STUDIES ON THE MUTAGENIC AND CARCINOGENIC BEHAVIOR OF SOME

STEROIDS AND ALKALOIDS

DISSERTATION SUBMITTED

IN PARTIAL FULFILMENT FOR THE DEGREE OF

MASTER OF PHILOSOPHY

IN

BIOCHEMISTRY

BY

SHABANA ISLAM

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES ALIGARH MUSLIM UNIVERSITY

ALIGARH

1988

4m*>i>~.

DS1318

In the, loving mmoiy o^ my GKand^^atkdn. {Late. MA.. Moinuddin]

Vzdicatzd to My Vzan. Pan.e,nt6 (A Pfizcioui Glii o{i God)

DEPARTMENT OF BIOCHEMISTRY Faculty Of Life Sciences A L I G A R H M U S L I M U N I V E R S I T Y , ALIGARH-202002 U. P INDIA

TELEX ! 564230 CEA IN P H O N E OFF. -5741

CERTIFICATE

Thi6 i6 to ctfiti^y that the. Kt^ajxich woKk zmbodlzd In thi-i di66tfitation tntltltd "Studiti on the, mutagenic and zaMilnoQznlc. bzhavloi 0^ 6ome 6te.Kotdi, and alkaloidi>" 16 an on.iginal wofik, unlz66 otke.n.wii& 6tatzd, ca\n.izd oat by M-c6i Shabana lilam andtl my 6upeAvi6lon and li> suitable, {^on. 6ubml66ion {^on. the, awafid o^ M.Phil d^gite. in Biochzmi6tfiy.

Vate,d : 5.0^ W<«^,Sl mSOOV AHMAV

ACKNOdlLEVGEMENTS

I u)i6h to txfitii my mo6t 6inctK& and dtzp iitniz o;} gmtitudz to Vi. Maiood Ahmad {^oi ki6 unznding 6uppofit and gatdmce., conitMictlvd ciiticlim and ance.a6ing zncouH.agejne^t thtoughoat the. coun.6t o^ thii 6tady.

I am gfiatz^ul to P^o^. AM. Slddlql, Chaln.man, Vtpaltmmt o{i Blochtmi6tiy ioK hit, ptiiiitdnt intzte.^t and gzn(Lloui> availability o{i KZi>ta>ich {iacllltle.6. ^ ^, " , , , j ^, , ,

J am highly thank^^ul to Pio^fii] M. Salttmu.ddln, SM. Hadl, VK(6] A.H.K.Voa6ail, (M-t-i) Ellqttii Bano, fa6lh Ahmad, Saad Tayyab and Qayyum Hu6aln ^ofi thzlK htlp^iUl 6ugge.6tlon6.

My thanki an.t dxttndtd to all my lab. collzaguzi e^ptc-Cally V^{6] Imiana Naie^zm, Shallnl Mohan, Mz66z^6 Ta^lq Khan, Shahabaddln, Al6had Rthman and Mli6 Shabana Jqfian. ^on. thzln. amicable htlp and coope.fiatlon.

A token 0^ deep appfidclatlon to Javed bhal ^on. the. genuine and kind help during the coa>i6e o^ my 6tady.

My ilncen.e thanki aie alio to J6lam bhal, lehia apa and Sha^at ioK thel'i Immense help, encouragement and Ini^lnlte coopen.atlon.

I ieel i>hoH.t 0^ u)on.di to expiea my hean.tiul gratitude to my dear parents who lighted the academic lamp of, my life. I aZ6o unrmly acknowledge my brotheri and iliter, Naj'ml bhal, lla and Shaheen who contributed significantly with their affection and moral support.

Mr. Akmal deserves much praise for his quick and excellent word processing of this dissertation.

JfSTAV, Jeddah IK.S.A.], University Fellowship and ICMR, New Velhl [India] are greatly acknowledged for the financial support.

SHABANA ISLAM )

CONTENTS

Page No.

Chapter

Chapter

Chapter

Chapter

Chapter

I

II :

III :

IV :

V :

Certificate

Acknowledgements

List of Abbreviations

Preface

Review of Literature

General Materials and Methods

Ames Testing of Steroids

Role of SOS Repair anc BEDSC-Induced Injury: in vitro studies.

General Discussion

Future Plan

Bibliography

I Mutagenesis in In vivo and

i -

iii -

1 -

31 -

40 -

52 -

61 -

65

66 -

ii

V

30

39

51

60

64

78

LIST OF ABBREVIATIONS

arg 'Arginine

bio Biotin

CFU Colony forming units

dsb Double strand breaks

F~ Female

g/1 Grams-litre"-"-

h Hour

his Histidine

lac Lactose .s

A Lambda sensitive

leu Leucine

met Methionine

)jig Microgram

ji] Microlitre

rain Minutes

MM Minimal medium

0/N Overnight

PFU Plaque forming units

rfa deep rough mutation

sec Seconds

ssb Single strand breaks

ssDNA Single stranded DNA

str^ Streptomycin resistant

TA Soft agar

11

thi

thy

V.B. Salts

w.t.

Thiamine

Thymine

Vogel Bonner Salts

Wild-type

Steroids :

ACPIA

BEDSC

BEDTC

BESSC

CCPIA

CDBED

CEDTC

CESTC

lEDTC

lESiTC

lESoTC

3?-Acetoxy-5oC-cholestano(5, 6-b)-N-phthalimi-doaziridine

3p-Bromo-6, 6-ethylene-disulfonyl-5/.-cholestane

3P'-Brorao-6, 6-ethylenedithio-5(p<.-cholestane

3P>-Bromo-6, 6-ethylene-o(.-sulfonyl-fV-Sulfinyl-Soircholestane

3^-Chloro-5pt-choLestai\o (5 , 6~b)-N-phthaLi-midoaziridine

5oi.-Cholestan-3, S-dione bis ethylene di thiolane

3f^Chloro-6, 6-ethylene dithio-5i/.-cholestane

3f^Chloro-6, 6-ethylene-b(.-sulfinyl- thio-5o(r cholestane

3^-Iodo-6, 6-ethylene dithio-5^cholestane

36-Iodo-6, 6-ethyleneVrsulfinyl-^-thio-5o6 cholestane

3^-Iodo-6, 6-ethylene-e -sulfonyl- -thio-5cC-cholestane

I \ L

PREFACE

Eversince the industrial revolution which led to the moderni­

zation of the world, we are constantly being forced to breathe in the

polluted air, to drink the contaminated water and to eat the

adulterated food. Especially, in the last three decades, there has

been an unparalled expansion in the chemical industry as witnessed by

the development of many new organic chemical products and increased

product applications (Ames, 1979).

Naturally occuring as well as synthetic steroids and alkaloids

have been constantly used in various industries (Fishbein, 1984).

Owing to their contact with man and their constant persistence in the

environment, these chemicals interact with the normal metabolic

processes occuring in the living system. Several steroids and

alkaloids have been reported to be mutagenic and carcinogenic on the

basis of short-term tests (DunkeL et al,. 1985). Systematic and

thorough screening of these chemicals especially those having some

biological activities, therefore, seems to be desirable not only for

understanding their toxic behavior but also for understanding their

mechanism of carcinogenesis.

