It

was noticed, that a higher concentration i.e. IQiuq/fni of

captan, the wildtype cells also exhibited a decrease in

the survival with a proportional reduction in the

survival of mutant strains.

60

100

>

>

3 w

c 0) o >-l

a> 0-,

10.0

Concentration ( g)

Fip. 6. Survival of SOS defective E. coli K-12 strains exposed to captan

Wild type (——)i recB (-H); recA (- ) ; lexA (-D—) ; pol (-X-); pol {-<>-) .

61

Genotoxicity of foltaf

A relatively higher amount of foltaf was used for

the genotoxicity assay. Fig. 7 shows the comparision of

the survival curves of various DNA repair defective

mutants with the repair proficient strains in a

concentration range of 0 to 15pqWof foltaf. At a sub­

lethal concentration of 5^g, a noticeable change in the

survival pattern of the mutants was observed. This

effect became more pronounced at a higher concentration

of i5^g foltaf resulting in percent reduction in the

colony forming ability of pol and recA mutants,

respectively. The highest toxic dose was found to be

25ug which causes significant loss in the viability of

all the strains and no differential response was

noticed at this concentration .

Genotoxicity of phosphamidon

Fig.8 shows the survival curves of recA , recB ,

uvrA, lexA and polA mutants upon treatment with

phosphamidon in a broad concentration range of 0-100u^W-

Relatively weak genotoxic activities were noticed even

at a higher concentration of 100 cj^,Hawever, certain

mutants including polA , recA and lexA were found to

be much more sensitive compared to uvrA. recB and wild

type strains. An almost linear dose dependent decrease

in the colony forming ability of polA mutant was

observed. On the other hand , the mutant recA~ and

lexA exhibited resistance against the higher

62

100

> •r-l

> O

C (1) o 0)

a.

20 .0 25 .0

Concentration ( g)

Fig. 7. Survival of SOS defective E. coli K12 strains exposed to foltaf.

Wild type (-"—); recB H ^ ) ; recA (-+"); lexA (—CH ;

pol^ (-^-); pol~{Q ) ; uvrA (^ ).

63

100

CO >

>

to

u c u QJ CL,

25.0 100.0

Concentration (;jg)

Fig. 8. Survival of SOS defective E. coli K12 strains exposed to phosphamidon

Wild type e-«-); recB {-\--) \ recA (- -) ; lexA (-D-) ;

pol^ (->^); pol"( <> ); uvrA {r^:^-).

64

concentration of phosphamidon. The wildtype polA-<- and

AB1157 also showed almost similar response in the test

range.

Genotoxicity of furadan

The genotoxicity of furadan was evaluated at a

much higher concentration range, ranging form 0-300 ygftnt.

As shown in Fig. 9 , the relationship between the

percent survival and the dose of furadan represents a

weak genotoxic ability of furadan. Even the higest

concentration of 300^g was found to be insufficient to

induce significant DNA damage which could result in the

loss of viability of the bacterial strains.

Nevertheless, the differential pattern of the survival

curve, as observed at 100pg concentration of furdan,

indicates that pol , recA and lexA ars relatively

more sensitive strains as compared to the recB mutant

and the wiIdtype-strains.

Discussion

Bacterial injury caused by UV and ionizing

radiations has been well documented (Auerbach, 1976;

Howard-Flanders, 1968; Witkin, 1976; Friedberg, 1985).

Cellular DNA damage occurs following exposure to

ultraviolet light, ionizing radiation, various

environmental chemicals and reactive oxygen species. If

left uncorrected, it can result in cell death, mutation

and neoplastic transformation. Prokaryotes and

eukaryotes have evolved systems for reversing DNA

65

100.0 200.0 300.0

Concentration (jag)

Fig. 9. Survival of SOS defective E. coli K12 strains exposed to furadan

Wild type { — ) ; recB (- i ); recA (-H); lexA (-O-) ; pol* (-><-); pol~( O ).

66

damage, and several classes of DNA repair enzymes have

been identified (Friedberg and Bridges, 1983).

The pesticides which were detected to be mutagenic

in the Ames testing system were also tested with SOS

repair defective recA , recB , lexA , uvrA and polA

mutants of E.coli K-12. It is postulated that the

inducible error—prone repair pathway could potentially

operate on all types of lesions in DNA whether produced

by radiation or by other agents (Walker 1785). These

mutants were found to be highly sensitive to the

pesticides (Figures 6, 7, 8 and 9) suggesting thereby,

the damage to the DNA of the exposed cells as well as

the role of polA, recA, recB. uvrA and lexA genes to

cope with the hazardous effect of the pesticide. polA

mutant was found to be most sensitive to all the four

pesticides. The role of recA and lexA gene in

initiating the error prone repair in E.coli is well

documented (Witkin, 1976; Walker, 1985)This idea gains

further support by Ames testing studies (Figures 1, 2,

3 ,4 and 5) with Salmonella strains triggering the

error prone SOS response.