Cancer is the second largest cause of death with about one-

quarter of the population in Western Society developing malignancies.

Epidemiological studies of different populations and of migrants

moving from one area to another suggest the important role of

environmental factors in the causation of cancer. Prevention of cancer

to a significant measure thus could be possible by the appropriate

iV

identification of the responsible chemical (Sobels, 1984).

In view of the large number of compounds for screening, it is

very difficult, rather impossible, to thoroughly test the mutagenic

and carcinogenic potential of these biologically and industrially

important compounds. Because of both, the involvement of high cost and

relatively more time required for the screening of particular

biological activity, short-term bacterial tests such as 'Ames Testing

System' can fulfil the demand to some extent. Alkaloids and steroids^

because of their well-known phainiiaceutical and medicinal importance as

well as their relationship with the promotion of neoplastic

transformation in certain cases have been selected for their potential

mutagenicity and carcinogenicity.

These chemicals were easily available from the Chemistry

Department of our University. The major objective for the synthesis of

these chemicals was to develop pharmaceutically important compound

having diversified type of biological activities. These chemicals

would therefore undergo a thorough screening before they could serve

as the potential drug. It seemed worthvdiile to test their mutagenic

activity at the very begining. We have, therefore, carried out the

mutagenicity testing employing Ames-testing as well as E.coli and

lambda systems. A preliminary work was also done on the mutagen-DNA

interaction using BND-cellulose chromatography. A detailed and

thorough study is intented during the course of our Ph.D. work to find

out a plausible mechanism of carcinogenesis induced by these

chemicals.

In the first chapter of this dissertation, review of literature

is presented to become acquainted with the latest trends in the field

of mutagenicity testing.

The second chapter describes some general methodology, bacterial

and phage strains, composition of media and buffers etc. employed

in these investigations.

Third chapter incorporates the data on the Ames testing of

steroids.

Survival of SOS defective E.coli K-12 strains, induction of

lysogen and in vitro study employing BND-cellulose chromatography has

been presented in the fourth chapter.

Fifth chapter is devoted for general discussion and conclusion

drawn from the experimental data. In the last, bibliography is

documented.

CHAPTER I : REVIEW OF LITERATURE

Cancer is derived from the Greek word 'Karkinos' meaning crab.

It is a condition resulting from the uncontrolled reproduction of

cells. The cells in a cancer do not multiply at a faster rate than

normal cells but they do multiply more frequently as the time between

divisions diminishes. They escape controlling factors and eventually

form a cluster of cells. Soon the cluster becomes an abnormal

functionless mass of cells called a tumor. The body normally responds

to a tumor by surrounding it with a capsule of connective tissue. Such

a tumor is said to be benign. However, if the cells multiply too

rapidly to be restricted, or if they break out of the capsule and

metastasize, the tumor is described as malignant. The condition is

now called cancer, meaning that the cells are radiating out like the

'arms of a crab' (Alcamo, 1987).

It is suggested that cancer essentially is increased entropy,

randomness and disorder. The cancer cell is unable to arrest growth

because it is unable to inactivate its glyoxalase, which destroys the

ketone-aldehyde that keeps the cell at rest and in the radical state

(Szent-Gyorgyi, 1977).

One of the most striking properties of the neoplastic state is

its heritable nature. Ihis is also true of the preneoplastic state.

Ihe heritable nature of both the preneoplastic and the neoplastic

state has focussed speculation that the pathogenesis of cancer

involves permanent alterations in gene expression perhaps accompanied

by permanent alterations in gene structure, i.e. mutation (Friedberg,

1985^. These loosely knit concepts are embodied in the general theory

of carcinogenesis called the somatic mutation theory (Florey, 1962;

Lloyd-Luke et al., 1978) which dates back to observations by Boveri

(1914) of altered nuclear morphology in cancer cells. The somatic

mutation theory may be stated as follows: Agents that initiate

neoplastic transformation do so by interacting with the DNA of cells,

causing damage. The results with the Ames test strongly support the

somatic-cell mutation theory of cancer (Flessel £tjal., 1987).

The oncogene theory which is also a well documented one, was

first proposed by Huebnor and Todaro in 1969. This theory suggests

that the genes to transform a cell, the oncogenes, reside in the

chromosomal DNA of a normal cell. When a genetic change occurs, such

as by introduction of a virus, the oncogenes transform the cell to a

cancer cell (Cooper, 1982; Fink, 1984). Genetic changes which

contribute to cancer include altered gene-function, altered expression

of genes or their loss (Ponder, 1988).

For some years, mutations of dominantly acting oncogenes have

been the centre of attention, but the recent description of the

genetic events in the development of retinoblastoma has focussed

interest once again on losses of gene function (Ponder, 1988). A

report by Vogelstein et al (1988) of multiple genetic losses in

colonic cancers is the latest in a series of such findings. The

development of retinoblastoma has been shown to involve loss of both

alleles at a single locus, mapped to the long arm of chromosome 13(13q

14), and individuals with the hereditary forms of retinoblastoma have

been shown to have inherited the loss of one copy of the gene in the

germ line (Hansen and Canenee, 1988). The retinoblastoma gene is

regarded as the prototype of a class of tumor-suppressor genes whose

presence in normal cells is required to prevent the emergence of a

tumor and which seem likely to have important functions in the

regulation of normal cells, proliferation and development.

It has long been known that cancer can arise as the 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 produce cancer (Miller, 1978). Damage to WA by environmental

mutagens may be the main cause of death and disability in advanced

societies (Cairns, 1975b). It is believed that this damage,

accumulating during our 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 al., 1975).

Since a large proportion of human cancer may arise from chemical

causes, cancer prevention will depend to a large extent upon the

recognition and possible elimination of environmental and endogenous

exposure to carcinogenic agents (Doll and Peto, 1981). To this end,

surveys of available and relevant data on potential carcinogens to

^ich man may be exposed are being published (Bowman, 1982; lARC

Monograj^s, 1972-1984). Since some degree of exposure is unavoidable,

particularly that arising from natural sources, effective prevention

must also depend upon methods to prevent the development of neoplasia

following exposure. Ultimately this may only be attainable through a

thorough understanding of each sequential stage of the carcinogenic

process (Saffhill et al., 1985). Rapid and accurate, in vitro tests

such as the Salmonella/mjcrosome test, should play a crucial role in

the identification of environmental mutagens and minimizing human

exposures (McCann and Ames, 1976).

Carcinogenic Agents And their Mechanism of Action

A high proportion of human cancers are attributed to environmen­

tal agents, mainly the chemicals. Epidemiological evidence strongly

suggests that there is a major 'environmental' factor in the occurence

of cancer. This factor is a combination of all aspects of our

lifestyles, including social and cultural habits, diet, agricultural

practices and exposure to manmade pollutants. Many estimates of the

proportions of cancers caused by specific environmental factors have

been made (Farmer, 1982).