67

CHAPTER-V

DISCUSSION

DISCUSSION

Our studies on the mutagenicity and genotoxicity

of the pesticides viz captan, foltaf, phosphamidon and

furadan have provided, a complete profile of the

mutagenic potential as well as insights into their

mechanism of repair in bacteria. It is generally

believed that a large proportion of human cancer is

mainly due to the exposure to genotoxic chemicals

present in the environment. Ironically, very few of

them have been tested for their carcinogenic and /or

mutangenic activities. It is, therefore, emphasized to

identify the entire spectrum of chemical carcinogens by

performing the short-term mutagenicity and

carcinogenicity assays in combination with

epidemiological investigations. Among the plethora of

environmental mutagens, pesticides are considered to

belong to a noxious chemical class of compounds, posing

a major threat to the global environment, A majority of

them have been reported to be mutagenic, carcinogenic

and teratogenic (Lucier and Rambush, 1983; M£tzler,

1984; Dunkel et. al., 1985). Unfortunately due to lack of

a national database, no accurate or current statistics

are available regarding the incidence of acute

pesticide poisoning in this country. However, in the

United States atleast 30 to 40 persons die of acute

poisoning each year and approximately 20,000 persons

Are taken to emergency room each year for actual or

68

suspected pesticides poisoning (Weisenburger, 1993). In

fact acute poisoning is thought to be a major global

health problem with approximately 25 million

occupational poisoning per year (Jeyaratnam, 1990).

Most of these occur in third-world countries where

pesticide education and monitoring and the use of

safety equipments are largely non-existent.

The epidemiological data have indicated that most

of the cancer is due to the environmental factors

(Ramel, 1991). Consequently, the identification of

carcinogens in the environment is of fundamental

importance in cancer prevention. This has actually

prompted us to evaluate the genotoxic and mutagenic

potential of certain pesticides. The four selected

pesticides tested in this study have shown a

differential mutagenic response in the following order,

capton > foltaf > phosphamidon > furadan. As indicated,

captan, a highly effective broad spectrum antimicrobial

agent (Dillwith and Lewis, 1982a) was found to be most

mutagenic among all the pesticides tested. However, its

mutagenic activity was observed to be decreased in

presence of S9 mix suggesting that captan could be a

direct acting mutagen and does not require any

metabolic activation. In fact, its catabolism resulted

in the production of relatively less mutagenic

intermediates as evident in Fig. 2. This attenuated

mutagenic response of captan in presence of S9 mix is

in accordance with the earlier reported results (

69

Moriya et. aj_ 1978). Captan was reported to be mutagenic

with the strains that detect the mutagens damaging

G > C base pairs (Bridges et, al_j 1972; Xu and

Schurr 1990). It is now clear from our results that

captan also exhibits positive mutagenic response with

the strain TA104, which suggests that it could also

induce A > T base pair substitution. We have also

noticed about 1.6 to 13 times higher mutagenic response

of captan with different tester strains under our

experimental conditions; compared to the reported

values . As expected captan was found to be relatively

less mutagenic in strain TA102, the strain with intact

excision repair system, suggesting that the captan

induced DNA lesions are repairable by the enzymatic

repair mechanism.

Other pesticides including foltaf, phosphamidon,

and furadan have been identified as comparatively weak

mutagens. Although, all of them have exhibited

mutagenic response to a certain extent in strains TA104

and TA102, none of them were significantly responsive

with strain TA100. Comoarison of the test pesticides on

the basis of their structure-activity relationship

revealed that the tricnloromethy1thio moiety of captan,

is primarily responsible for the formation of adducts

with DNA. Although, foltaf has a reactive group similar

to captan, but the substitution of its terminal CHCI2

group seems to be more difficult than in captan.

70

However, the mutagenic profile of foltaf suggests its

broad specificity for inducing both the transition and

frameshift mutations in DNA. Our studies revealed that

the most sensitive loci were his6428 and hi5(ai242 in

the multi-copy plasmid pkMi01 inside the tester strains

TA102 and TA97a respectively. Since Foltaf exhibited

maximum mutagenic response in excision repair

proficient strain TA102, it could possibly be due to

the partial removal of lesions by uvrABC excinuclease

and might have involved the error prone type of

mechanisms in repairing the damage at the expense of

mutations at the repaired sites.

Similarly, phosphamidon contains only one chlorine

2

atom a t t a c h e d t o sp - c a r b o n ( v i n y l i c c h l o r i d e ) and

t h e r e f o r e , i t s s u b s t i t u t i o n w i l l be even more d i f f i c u l t

than in f o l t a f . Moreover , t h e p l a u s i b l e r e a s o n fo r l e s s

mutagenic a c t i v i t y in c a s e of fu radan c o u l d be t h e

absence of hydrogen m o e i t y . A p p a r e n t l y , t h e on ly

r e p l a c e a b l e g r o u p i s -NHCH3, which i s supposed t o be a

poor l e a v i n g g r o u p . The a v a i l a b i l i t y of an e a s i l y

r e p l a c e a b l e e l e c t r o p h i l i c f u n c t i o n a l g r o u p in a

c a r c i n o g e n i s t h e r e f o r e an i m p o r t a n t f a c t o r in

n u c l e o p h i l i c s u b s t i t u t i o n r e a c t i o n w i t h DNA. Thus u s i n g

a s e t of S a l m o n e l l a typhimur ium s t r a i n s we have ,

e s t a b l i s h e d t h e m u t a t i o n a l s p e c i f i c i t y of t h e t e s t

p e s t i c i d e s w i t h o u t i n v o l v i n g any o t h e r complementary

a s s a y .

71

Our studies on the genotDxic effect of the

pesticides on DNA repair defective mutants of E . coli.

have also clearly suggested the role of pol. recA and

lexft genes in the repair of the pesticide induced DNA

damage . The mutants polA , recA and lexA have shown

a significant decrease in their survival upon exposure

to the pesticides. In the case of captan, foltaf and

furadan, the wild-type AB1157 has also exhibited a

decline in the survival curve particularly at a higher

concentration range which could be due to the

inhibition of the cellular metabolic machinery. There

a.re reports indicating that captan inhibits the

activity of DNA polymerase I (Dillwith and Lewis,

1982b), It also inhibits both the DNA and RNA synthesis

(Dillwith and Lewis, 1982b) by blocking the binding of

polymerase to the DNA template. The reduced survival of

wild type strain upon treatment with foltaf, furadan

and phosphamidon could be due to similar reasons. Since

the role of recA and lexA genes in initiating the

error—prone repair in E^. col i is well documented

(Witkin, 1976; Walker, 1985), the involvement of these

genes in our repair study led us to suggest the role of

inducible error—prone repair pathways in the enzymatic

removal of pesticides induced lesions in DNA.

72

CHAPTER-VI

REFERENCES

REFERENCES

Ames, B.N. (1971)- In"Chemical Mutagens: Principles and methods for thier detection". Hollaender, A; Plenum Press. N.Y. 267.

Ames, B.N.; Lee, F.D. and Durston, W.E. (1973)- Proc. Natl. Acad. Sci.(U.S.A.) 72, 2423.

Ames, B.N.; McCann, J. and Yamasaki, E. (1975)- Mutat. Res. 31, 347.

Ames, B.N. (1979)-Science, 204. 587.

Ames , B.N. (1984) -Citation Classic Curr. Cont. 27 No. 12., 18.

Ames, B.N.; liagaw, R. and Gold, L.S. (1987 )-Science, 236. 271.

Ames, B.N. (1989)- ACS Symp. Ser. 414. 223.

Anon (1973)- FAG Agricultural Series, No.90, P47.

Ashby, J. and Tennant, R.W. (1988)- Mutat. Res. 204. 17.

Auerbach, C. (1976)- In "Mutation Research." John Wiley and Sons, N.Y.

Barnes, W.; Tuley, E. and Eisenstadt, E. (1982)- In "13th Annual Meeting of the Environmental Mutagen Society Prog, and Abstracts. No. Aa - 1, 59.

Bartsch, H. ; MalaveiUe, C ; Camus, R.A. M.; Martel-Planche, G.; Brun, G.; Hantefeuille. A.; Sabadie, N.; Barbin, A.; Kuroki, T.; Drevon, C ; Picco^fi, C. and Montesano, R. (1980)- Mutat Res. 76., 1.

Bebenek, K. and Janion, C. (1985) - Mol. Gen. Genet. 201. 519.

Benbow, R.; Zuccarelli, A. and Sinsheimer, R.L. (1974) - J Mol. Biol. 88, 629.

Bishop, M. (1991)- Cel. 64.: 235.

73

Blair, A.; Axelson, 0. ; Franklin, C. et, AI_. (1990)-Carcinogenic effect of pesticides in Baker, S. R, Wilkinson, C.F (eds): The effect of pesticides on Human Health, Advances in Modern Environmental Toxicology, Princeton, NS, Princeton Scientific, 18.: 201.

Blair, A. and Zahm, S.H. (1991)- Cancer among farmers in Cordis, D.H.; Rea, D.F. (eds) Health hazards of farming. Dccupatinal Medicine: State of Art Reviews. b_; 335.

Bohr, V.A.; Smith, C.A.; Okumoto, D.S. and Hanawalt, P.C. (1985)- cell. 40, 359.

Bohr ,V.A.; Okumoto, D.S. and Hanawalt, P.C. (19e6a)-Proc. Natl. Acad. Sci. (USA). 83., 3830.

Bohr, V.A.; Okumoto, D.S.; Ho, L. and Hanawalt, P.C. (1986b)- J. Biol. Chem. 262. 16666.

Bohr, V. A. (1991)- Carcinogenesis. 12., 1983.

Boiteux, S.; Villani, G.; Spadari, S.; Zambrano, F.and Radman, M. (1978) - In "DNA Repair Mechanisms" Symp. Mol. Cell. Biol. (eds). P.C. Hanawalt; E.C. Friedberg and C.F. Fox Academic Press. N.Y. =•• 73.

Boone, C.W.; Kelloff, G.J. and Steele, V.E. (1992)-Cancer Res. 52. , 1651.

Boveri, T. (1914) - Zur. Frage. der. Entstehung. Malign. Tumoreu. Jena. Gustav Fisher.

Bowman, M.C. (ed.-1982)- In "Handbook of Carcinogens and Hazardous Substances". Marcel Dekker, Inc. New York and Basel.

Boyce, R.P. and Howard - Flanders, P. (1964) - Proc. Natl. Acad. Sci. (U.S.A.) 51., 293.

Boyle, W.J. (1992)- Curr. Opin. Oncol. 4, 156.

Brent, R. and Ptashna, M. (1981) - Proc. Natl. Acad. Sci. (U.S.A.) 78 , 4204.

Bridges, B. A.; Moltershead, R.P. ; Anne Rothwell, A.M. and Green, M.H.L.(1972) - Chem. Biol. Interact. 5, 77

74

Burnet, M. (1974) - In "Intrinsic Mutagenesis" Medical and Technical Publ. Co. Ltd. Lancaster, U.K.