It is frequently assumed that ESMA -damaging agents are

carcinogenic because they induce mutations. However, another strong

possibility is that the damage leads to heritable changes in the

methylation of cytosine in EJ A. Considerable evidence exists that gene

expression in mammalian cells is in part controlled by methylation of

specific DNA sequences (Robin, 1987). Carcinogens may act by altering

the normal epigenetic controls of gene activity in specialised cells

and thereby produce aberrant heritable phenotypes. It is known that

agents which inhibit DNA methylation can be carcinogenic and that

tumor cells are altered in DNA methylation (Robin, 1987).

One of the theories at etiology of cancer, which is being widely

accepted, holds that the major cause is damage to DNA by

oxygen-radicals and lipid peroxidation (Totter, 1980; Ames, 1983).

Ihere is an increasing amount of data which suggests that chemical

carcinogens may cause both direct Wk damage, i.e., carcinogen-DNA

adducts, and indirect DNA damage by causing formation of free radicals

and superoxides that react with DNA and cause molecular lesions

(Cerutti, 1985).

Most chemical carcinogens are alkylating or acylating agents

(electrophilic reagents) or produce such compounds following

metabolism in the body. This means that they have electron deficient

centres and will combine with electron-rich centres within the cell.

Reaction with EWA is the most important process in the initiation of

mutagenesis and carcinogenesis, although reaction with certain

amino-acids in proteins will also occur (Farmer, 1982). Alkylating

agents are able to react at many sites in EWA, in particular at the

ring nitrogen and the exocyclic oxygen atoms of the DNA bases and at

the oxygen atoms of the phosphate internucleotide linkages (Margison

and O'Connor, 1979; Singer and Kusmierek, 1982; Pegg, 1983).

Substitution at the N-7 position of guanine in DNA is usually the

major reaction product with alkylating carcinogens although many other

products such as 3-alkyladenine, 3-alkylguanine, 0 -alkylguanine,

alky1thymidine, 3-alkylcytosine and the exocyclic N -alkylguanine are

also formed (Farmer, 1982).

The majority of chemical carcinogens are known to form covalent

adducts with DNA (Miller, 1978; O'Connor, 1981) and there is now a

large body of evidence implicating DNA as a critical target in

chemically induced cancer. Studies with many alkylating agents have

indicated that it is their capacity to react with certain oxygen-atoms

in EWA, in particular those associated with promutagenicity, v ich

provides an indication of their carcinogenic potential (Loveless,

1969; Saffhill, 1985). Many compounds, however, do differ in their

extents of reaction with the EWA of various tissues ^ vivo,

reflecting both the distribution of the compound within the animal and

the need for some compounds to be metabolized before they can undergo

reactions leading to the alkylation of DNA (O'Connor et al, 1979;

Pegg, 1983).

During the past few years it has been established that one of

the mechanisms of chemical carcinogenesis is induction of mutation

that "activate" the protooncogene of mammalian genomes (Meisler,

1987).

Certain promoters of carcinogenesis act by generation of oxygen

radicals; this being a common property of these substances. Many

carcinogens which do not require the action of promoters and are by

themselves able to induce carcinogenesis also produce oxygen radicals

(Demopoulos et al., 1980). The mechanism of action of promoters

involves the expression of recessive genes and an increase in gene

copy number through chromosome breaks and creation of hemizygosity

(Varshavsky, 1981; Kinsella, 1982). Promoters also cause modification

of prostaglandins which are intimately involved in cell-division,

differentiation and tumor growth (Fisher et a]^., 1982).

Most data on radical damage to biological macromolecules concern

with the effects of radiation on nucleic acids because of the possible

genetic effects (Wolff et al ., 1986). Much of the toxic effect of

ionizing radiation damage to DNA is also due to the formation of

oxygen radicals (Totter, 1980).

Since virtually every chemical known to cause cancer in humans

also causes cancer in animals (lARC Monographsm, 1972-1975; Tomatis et

al., 1973; Epstein, 1974) the simplest assumption is that any chemical

which is a carcinogen in an animal test is likely to be a human

carcinogen, though, there are mapy uncertainities in determining the

risk to humans from animal data (Epstein, 1974; Mantel and

Schneiderman, 1975; Ames et al, 1987). In general chemicals

carcinogenic in one species are carcinogenic in other species (lARC

Monographs, 1972-1975; Tomatis, 1973) although the carcinogenic

potency of a particular chemical can vary considerably depending upon

the animal species in which it is tested and the manner in which the

chemical is administered (lARC Monographs, 1972-1975; Weisburger,

1973, 1975; Sontag £t al., 1975). Chemicals of very similar structure

can also differ greatly in carcinogenic potency (McCann et al.,

1975a).

Correlation of Carcinogenesis And Mutagenesis

Ames test has been widely used to investigate the mutagenic

potential because many carcinogens are also mutagenic (Flessel et al.,

1987). Although a high degree of variation was obtained with regard to

the sensitivity of Ames test ranging from 45% to more than 90% (McCann

et_ al., 1975b; McCann and Ames, 1976; Purchase et al., 1976; Sugiraura

et_al., 1976; Heddle and Bruce, 1977; Levin etal., 1984; Tennant et

al., 1987; Zeiger et al., 1987). It was also suggested that a high

level of sensitivity is often more a characteristic of the composition

of the test material than that of the test system (Dyrby and

Ingvardsen, 1983 ). It is also noteworthy that as the time have

elapsed from the first Ames test conducted by McCann et al (1975a) to

correlate the mutagenic potential of a carcinogen upto the work of

Tennant et al (1987) there appears a continuous decrease in the degree

of sensitivity by Ames testing. Zeiger et al (1987) reported that the

Ames test has a sensitivity (percentage of carcinogens identified as

mutagens) of only 54% and a specificity (percentage of non-carcinogens

identified as non-mutagens) of only 70%. Even worse, the same year

Tennant et al (1987) found that the Salmonella assay would only

identify about 45% of carcinogens. These results are at variance with

the observations made a decade ago when sensitivities and

specificities of 90% or more were claimed for the Ames test (McCann et

al., 1975a; McCann and Ames, 1976).

Nestraann (1986) stated that a mutagen is a mutagen, not

necessarily a carcinogen. For instance caramel, a sugar derivative

which is widely used as a food coloring and flavoring agent, is also

mutagenic in Salmonella test system, but has no carcinogenic effect

when fed to rats as 6% of the diet for 2 years (Evan et al., 1977).

Cancer And WA Damage

Damage to DNA is likely to be a major cause of cancer and other

diseases (Hiatt et al., 1977; Ames, 1979). There is an evidence which

support that carcinogens and radiations likely to initiate most human

cancers and genetic defects do so by damage to DNA (McCann et al.,

1975a). Defined precisely, DNA damage is an alteration that constitute

a stumbling block for the replication machinery and hence hampers the

replication of WA, endangering the survival of the cell (Devoret,

1979).

The correlation between radiation-induced damage and cancer has

long been apparent to radiation biologists but through molecular

evidence it has been found that DNA damage is a direct cause of

cancer. The evidence comes from patients suffering from xeroderma

pigmentosum (Devoret, 1979). With certain eukaryotic cells, the

consequences of EWA damage can also be assessed by cytogenetic

analysis. The human disorders atoxia telangiectasia, Blooms syndrome

and Fanconi's anemia are genetic diseases characterized by an

increased susceptibility to cancer (Walker et al., 1985).

Studies with Poecilia formosa showed an increased incidence of

tumors in recipients bearing cells maintained under non-DNA repair

conditions thereby implicating EWA damage as a specific etiological

factor in the neoplastic transformation (Hart and Setlow,

1975; Woodhead and Scully, 1977; Woodhead, et al., 1977). Study with

Syrian hamster (DiPaolo and Dortovan, 1978; Doniger et al., 1981) also

suggested a causal relation between EWA damage and neoplasia. The

10

demonstration of the photoactivation of UV-induced ovabian-resistant

mutations in haploid frog cells in vitro adds further evidence that

pyrimidine diraers are mutagenic lesions in vertebrates (Massey et al.,

1976).

Mutation (or DNA damage) as one stage of carcinogenic process is

supported by various lines of evidence: association of active forms of

carcinogens with mutagens (Ames and McCann, 1981), the changes in EWA

sequence of oncogenes (Weinberg, 1985), genetic predisposition to

cancer in human diseases such as retinoblastoma (Knudson, 1985) or

DNA-repair deficiency diseases such as xeroderma pigmentosum (Cleaver,

1984).

According to McCann and Ames (1976) and other evidences listed

below, it is compelling to propose that radiations and chemical

carcinogens cause cancer through damage to EWA (somatic mutation), (i)

It is known that cell regulation can be altered by mutation, and that

a heritable change in cell regulation is a characteristic property of

a cancer cell, (ii) The theory is simple and consistent with facts in

cancer biology (Cairns, 1975a, b) (iii) It is supported by studies on

the genetics of cancer (Knudson, 1975). (iv) There are human mutants

lacking DNA repair systems that are extremely prone to cancer (Cleaver

and Bootsma, 1975). (v) There is a correlation between capacity for

repair of DNA damage and the occurence of organ-specific cancer (Goth

and Rajewsky, 1974; KleiLues and Margison, 1974; Nicoll et al., 1975).

(vi) Active forms of many carcinogens are electrophiles capable of

interacting with EWA (Miller and Miller, 1971). (vii) Almost all

11

carcinogens tested have been shown to be mutagens (McCann et al.,

1975a). (viii) Many potent aromatic carcinogens are unusually

frameshift mutagens and the structural basis is the presence of an

aromatic ring system capable of strong stacking interaction with ESMA

and an electrophilic moiety (Ames and Whitfield, 1966; Ames et al.,

1972; Ames et al., 1973b). (ix) In addition to the many chemical

carcinogens as mentioned above there is a diverse collection of

carcinogens such as asbestos (Sincock and Seabright, 1975), metal

carcinogens (Nishioka, 1975), and a variety of radiations that have no

obvious connection other than their ability to damage DNA.

DNA Repair

DNA is the primary carrier of genetic information and the

structural integrity of EWA is a prerequisite for gene expression

(Modak, 1972). It is a known fact that primary structure of WA is

dynamic and subject to a constant change (Friedberg, 1985b). Ionizing

and UV-radiations as well as 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.

Ihe living systems have, therefore, evolved repair processes to

maintain stinactural and functional fidelity of EWA against a large

range of insult (Friedberg, 1985b). The molecular mechanisms involved

in repair of damaged portions of EWA and their restriction into

functionally intact informational units is fundamental to the

12

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 ptieuotype, or neoplastic

transformation. Repair at the cellular and macromolecular level is

multiple in its form and varies as a function of species, tissue and

stage 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 WA

damage have established the existence of DNA repair pathways.

Moreover, the genetic studies have identified a large number of genes

participating in the repair of damaged EWA (Walker, 1985).

The earliest suggestion on recovery of bacteria after exposure

to ultraviolet (UV) light was made by Hollender 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. He coined the term 'photoreactivation'. 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 viild-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

13

damage induced in WA by carcinogens, mutagens and other hazardous

chemicals (Ishi and Kondo, 1975; Auerbach, 1976; Seeberg, 1981;,

Friedberg, 1985l>). 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:

Photoreactivation : The simplest class of repair pathway is

photoreactivation that directly rectifies the cyclobutane type

pyrimidine dimers in UV-irradiated EWA without the formation of new

phosphodiester bonds (Walker £t al., 1985). This type of recovery

process requires the exposure of cell to visible light. It was the

first system to be observed in vitro (Rupert et al., 1958) and was the

first to be characterized with regard to mechanism (Rupert, 1952a, b).

Hiotoreactivation is a universal phenomenon because it is known to

occur in E.coli, 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 enzjmies, and the subsequent

reconstruction of the twin helix by repair enzymes that make 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

dark by excision repair system. Mechanism of repair of DNA by excision

has been studied extensively using mutants of E.coli sensitive to

14

ultraviolet radiations (Setlow and Carrier, 1964; Boyce and

Howard-Flanders, 1964; Howard-Flanders et al., 1966).

Excision repair is the process in which the lesions in DNA are

removed and the gaps are refilled with correct base sequences using

the opposite intact strand as template. Excision repair enzjnnes have

also been demonstrated to act upon the DNA damaged by chemical

mutagens and carcinogens (Kondo et al., 1970; Miller and Heflich,

1982). Ihis repair system could have at least four steps viz.

incision, excision, gap filling and sealing.

Post Replication Recombinational Repair : The DNA lesions, especially

UV-induced pyrimidine dimers that are neither split photoenzymatically

nor removed from EWA by excision repair, block the continuous progress

of the DNA replication fork. However, they do not prevent the

reinitiation of DNA synthesis at a point beyond the diraer (Rupp and

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

strand opposite the lesions. Ihe continuity of daughter strand is

interrupted by gaps of obout 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. Ihis was first

demonstrated in E.coli by Rupp and Howard-Flanders (1968).

Inducible Error prone SOS Repair : The term 'SOS' (international

distress signal) implies to an error prone repair, induced under

enormously stressed condition, of growth as a last resort for the

survival of cells. The existence of 'SOS' network was clearly

15

postulated by Defais et_ al (1971) and was further developed and

amplified by Radman (1974; 1975) and Witkin (1976). The exposure of

E.coli to the agents that damage EWA 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^^l., 1971). 'SOS' repair is a

highly integrated and sophisticated regulatory network that require de

novo protein synthesis for expression (Kovel, 1986). It is an

inducible repair process and is believed to be responsible for a

conmon mutagenic pathways (Radman, 1974; Witkin, 1976; Walker, 1985).