Cairns, J. (1975) - Sci. Am. 233. 64.

Cardona, R.A. (1987)- In "Pesticides-Minimizing the Risks". ACS Symp. Ser. 336. Amer. Chem. Sac., Washington, D.C.

Carere, A. and Mospurgo, 6.(1981) - Prog. Mutat. Res. 2, 87.

CFR 40 162.3 (1986) - Protection of Envircnment, Office of the Federal Register, National Archives and Records Administration, U.S. Govt. P-inting Office, Washington, D.C.

Chaudhury, A.M. and Smith, G.R. (1985)- Mol. Gen. Genet. 201,525.

Clarke, C.H. (1971) - Mutat. Res. 11., 247.

Cohn, S.M. (1983) - Diss. Abstr. Int. B. 43. , 27^3.

Cooper, 6.M. (1982) - Science, 217, 801.

Crawford, B.D. (1985) - In "Advances in Modern

Environmental Toxicology" (eds.) W.6. Flames and R.J. Lorentzen. Princeton Scientific. Prirceton, N.J. p.13-39.

CuUiney, T.W. ; Pimental, D. and Pimentel , M.H. (1992) - Agric. Ecosystems Environ. 41_ , 297.

Defais, M. ; Foquet, P. ; Radman, M, and Erre-^a, M. (1971) - Virology, 43 , 495.

Deng, G. ; Chen, Ling-chun; Schott, D.R.; Thor, A. ; Bhargava, V.; Ljung, B.M.; Chew, K and Smit", H.S. (1994)- Cancer Research. 54 : 499.

deSerres, F.J. & Ashby, J. (eds.).(1981 . In "Evaluation of short-term tests for carcinogens." Reports of the International collabcrative Program. Elsevier/North-Holland, Amsterdam.

Devoret, R. (1979) - Sci. Am. 241 ,20.

Dillwith, J.W and Lewis, R.A. (1982a)- Biochem. Biophys. Acta. 696. 245.

75

Dillwith, J.W. and Lewis, R.A. (1782b)- Biochem. J. 201. 145.

Doll, R.and Peto, R. (1981) - J. Natl. Cancer Inst. 66. 1129.

Donna, A. ; Crosignani, P. and Robutti, F. (1989)-Scand. J. Work Environ. Health. 1_5., 47.

Dunn, K.; Chrysogelos, S. and Briffith, J. (1982) Cell, 28., 575.

Dunkel, V.C.; Zieger, E; Brusick, D; Mccoy, E; McGregor, D; Mortelmans, K; Rosenkranz, H. S. and Simmon, V.F. (1985)- Environ Mutagen 1 , 1.

Durstan, W.E. ; Yamasaki, E. and Lee, F.D. (1973) Proc. Natl. Acad. Sci. (U.S.A.). p 2281.

Dyrby, T. and Ingvardsen, P. (1983) - Mutat. Res. 123 , 47.

Elkind, M.M. and Whitmore, 6.F. (1967)- In "The Radiobiology of Cultural Mammalin cells. Gordan and Braden Pub., N.Y.

Farmer, P.B. (1982) - Chemistry in Britain, 18. , 790.

Finch, D.W.; Chambers, P. and Emmerson, P.T. (1985) J. Bacterial. 164 ,653.

Fink, L. (1984) - Bioscience, 34. , 75.

Flessel, P.Y. ; Wang, Y. ; Kuo In Chang and Wesolowski, J.J. (1987) - J. Chem. Education, 64 , 391 .

Florey, H. (1962) - General Pathology, 3rd eaition. London .

Flory, J. and Padding, C M . (1982) - Cell, 28, 7^7.

Friedberg, E.C. and Bridges, B (eds.) (1983)- Cellular Response to DNA Damages. Liss, New York.

Friedberg, E.C. (1985) - "DNA Repair", p 614.

Garrett, C.T. (1986)- Clin. Chim. Acta. 156. 1.

76

Barrett, N.E.; Sandhu,S.S.; Slack, H.F.and Waters, M,D. (1987) - U.S. Environ. Prat. Agency Res. Dev. EPA . EPA/600 D-87/153, 24 PP.

Georgian, L. (1975) - Mutat. Res. 31., 103.

Gocke, E. ; King, N.T.; Eckhardt, K. and Wild, D. (1981) - Mutat. Res. 90. , 91.

Sottesman, S.(1981) - Cell, 23, 1.

Gupta, B.N. (1989) - Indian J. Environ. Prot. 9, 801.

Hansen, M.F. and Cancenee, W.K. (1988) - Trends Genet. 4, 125.

Harris, C.C. (1985) - Environ. Health Perspect. 6^ , 185.

Hart, R.W.; Steven, M.; O'Ambrosio ; Kwokei, J.N.G.5 Sohan, P. and Modak, S.P. (1979) - Mechanisms of Aging and Development, 9 , 203.

Hatch, F.T.; Felton, J.S. ; Stuermer, D.H. and Bjeldanes, L.F. (1984) - In "Chemical Mutagen". Principles and Methods for their Detection (ed.) F.D. de Serres. (Plenum, N.Y.) 9, 111.

Hay, A. (1988) - Nature, 322. , 782.

Hayashi, Y. (1992)- Exp. Toxic. Pathol. 44., 465.

Hermanowicz, A. and Kossman, S. (1984)- Clin. Immunol. Immunopathol . 33., 13.