In response to DNA damages calling for the SOS repair, EWA repair

systems in E.coli are activated, cell division is altered, integrated

viruses are induced and respiration is blocked (Witkin, 1976; Little

and Mount, 1982).

Several bacterial genes have been identified v^ich coordinately

function in 'SOS' repair. These are uv^and uvrB (EWA repair), umuC

(mutagenesis), sfiA (filamentation), himA (site specific recombina­

tion) and several din genes with unknown functions in addition

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 £t al., 1985).

RecA and iexA genes are the regulators which control 'SOS'

response. Under normal conditions lexA protein represses the

subordinate genes of the systepi and recA protein derepresses these

loci in response to DNA damage (Kenyon, 1983). LexA protein is a self

16

repressor and also binds to similar operator sequences in each gene

(Littlest al., 1981; Sancer jet al., 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

acquires a specific protease activity to form recA when it interacts

with an 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 resumes (Brent and Ptashna, 1981; Little et ad, 1981;

Sancer£t al., 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'.

Mutagenesis resulting from 'SOS' processing of damaged DNA

template is targeted and is not due to the induction of some random

mutator activity (Miller, 1983; Walker, 1984). Radman (1974, 1975)

suggested that the appearance of mutation during 'SOS' processing

17

might 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 WA polymerases (Radman et

_al., 1977; Boiteux _et _al., 1978; Villani _et _al., 1978). Moreover, the

lexA dependent conversion involving theesubunit of K^A polymerase III

holoenzyme to 'SOS' polymerase has also been postulated (Scheuermann

_et _al., 1983; Piechocki _et _al., 1986). Lu _et (1986) in fact, have

demonstrated the recA mediated inhibition of '3 -^5' nuclease activity

of isolated c subunit of DNA polymerase III and was suggested to be

responsible for targeted mutagenesis.

Theories of Chemical Carcinogenesis

One of the major objectives of cancer research is to determine

the molecular mechanisms by which chemical carcinogens contribute to

the development of neoplasia. Evidence for a mutational basis of at

least some neoplasia is provided by studies of transforming genes of

the ras family (Yuasa et al., 1984). Activation of ras protooncogenes

in human tumors is apparently often due to point mutations in the ras

coding sequence. In these cases, an error-prone repair process might

play a direct role in the initiation of cancer after exposure to a

chemical carcinogen and error-free repair might play a direct role in

its prevention (Walker et al., 1985).

However, some carcinogens may act epigenetically to change gene

expression. Certain observations are consistent with the hypothesis

that at least some effects of chemical carcinogens are due to

generalized effects on gene expression caused by alterations in the

degree of cytosine raethylation. The correlation between

hypomethylation of cytosine residues and active gene expression in

eukaryotic cells has been well documented (Razin and Riggs, 1980;

Ehrlich and Wang, 1981). Wilson and Jones (1983) suggested that

alterations in specific raethylation patterns could give rise to

oncogene transformation by activating quiescent genes. This view is

supported by the observations that the metallothionein-1 gene is

activated by UV-irradiation of mouse cells and that this gene

expression is associated with extensive demethylation of the

metallothionein-1 gene (Lieberman et al., 1983). Mechanisms by which

DNA damage could lead to a heritable loss of raethylation pattern

include direct modification and inactivation of the methyltransferase

enzyme (Drahovsky and Wacker, 1975; Cox, 1980; Wilson and Jones, 1983)

and/or slow and incomplete raethylation of deoxycytosine incorporated

during excision-repair synthesis (Kastan et al., 1982).

Carcinogenicity Testing Systems

It has become increasingly apparent that the traditional methods

for identifying carcinogens by using long-term studies in rodents are

unable to meet demands for a quick, sure and inexpensive

identification of environmental carcinogens. This has brought about an

intensive search for appropriate test systeras and over the last few

decades a series of short-term tests have been published (Dyrby and

19

Ingvardsen, 1983).

Long-term tests, however are expensive and time consuming they

have given considerable evidence in estimating the potency of a

carcinogen (Farmer, 1982). But, there is a problem of predicting

whether humans will respond to the carcinogen in the same way as

animals (Farmer, 1982). There are several instances in which only one

species like mouse was found to respond to the test and the rat was

ineffective. Then how can we extrapolate the risk from rodents to

humans, a very dissimilar long-lived species. Opposite was also found

in v^ich a chemical was found to be carcinogenic by epidemiological

studies but was non-carcinogenic in rodents (Ames et al., 1987).

Short-term tests (STTs) for genotoxic chemicals were originally

developed to study mechanisms of chemically induced DNA damage and to

assess the potential genetic hazard of chemicals to humans. The four

better known short-term tests are chromosome aberration, sister

chromatid exchange, mutagenicity assay using mouse lymphoma cells and

the Ames test. Ihe chromosome abberations are the result of effects

that occur anywhere in the genome therefore, 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 sensitive method for determining, whether or not

environmental agent can interact with the genetic material (Wolff,

1984). For the majority of chemical mutagens which are S-dependent

agents the sister chromatid exchange (SCE) has proved to be even more

20

sensitive than the induction of aberrations. The SCEs, like chromosome

aberrations induced by S- dependent agents, are formed by unrepaired

lesions that are present when the cell passes through S and the

chromosomes replicate. SCE method has been used as a test system to

tell v iether or not a chemical is potentially dangerous and to

determine which of the metabolites of a premutagenic and

precarcinogenic agent might be the likely ones to interact with DNA

and cause its effect (Wolff, 1984). Ashby and Tennant (1988) observed

that no combination of the four STTs is any better than Salmonella on

its own for flagging probable carcinogens. The role of these tests has

increased however because of accumulating evidence in support of the

somatic mutation theory of carcinogens (Straus, 1981; Crawford, 1985;

Ames et al., 1987) and because of reports that many rodent carcinogens

are 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 and do not involve testing in animals. Early studies of con

cordance between results from in vitro STTs and rodent carcinogenicity

tests were highly encouraging (IXirston et al., 1973; Sugimura et al.,

1976; Devoret, 1979). Sensitivities (percentages of carcinogens

identified as mutagens) and specificities (percentages of

non-carcinogens identified as non-mutagens) of 90% or better were

reported, especially for the Ames Salmonella mutagenicity assay

(McCann et al., 1975a; Purchase et al., 1976).

Wide use of short-term tests for detecting mutagens (Ames, 1979)

21

and a number of animal cancer tests on plant substances have

contributed to the identification of many natural mutagens and

carcinogens in the human diet (Kapadia (ed) 1982). There is a range of

applications in v^ich STTs have been used successfully, from the

identification of mutagenic fractions in complex mixtures such as

cooked meat (Hatch etal., 1984; Sugimura, 1985) or air pollutants

(Schuetzle and Lewtas, 1986) to the early identification of genetic

toxicity in the development of new chemical products (Tassignon,

1985).

On the basis of a literature-derived study of the performance of

STTs (Waters and Auletta, 1981), it became apparent that there were

two impediments to a thorough evaluation of the ability of these tests

to predict rodent carcinogenicity : for most STTs there was a dearth

of results for documented noncarcinogens (Shelby and Stasiewiez, 1984;

Kier et_ al., 1986; Ray et al., 1987) and too few chemicals had been

tested in multiple STTs to permit meaningful comparisons of the

ability of different STTs and STT combinations to predict carcinogens.

Even with a battery of assays, not all rodent carcinogens are in vitro

mutagens nor are all in vitro mutagens rodent carcinogens. STTs do,

however, contribute to offer an economical, rapid and dependable means

to detect genotoxic chemicals (Tennant et al., 1987).

Ames Testing System

Of the various bacterial manifestations of DNA damage, Ames

chose mutagenesis as the basis of his pioneering work to develop a

test for potential carcinogens (Devoret, 1979). The

22

Salmonella-mutagenicity test (Ames et al., 1975), along with other

short-term assays (Hollstein al., 1979), is being extensively used

to survey a variety of substances in our environment for mutagenic

activities (Ames, 1984). The test measures back mutation in several

specially constructed mutants of Salmonella.

The test has been adapted for use in detecting chemicals which

are potential human carcinogens or mutagens by adding homogenates of

rat liver (or other mammalian tissue) directly to the petri-plates as

an approximation of mammalian metabolism into the in vitro test (Ames

et al., 1973a). Ihis test is also instrumental in understanding the

primary events of the carcinogenic process initiated by chemicals

(Devoret, 1979). Ihe compounds are tested on petri-plates with several

specially constructed mutants of Salmonella typhimurium LT2 type

selected for sensitivity and specificity in being reverted from a

histidine requirement back to prototrophy by a wide variety of

mutagens (Ames et al., 1973a).

Several histidine- requiring mutants in the standard set of

Salmonella tester strains have GC base pairs at the critical site for

reversion e.g. -C-C-C- in the base-pair substitution strain, TAIOO

(Barnes et al., 1982), -C-C-C-C-C-C- in the frameshift tester strain,

TA97a (Levin et al., 1982a) and -C-G-C-G-C-G-C-G- in the frameshift

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 which were

not detected in the standard tester srains (Levin et al., 1984).

23

Steroids ; Uses And Applications

Naturally occuring as well as synthetic steroids have been

conmonly used in various industries (Fishbein, 1984). Most of them are

used as medicine and pharmaceuticals (Wilpart et al., 1985).

Homo-aza-steroidal esters are known for their antineoplastic activity

(Pairas et^ al., 1986). Certain steroids have also been reported to

control energy metabolism throughout pregnancy (Baird et al., 1985).

Steroid-alkaloid formulation has been used for skin disorder treatment

(Chain £t £l,, 1984). Moreover, some oxazoles and their derivatives

have been reported to possess anti-inflammatory, analgesic,

antibacterial, antimicrobial and antiviral activities (Turchi and

Dewar, 1975). Also, there are some steroidal compounds which increase

resistance against drugs and toxic agents (Kourounakis, 1986). It has

been found that medroxyprogesterone acetate inhibits growth of

hormone-dependent mammary carcinoma cells (Costa et al., 1986).

Mechanisms of antitumor action of gestagens on endometrial cancer has

also been investigated (Nishida et al., 1986). As pharmaceuticals,

oxazoline are useful as antihypertensive as well as central nervous

system regulators (Levitt, 1970 a,b). Fluorouracil-estradiol conjugate

(Asano et al., 1978) and modified steroid-alkylating agents (Panayotis

et al., 1983) have been reported to possess antitumor and antimutator

activities respectively.

Steroids As Potential Carcinogens

There is a high risk of the development of cancer with the use

of steroids. Several steroids have been reported to possess

24

tumorigenic and carcinogenJLc activities. Increased rate of oesophagal

cancer has been attributed to the presence of these potentially

carcinogenic chemicals present in our food (Allen £t£l., 1981; Kay,

1981; Candrian^t al., 1984).

Several steroids have been reported to be mutagenic and

carcinogenic on the basis of short-term tests (IXinkel et al., 1985).

Various steroids have been shown to play an active role in human

carcinogenesis (Lipsett, 1986). Especially the steroidal hormones and

their derivatives have been demonstrated to be mutagenic and

carcinogenic in the bacterial as well as in the animal testing systems

(Kay, 1981; McKillop et_a_l., 1983; Metzler, 1984).

Estrogenic hormones have been shown to possess the tumorigenic

activity and are assumed to serve as regulators of tumor growth. These

hormones probably maintain the neoplastic state of the cells in a

variety of well-characterized experimental animal tumor systems

(Katzenellenbogen, 1986). Growth promoting effect on hepatocarcinoma

has also been demonstrated to be mediated by estrogen-receptor in the

male rats (Kohigashi et al., 1986). Carcinomas in ovary tumors are

more often estrogen positive than benign tumors (Lantta and Acta,

1984).

The mechanism of action of steroid hormones involves their

interaction with tissue-specific binding sites and results in a

precise modulation of gene expression by regulating some RNA

processing events. It has been found that the majority of the human

primary breast tumors contained detectable levels of steroids in the

25

homogenate or cytosol fractions (Prakash et al., 1986). Tissue

steroids have been found to play a role in regulating aromatase

activities in breast and endometrial cancer (James et al., 1986).

Steroid alcohol sulfotransferases have been isolated from the cytosol

of a human breast carcinoma cell line MCF-7 (Rozhim £t £l., 1986).

Moreover, the steriochemical complementarity of WA and reproductive

steroid hormones have been found to correlate with biological activity

(Lawrence £t £l., 1986).

Contraceptive steroids have been assessed for toxicological and

carcinogenic hazards (Heywood, 1986). Oral contraceptives are believed

to pose a high carcinogenic risk with regards to the mammary gland and

reproductive gland (Heywood, 1986). They have also been shown to

enhance liver tumor in the Egyptian toad (Sadek and Abdelmeguid,1986).

Contraceptive steroids have been shown to enhance mutagenic activity

in Salmonella (Rao et al., 1983) and were also found to be the

promoter of tumorous growth in rat liver (Heike et al., 1986).

Alkaloids ; Uses And Applications

Naturally occuring as well as synthetic alkaloids have been

commonly used in various industries (Fishbein, 1984). Most of them are

used as medicines and pharmaceuticals. Polyindoline alkaloids of

Psychotriaforsteriana is currently been used in antitumor chemotherapy

(Roth et al., 1986). Crinasiatine and phenanthridone alkaloid have

bacteriostatic and tumor inhibiting activity (Ghosal et al., 1985).