Hollstein, M.J.; Mc Cann, J.; Angelosanto, F.A. and Nichols, W.W. (1979)- Mutat. Res. 69., 173.

Heyneker, H.L. and Klenow, H.(1975) - In 'Molecular Mechanisms for Repair of DNA" (eds.) P.C. Hanawalt and R.B. Setlow. Part A, Plenum Press, MY, P219.

Hill, R.F. (1958) - Biochim. Biophys. Acta, 32., 636.

Hollaender, A. and Curtis, J.T. (1935) - Froc. Soc. Exp. Biol. Med., 33^, 61.

Hollstein, M. ; fIcCann, J. ; Angelosanto, F.A. and Nichols, W.W. (1979) - Mutat. Res. 65, 133.

77 r Ace No. V

Hoover, R.N. and Blair, A. (1991)- Pesticides and Cancer, in Devita, V. ; Helmans, Rosenberg, S. (eds.). Cancer Prevention, P.A. 1.

Houk, V.S.and De Marini, D.M. (1987) - Mutat. Res., 182 , 193.

Howard-Flanders, P.; Boyce, R.P. and Theriot, L. (1966) - Genetics, 53., 1119.

Howard - Flanders, P. (1968) - Ann. Rev. Biochem. 37. 175.

Howard - Flanders, P.; Cassuto, E. and Ross, , P.(1981) -Genet. Kongr. i£, 148.

Husain, I.; van Houten, B.; Thomas, D.C. ; Abdel-Monem, M. and Sancar, A.(1985) - Proc. Natl. Acad. Sci. 82. , 6774.

lARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man (1972-1984) vols. 1-35.

Innes, J.R.M.; Ulland, B. M.; Valerio, M.G.; Petrucelli, L. ; Fishbein, L.; Hart, E.R. ; Pallota, A.J.; Bates, R.R. ; Falk, H. L.; Gart, J. T.; Klein, M. ; Mitchell, I. and Peters, J. (1969) - J. Natl. Cancer. Inst. 42., 1101.

IPCS (1990a) - International Programme on Chemical Safety. Health and Safety Guide No.49 - CAPTAFOL. Pub. WHO for the IPCS, Geneva.

IPCS (1990b) - International Programme on Chemical Safety. Health and Safety Guide No. 50 -CAPTAN, Pub. WHO for the IPCS, Geneva.

Ishi, V. and Kondo, S. (1974) - Mutat. Res. ZL^ 27.

Isono, K. and Yourno, J. (1974) - Proc. Natl. Acad. Sci. /ll, 1612.

lyaniwara, T.T. (1991) - Rev. Environ. Health, 9 , 47.

Iyer , V.N. and Rupp, W.D.(1971) - Biochim. Biophys. Acta, 228., 117.

Jain, V. (1992) - Environ. Sci. Technol . 26., 226.

78

Jeyaratnam, T. (1990)- World Health Stat Q. 43, 139.

Kada, T.; Moriya, M. and Shirasu, Y. (1974) - Mutat. Res. Z6, 243.

Kannan, K.; Tanabe, S.; Ramesh, A.; Subramanian, A. and Tatsukawa, R. (1992) - J. Agric. Food Chem. 40. 518.

Kapadia, G.J.(1982) - Oncology overview on naturally occurring dietary carcinogens of plant origin. International Cancer Research Data Bank Prog. Natl. Cancer Inst., Bethesda, Maryland.

Kaufmann, W.K. and Kaufman, D.G. (1993)- The FASEB Journal. 7, 1188.

Kelner, A. (1949) - J. Bacteriol. 58, 551.

Kenyan, C.J. (1983) - TIBS, 8, 84.

Khosravi- Far, R. and Der, C.J. (1994)- Cancer and Metastasis Reviews . 1^, 67.

Kier, L.D.; Brusick, D.J. ; Auletta, A.E.; von Halle, E.S.; Brown, M.M.; Simmon, V.F.; Dunkel, V.; McCann, J.; Mortelmans, K.; Prival, M.; Rao, T.K. and Ray, V.(1986) - Mutat. Res. 168, 69.

Knudson, A.G, (1971)- Proc. Natl. Acad. Sci. (USA). 68., 820.

Kondo, S.; Ichikawa, H.; Iwok and Kato, T.(l?70) Genetics, 66., 187.

Kovel, T.M. (1986) - Mutat, Res. 173., 291.

Kross, B.C.; Vergara,A. and Raue, L.E. (1992) - J. Toxicol. Environ. Health. 37., 149.

Levin, D.E.; Yamasaki, E.and Ames, B.N. (1982a) Mutat. Res. 94., 315.

Levin, D.E.; Hollstein M. ; Christman, M.; Schwier, E.A. and Ames, B. N. (1982b)- Proc. Natl. Acad. Sci. (USA) 79_. 7445.

Levin, D.E.; Hollstein, M.; Christman, M.F. and Ames, B.N. (1984) - Methods in Enzymol., 105. 249.

79

Little, J.W.; Mount, D.W. and Yanisch - Perron, C.R. (1981) - Proc. Natl. Acad. Sci (U.S.A). 78, 4199.

Little, J.W. and Mount, D.W. (1982) - Cell, 29, 11.

Little, J.W. (1984) - Proc. Natl. Acad. Sci. (U.S.A.) 81. 1375.

Lloyd-Lude; Trosko, J.E. and Chang, C.C. (1978) Photochem. Photobiol . Rev. Z_, 135.

Lohman, P. H. M and Bootsma, D. (1974)- Mutat. Res. 23. 147.

Lowengart, R.A.; Peters, J.M. and Cicioni, C. (1987)-J. Natl. Cancer Inst. 79., 39.

Lu, A.Y.H.; Kuntzman, R.; West, S.; Jacobson, M. and Conney, A.H. (1972) - J. Biol. Chem. 247. 1727.

Lu, C ; Scheuermann, R.H. and Echols, H. (1986) Proc. Natl. Acad. Sci. (U.S.A.) 83., 619.

Lucier, 6.W. and Rambush, R.C. (1983)- lARC. Sci. Publ . 51. 49.

Mahr, U. and Miltenburger, H.G. (1976) - Mutat. Res. 40. 107.

Manske, D. D. and Johnson, R.D. (1977) - Pestic. Monit. J. 10., 134.

Maron, D.M. and Ames, B.N. (1983)- Mutat. Res. 113. 173.

Marshall, C.J. (1991)- Cell. 61, 313.

McCann, J.; Choi, E.; Yamasaki, E. and Ames, B.N. (1975) - Proc. Natl. Acad. Sci. (U.S.A.) 72, 5135.

McCann, J. and Ames, B.N. (1976) - Proc. Natl. Acad. Sci. (U.S.A.) - 73, 950.

McCann J. and Ames, B.N.(1977). In "Origins of Human Cancer".(eds.) Hiott, H.H.; Watson, J.D. and Winsten, J. A; Cold Spring Harbor Lab. N.Y. 1431.

McCormick, A. G. (1989)- Cell, 56., 5.

80

Mellon, I.; Bohr, V.A.; Smith, C.A. and Hanawalt, P.C. (1986)-Proc. Natl. Acad. Sci (USA) 83, 8878.

Mellon, I.; Spivak, G. and Hanawalt, P.C. (1987)- Cell. 51, 241.

Mellon, I. and Hanawalt, P.C. (1787)-Nature. 342. 75.

Metzler, M. (1784). Arch. Toxocol . 55., 104.

Miller, E.C. (1778) - Cancer. Res. 38., 1477.

Miller, J.A. and Miller, E.C. (1777) - In "Origins of Human Cancer", Cold Spring Harbor, N.Y. 605.

Miller, J.M. and Heflich, R.H. (1782) - Chem. Biol. Interact. 37., 45.

Miller, J.M. (1783) - Ann. Rev. Genet. 12., 215.

Modak, S.P. (1772) - Cell Differentiation (eds. ) R. Harris; P. Allin and D. Viza. p 437-442.

Moriya, M.; Kato, K. and Shirasu, Y. (1778) - Mutat. Res. 5Z, 257.

Moriya, M.; Ohta, T.; Watanabe, K. ; Miyazawa, T.; Kato, K. and Shirasu, Y. (1783) - Mutat. Res. 116. 185.

Mosbaugh, D.W. and Linn, S. (1782) - J. Biol. chem. 257. 575.

Miura, A. and Tomizawa, J. (1768) - Mol.Gen. Genet. 103. 1

Mottram, J.C. (1744)- J. Pathol. Bacterial. 56., 181.

Nestman, E.R.; Renata , L.B.; Marcia, P.; McPherson and Luria, K.M. (1787) - Environ. Mol. Mutagen. 10. 167.

Newbold, R.F. (1785)-, Multistep malignant transformation of mammalian cells by carcinogens, induction of immortality as a key event. In carcinogenests (Barrettt, J.C. and Tennant, R.W., (eds) Vol. 7, 17 Raven press, N. York.

Nishimoto, T.; Uzawa, S. and Schlegel, R. (1972)- Curr. Opin. Cell. Biol. 4, 174.

81

Patankar, N. and Vaidya, V.6. (1980) - Indian J. Exp. Biol, ra, 1145.

Pearson, A.; Wang, A.; Solez, K. and Haplinstall, R.H.(1975) -Am. J. Pathol. 81., 379.

Pednekar, M.D.; Bandhi, S.R. and Netrawali, M.S. (1987) - Bull. Environ. Contam. Toxicol.38,, 925.

Perera., F. (1988) - Science, 239. 1227.

Piechocki, R.; Kupper, D.; Quinones, A. and Langhammer, R. (1986) - Mol. Gen. Genet. 202, 162.

Pitot, H.C. (1993)- Cancer suppl . 72. , 962.

Ponder, B. (1988) - Nature, 335, 578.

Price, G.B. & Makinodan, T. (1973)- Gerontologia. 19. 58.

Prins, R.A. (1978) -In "Nutrition and Drug Interrelations", (eds.) J.N. Hathcock and J. Coon. Academic Press, New York P. 189.

Purchase, I.F.H.; Longstaff, E.; Ashby, J.; Styles, J.A.; Anderson, D.; Lefevre, P.A. and Westwood, F.R. (1976) - Nature, 264. 624.

Radman, M. (1974) - In "Molecular and Enviramental Aspects of Mutagenesis" (eds.) L. Prokash; F. Sherman; M. Millor; C. Lawrence and H.W. Tacor. p 128.

Radman, M. (1975) - In "Molecular Mechanisms for Repair of DNA" (eds.) P.C. Hanawalt and R.B. Setlow Part A. Plenum Press. N.Y. p. 355.