Chromone alkaloid and its salts have been reported to have excellant

analgesic and iramunomodulating activity in vivo and in vitro (Vasudev

26

et al., 1985). Studies on the anticancer activities and mechanism of

mistletoe and its alkaloids have also been proposed (Tasneera eit al.,

1986). Quinoline alkaloids are used as antibacterial agents (Willems,

1986). Some clavine-type ergot alkaloids have been shown to possess

antimicrobial activity against certain human pathogenic bacteria (Eich

^ al., 1985). Indole-alkaloid derivatives have been tested for their

antibacterial activity (Lumonadio et al., 1986). Studies of some

medicinal plants have resulted in the isolation of a series of

antimicrobially active indole alkaloids. Some of them are toxferine,

secamine and ibogam-type alkaloids (Verpoorte, 1986). Some quaternary

alkaloids are found to be responsible for the inotropic activity on

the heart (Cave et al., 1984). Some butyrated alkaloids hydroxyanisole

have been evaluated as protective agents against pyrrolizidine

alkaloid toxicity in rat (Garret and Checke, 1984). On the other hand,

pyrrolizidine alkaloids isolated from Heliotropium marifolium are

known for their significant antitumor activity (Satish et al., 1986).

Moreover, there are some alkaloids which are used as coloring and

preservative agents (Osawa et al., 1981).

Alkaloids As Potential Carcinogens

Several alkaloids have been reported to possess tumorigenic and

carcinogenic activities. Increased rate of oesophagal cancer has been

attributed to the presence of these potentially carcinogenic chemicals

present in our food (Allen et al., 1981; Kay, 1981; Candrian, 1984).

Pyrrolizidine alkaloids are naturally occuring carcinogens and they

have been found in some fifty species of the families Compositae,

27

Boraginaceae and Leguminosae (Schoental, 1982) which are used as foods

or herbal remedies. Indole alkaloids from various sources have been

reported to be tumor promoters (Fujiki £t aj * > 1983). Mutagens in

opium pyrolyzates possibly implicated in oesofiiagal cancer have been

characterized and identified through epidemiological and environmental

field studies (Bartsch £t al., 1984). Moreover, several pyrrolizidine

alkaloids and their metabolites have also been demonstrated to be

mutagenic, carcinogenic and antimitotic in bacterial and animal

testing systems as well as in plants (Alderson and Clark, 1956;

Malaveille et al., 1982; Griffin et al., 1986).

Recent Advances In Carcinogenicity Testing System

Ashby and Tennant (1988) concluded from their survey of around

222 carcinogens and non-carcinogens that the Salmonella assay (the

so-called Ames test) does not identify all carcinogens. It does work

for certain chemicals particularly those that become reactive

electro{iiiles and attach to DNA(Miller and Miller, 1977), but it

certainly does not work for all of them (Tennant, 1987). Moreover,

Tennant (1987) observed that three of the better-known short-term

tests f those for chromosome aberrations, for sister chromatid

exchange and a mutagenicity assay using mouse lymphoma cells) although

each was more sensitive than the Ames test,but they were all less

specific, and therefore falsely labelled more non-carcinogens as

probable carcinogens. He further observed that no combination of the

four tests is any better than Salmonella on its own for flagging

probable carcinogens. A second genotoxic assay conducted in vivo was

28

suggested by Ashby and Tennant, (1988). The test is the mouse

micronucleus assay which reacts on cells with chemically induced

chromosome aberrations having an unusual distribution of chromatin

during cell division which can be observed as distinct micronuclei in

cytoplasm. Validation of this genotoxicity assay, is likely to be of

more use than in vitro tests which do not aid identification of

carcinogens.

The reliable and sensitive detection of characteristic molecu­

lar markers for human exposure to chemical carcinogens is no longer

barrier to further study, as several sophisticated analytical

techniques such as tandem mass spectrometry and fluorescence line-

width narrowing spectrophotometry are now available (Shuller, 1987).

A new Salmonella mutagenicity test method is under development

to test a chemical with more than one strain simultaneously (the

"SIMULTEST") that is, different Salmonella typhimurium tester strains

with R-plasmid and without plasmid are used in combination on the same

plate. This approach may be useful in reducing the workload associated

with mutagenicity testing with Salmonella (Nestman et al, 1987). The

process of chemical carcinogenesis is well understood to support a

policy distinction between genotoxic and nongenotoxic carcinogens.

Conservative Mathematical model could account for the possibility that

a carcinogenic chemical would "add on" to the background of cancer

(Perera, 1988).

Recently Ames et al (1987) have invited a great deal of

controversy over the popular view that synthetic carcinogens are

29

posing a higher risk than the natural ones. According to this review

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. However the authors have put a

question mark on the degree of importance on the natural exposures.

Animal models provide invaluable information in studies of

carcinogenesis (Harris, 1985; tky, 1988). Extrapolation of this

information from experimental animals to humans remains, however, a

problematic endeavor. Most scientists consider the qualitative

extrapolation to be accurate, i.e., a chemical that is 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 u^ether such

association exists in human carcinogenesis (Harris, 1985). Lifetime

carcinogenicity studies will still be needed for those nongenotoxic

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).

Definition of the Problem and the Objectives of the M.Phil Work

Owing to the biological activities of naturally occuring as well

as synthetic steroids and alkaloids these compounds are extensively

being used for their clinical trials. Moreover, the compounds with the

steroidal and alkaloidal nuclei are already available in the market in

30

the form of medicine and other household materials and thus are coming

in direct contact with man. It is therefore, essential to investigate

their hazardous nature too. Increasing incidence of certain t3^es of

occupational cancer also calls for particular attention to screen the

potentially carcinogenic compounds present in the environment. The

idea of the routine and systematic study of steroidal and alkaloidal

compounds for their potentially mutagenic and carcinogenic behavior

gains further support in view of their tumorigenic and carcinogenic

activities. The short term assay would obviously be the first step

forward in the direction of minimizing the exposure to the potentially

toxic steroids, as prevention is always better than cure.

In view of the present literature, we initiated the project on

the diagnosis of some available steroids for mutagenicity and

carcinogenicity employing Ames testing system.

The major objectives were as follows :

(i) Screening of available steroids for mutagenicity by means of

Ames testing system (Spot-test),

(ii) Confirmation of mutagenicity of the steroids by performing plate

incorporation assay. The work with a single potent steroid and

its parent compound to be included to determine the effect of

the substituted group,

(iii) The potent steroid to put to test by SOS repair defective

strains of E.coli and E.coli-lambda systems,

(iv) In vitro study to put forward a mechanism for DNA carcinogen

interaction.

CHAPTER II : GENERAL MATERIALS AND METHODS

31

Table 1. The Salmonella t3^himurium and Escherichia coli strains

used in this study have been tabulated as under:

Strain

designation

Relevant genetic markers Source

TA97a

TA98

TAIOO

TA102

TA104

AB1157

AB2463

AB2494

Ames Strains

uvrB, hisD6610, bio, rfa,

R-factor plasmid-pkMlOl

uvrB, hisD3052, bio, rfa,

R-factor plasmid-pkMlOl

uvrB, hisG46, bio, rfa,

R-factor plasmid-pkMlOl

uvrB, hisG428, bio, rfa,

R-factor plasmid-pkMlOl

multicopy plasmid-pAQl

uvrB, hisG428, rfa,

R-factor plasmid-pkMlOl

E.coli K-12 strains

thi-1, argE3, thr-1, leuB6,

r s proA2, hisG4, lacYl, F, Str, ^

recA13, thi-1, argE3, thr-1,

leuB6, proA2, hisG4, F, Str, A

lexA, thi-1, thr-1, leuB6,

proA2, hisG4, metB, lacYl,

r s F, Str, A

Ames, B.N.