Radman, M. ; Villani, G.; Boiteux, S.; Defais, H.; Caillet, F.P. and Spadari, S. (1977) - In "Origins of Human Cancer" (eds.) H. Hiatt; J.D. Watson and J.A. Winsten. Book B. N.Y. p 903.

Ramel, C. (1991)- Reviews in Oncology. 4, 27.

Rashid, A.K. and Ralph, O.M. (1986) - J. Environ. Sci. Health. B21. 319.

82

Rasquinha, I.A.; Wildeman, A.G. and Nazar, R.N. (1988) - Mutat. Res. 177. 261.

Rastogi, S.K.; Gupta, B.N.; Husain, T. (1989)- Am. J. Indust. Med. 16., 529.

Ray, P.K. (1986) - Environmental Pollution and Cancer. The Prof. S.C. Roy Commemoration Medal Lecture delivered at the Life Sciences Centre, University of Calcutta.

Ray, V.A. et. aJL, (1987) - ibid, 185. 197.

Register III, J.C. and Griffith, J. (1986) - Proc. Natl. Acad. Sci. (U.S.A.) 83., 624.

Reuber, M.D. (1989) - J. Environ. Pathol. Tcxicol. Oncol. 9, 127.

Rosenkranz, H.S.; Klopman, G.; Chankong, V.; Pet, J. and Haimes, Y.Y. (1984) - Environ. Mutagen. 3., 231,

Rosenkranz, H.S. and Ennever, F.K. (1990) - Mutat. Res. 244. 61.

Rous, P. and Kidd, J.G. (1941)- J. Exp. Med. 73., 365.

Rozengurt, E. (1992)- Curr. Opin. Cell. Biol. 4, 161.

Rupert, C.S.; Goodgal, S.H. and Herriott, P.M. (1=58) -J. Gen. Physiol. 41_, 451.

Rupert, C.S. (1962) - J. Gen. Physiol. 45., 724.

Rupp, W.D. and Howard - Flanders, P. (1968) - J. Mol . Biol. 31, 291.

Ryberg, D. ; Kure, E.; Lystad, S.; Skang, V.; Etange land, L.; Mercy, I. ; Borresen, A.L. and Hangen, A. (1994) Cancer Res. 5£, 1551.

Saffhill, R.; Margison, G.P. and O'Connor, P.J. .1985) - Biochim. Biophys. Acta, 823. 111.

Salles, B.; Paoletti, C. and ViUani, G. (1983) - Mol. Gen. Genet. 189. 175.

Salles, B. and Defais, M. (1984)- Mutat. Res. 13., 53.

83

Sancar, G.B.; Sancar, A.; Little, J.W. and Rupp, W.D. (1982) - Cell, 28., 523.

Scheuermann, R.; Tam, S.; Bergers, P.M.J.} Lu, C. and Echols, H. (1983) - Proc. Natl. Acad. Sci. (U.S.A.) 80, 7085.

Schild, D.; Johnson, J.; Chang, C. and Mortimer, R.K. (1984) - Mol. Cell. Biol., 4, 1864.

Schmandt, R.and Mills, G.B. (1993)- Clin. Chem. 39, 2375.

Schuetzle, D. and Lewtas, J. (1986) - J. Anal. Chem. 58. 1060.

Seeberg, E. (1981) - Prog. Nucleic Acid Res. Mol. Biol., 26., 217.

Seibert, D.; Zimmermann, F.K. and Lemperle, E. (1970) -Mutat. Res. 10., 533.

Seller, J.P. (1973) - Experientia, 29., 622.

Setlow, R.B. and Carrier, W.L. (1964) - Proc. Natl. Acad. Sci. (U.S.A.) 5J , 226.

Shanabruch, W.G. and Walker, B.C. (1980)- Mol. Gen. Genet. 179., 327.

Sharma, V.K. and Kour, S. (1990)- Contact Sensitization by pesticides in farmers. Contact Dermatilis. 23., 77.

Shelby, M.D. and Stasiewiez, S. (1984) - Environ. Mutagen., 6., 871.

Shirasu, Y.; Moriya, M.; Kato, K.; Furuhashi, A. and Kada, T. (1976) - Mutat. Res. 40., 19.

Shu H e r , D.E.G. (1987) - Nature. 329.

Simmon, V.F. (1978) - In vivo and in vitrao mutagenicity assays of selected pesticides. In "A Rational Evaluation of Pesticidal Vs. Mutagenic/Carcinogenic Action." (Eds.) W. Hart.; H.F. Kraybill and F.J. de Serres. DHEW Publ. No. (NIH) 78 -1306.

84

Sobels, F.H. (1984) - In "Genetics: New Frontiers", Proceed-ings of the XV International Congress of Genetics. (eds.) V.L. Chopra; B.C. Joshi; R.P. Sharma and H.C. Bansal, 3., 63.

Stevnsner, T. and Bohr, V.A. (1993)- Carcinogenesis. 14. 1841.

Straus, D.S. (1981) - J. Natl. Cancer, Inst. 67., 233.

Sugimura, T.; Matsushima, Y.; Takauchi, M. and Kawachi, T. (1976) - In "Fundamentals in Cancer Prevention" (eds.) P.N. Magee et al. University of Tokyo, Press, Tokyo.

Sugimura, T. (1985) - Mutat. Res. 150, 33.

Sweasy, Joann B.; Wilkin, Evelyn M.; Sinha, N.; Reogner Maniscalco, V. (1990)- J. Bacterial 172. 3030.

Szent -Gyorgyi, A. (1977) - Proc. Natl. Acad. Sci. (U.S.A.) 74, 2844.

Takahashi, M.; Daune, n, and Schnarr, M. (1986) - FEBS Letters, 196. 215.

Tassignon, J.P. (1985) - Food Chem. Toxicol. 23., 5.

Temin, H.M. (1974)- Ann. Rev. Genet. 8., 155.

Tennant, R.W.; Margolin, B.H.; Shelby, M.D,; Zeiger, E.; Haseman, J.K.; Spalding, J.; Caspary, W.; Resnick, M. Stasiewicz, S.; Anderson, 3. and Minor, R. (1987) - Science, 236., 933.

Terlleth, C. ; van Sluis, C.A. and van de Putte, P. (1989)- Nucleic Acids Res. 17., 4433.

Thomas, D.C.; Morton, A.G.; Bohr, V.A. and Sancar, A. (1988)- Proc. Natl. Acad. Sci (USA) 85., 3723.

Thomas, P.T.; Busse, W.W. and Kerkvliet, N. I. (1990)-Immunologic effects of pesticides, in Baker, SR, Wilkinson, C.F.(eds): The effects of pesticides on Human Health. Advances in modern environmental Toxicology. 18., 261.

Umorasaite - Rancelience, V.; Pinto, R.I.; Barenfeld, L.S. and Cieminis, K.(1986) - Tositologiya, 2B., 86.

85

Usha Rani, M.V.; Reddi, O.S. and Reddy, P.P. (1980) -Bull. Environ. Contam. Toxicol. 25., 277.

van Houten, B. (1990)- Microbiological Rev. 54_, 18.

van der Hoeven, N.; Kooijman, S.A. L.M. and De Raat, W.K. (1990)- Mutat Res. 254. 289.

Venema, J. ; Mullenders, L.F. ; Natrajan, A.T.; van Zeeland, A.A. and Mayne, L.V. (1990)- Proc. Natl. Acad. Sci (USA) 87., 4707.

Venema, J.; van Hoffen, A. ; Natrajan, A.T.; van Zeeland, A.A. and Mullenders, L.H.F. (1991)- Mol. Cell. Biol. 11, 4128.

Villani, G.; Boiteux, 3. and Radman, M. (1978, - Proc. Natl. Acad. Sci. (U.S.A.) 7 ., 3037.

Vishwanath, R. and Ja.-nil, K. (1986) - Indian J. Exp. Biol. 2A_ 305.

Vogelstein, B. et. al (1988) - J. Med. 319. 525.

von Rumker, R.; Lawless, E.W.; Meiners, A.G.; Lawr-ence, K.A.; Kelso, 6. L. and Moray, F. (1975) - E=A 540/1-74-001. Washington, D.C., U.S.A.

Walker, B.C. (1984) - Microbiol. Rev. 48., 60.

Walker, G.C. (1985) - Ann. Rev. Biochem. 5£, -2-25.

Walker, G . C ; Marsh, L, and Dodson, L.A. (19S5) - Ann. Rev. Genet. i9_, 133.

Waters, M.D.; Simmon, V.F.; Mitchell, A.D.; Jorcenson, T.A. and Valencia. R (1980) - J. Environ. Sci. Health, B15, 867.

Waters, M.D. and Auletta, A.(1981) - J. Chem. Inf. Comput. Sci. 21, 35.

Weisenburger, D. D. (1993)- Perspectives in Pathology. 21, 571.

Wilkins, J.R. and Sinks, T. (1990) - Am.J. Epidemiol 132. 275.

Witkin, E.M. (1976). Bacteriol. Rev. 40, 869.

86

Wogan, G.N. and Gorlick, N.J. (1985) - Environ. Health. Perspect. 62., 5.

Wolff, P.S. (1984) - In"6enetics : New Frontiers". Proceedings of the XV International Congress of Genetics. (Eds.) V.L. Chopra; B.C. Joshi; P.P. Sharma and H.C. Bansal. Oxford and IBH Publ. Co. New Delhi, 3, 3.

Wong, P.K.; Wai, C.C. and Liong, E. (1989) Chemosphere, 18., 2413.

Xu, H.H. and Schurr, K.M.(1990) - Toxic. Assess. 5., 1.

Young, A.L. (1987) - In "Pesticides : Minimizing the Risks (ACS Symposium Series 336). American Chemical Society, Washington, D.C.

Younos, T.M. and Weigmann, D.L. (1988)- Journal WPCF. 60: 1199.

Zeiger, E. (1987)- Cancer Res. 47., 1287.

87

B I O G R A P H Y

Ms. Sangeeta Saxena was born at Dehradoon (Uttar

Pradesh) on March 15,1968. She passed her High School

Examination in 1983 from A.S.S. Inter College, Hardwar.

She received her B.Sc. (Hons.) degree in 1988 and

Masters degree in Microbiology in 1990 from Aligarh

Muslim University, Aligarh. She was enrolled as a Ph.D.

student in July, 1992 in the Interdisciplinary

Biotechnology Unit, Aligarh Muslim University, Aligarh.

She is a member of the Society of Biological Chemists

(India).

88