Ames, B.N.

Ames, B.N.

Ames, B.N.

Ames, B.N.

Howard-

Flanders, P.

Howard-

Flanders -P.

Howard-

Flanders-P.

C600 thr, leu, thi, lac, A Thomas, R.

32

Table 2 : The strains of iysogen (ACT857/HB101) used in this study

are tabulated as under.

ACI857

HBlOl

Conditionally defective cl mutant.

The strain codes for a temperature

sensitive immunity repressor. Lytic

cycle is operative at 42 C and lys-

ogenic at 32 C.

pro, leu, thi, lac

Srivastava,

B.S.

Srivastava,

B.S.

33

Methods

Maintenance and j rowth of bacteria : Each strain of Salmonella

tvF^imurium 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 and a lysogen 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 0/N at 37 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 and lysogen were raised

in nutrient broth at 37 C and 32 C respectively. Ihe culture was

diluted fifty times in fresh broth followed by shaking at 37 C and

32 C till the cell density reached to about 2 x 10^ viable counts ml"'-.

Such exponential cultures were used in all the experiments.

Media

Media for 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 :

34

20 ml

15 g per

50 ml

10 ml

6 ml

3.15 ml

0.25 ml

910 ml

Sterile 50 x VB Salts*

Sterile agar

Sterile 40% glucose

Sterile histidine.HCl.H2O

(2 g per 400 ml H2O)

Sterile 0.5 mM biotin

Sterile arapicillin solution

(8 mg/ml 0.02 N NaOH)

Sterile tetracycline solution''"'?

(8 mg/ml 0.02 N HCl)

Ihe above components were mixed with the molten agar to prepare

the plates. ''«'<retracycline was added only for use with TA102 vv ich is

tetracycline resistant.

'fStock solution of VB salts (IX) was prepared using the

following ingredients :

^feso^. yH^o 0.2 g/1

Citric acid monohydrate 2.0 g/1

K HPO (anhydrous) 10.0 g/1

NaHNH^P0^.4H20 3.4 g/1

Ihe salts were added in the order indicated to warm distilled

water and each salt was allowed to dissolve completely before adding

the next. Ihe solution was then autoclaved for 20 min at 121 C.

Minimal glucose plates for mutagenicity assay :

Sterile VB salts 20 ml

Sterile 40% glucose 50 ml

35

Sterile agar 15 g/930 ml distilled

water

The above components were mixed with the molten agar and then

30 ml was poured over each plate.

Top agar for mutagenicity assay : The top agar contained 0.6% Difco

agar and 0.5% NaCl. 10 ml of sterile solution of 0.5 mM histidine.

HCl/0.5 raM biotin was added to the molten agar and mixed thoroughly by

swirling.

0.5 mM histidine/biotin solution for mutagenicity assay ;

I> Biotin 30.9 njg

L- Histidine. HCl 2A.0 n^

Distilled water 250 ml

Dissolve the biotin by heating the water to the boiling point.

Then mix histidine to it. Autoclave for 20 min at 121 C.

0.2 M sodium phosphate buffer, pH-7.4 :

NaH PC . H O 13.8 g/500 ml

Na^HTO 14.2 g/500 ml 2 4

Adjust the pH-7.4 and then sterilize it at 121 C for 20 min.

Media for E.coli K-12 strains and lysogen :

Nutrient broth (13 g/1) : Nutrient broth obtained from Hi-media

(India) 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

36

In the nutrient broth obtained from Difco (U.S.A.)? NaCl was

also added to it

Nutrient broth 8 g/1

NaCl 5 g/1

Nutrient agar (Hard agar) :

Nutrient broth 13 g/1

(Hi-media)

Agar powder 15 g/1

(Hi-media)

Soft agar : The composition of soft agar used for lysogen work is as

under :

Nutrient broth 13 g/1

(Hi-media)

Agar powder 7 g/1

(Hi-media)

Buffers

MgSO . 7H 0 solution (O.OIM) ; For all dilutions, O.OIM MgSO

solution was used.

Buffers for BND-cellulose chromatography :

0.3M NET buffer : For eluting unbound DNA

0.3M NaCl 3.510 g

lO'^M EDTA 2.0 ml

10"2M Tris HCl 2.0 ml

Distilled water 195 ml

37

IM NET buffer : For eluting

1 M NaCl

-4 10 M EDTA

lO'^M Tris HCl

Distilled water

IM NET+507„ Formamide buffer

1 M NaCl

10~^M ECfTA

10~^M Tris HCl

Formamide

Distilled water

double

: For

: stranded DNA

eluting single

O.OIM TNE buffer : For preparing DNA solution

11.70 g

2.0 ml

2.0 ml

196 ml

-stranded DNA :

11.70 g

2.0 ml

2.0 ml

100 ml

96 ml

2 wg/ml solution of EWA was prepared in O.OIM TNE buffer.

Chemicals

The following chemicals were used

Chemicals

Agar powder

Ampicillin

Acetone

3, 4- Benzo 'a' pyrene

Biotin

BND-cellulose

Chloroform

Chlorampiien ico 1

Source

Hi-media, India

Ranbaxy, India

BDH, India

Koch-Light Ltd., England

Nutritional Biochemical

Corporation, U.S.A.

Sigma, U.S.A.

BDH, India

Biochem Pharmaceutical

Industries, India

38

Calf thymus DNA

Citric acid monohydrate

Dimethyl sulphoxide

Dipotassium hydrogen phosphate

(dibasic)

Disodium hydrogen phosphate

Ethylene diamine tetra-acetic acid

Ethyl methane sulphonate

Formaldehyde

Formamide

D- Glucose

Histidine monohydrochloride

Hydrochloric acid

Hydroxyapati te

Magnesium sulphate

Methylmethane sulfonate

Nutrient agar

Nutrient broth

Sodium ammonium phosphate

Sodium azide

Sodium chloride

Sodium dihydrogen phosphate

Sodium hydroxide

Tetracycline

Tris

Sigma, U.S.A.

BDH, India

BDH, India

Saraliiai,M. Chemicals,

India

Sarabhai, M. Chemicals,

India

S.d. Fine-Chem., India

Koch-Light Ltd., England

BDH, India

E.Merck, India

BDH, India

E. Merck, Germany

BDH, India

Sigma, U.S.A.

E. Merck, India

John Baker Inc., U.S.A.

Hi-media, India

Hi-media, India;

Difco, U.S.A.

E. Merck, Germany

Trucizna, Poland

Sarabhai, M. Chemicals,

India

BDH, India

BDH, India

IDPL, India

Sigma, U.S.A.

39

Steroids

3^-Acetoxy-5<<'-cholestano (5, 6-b)-

N-fii thai imidoazir idine

3 f-Broino-6, 6-ethylene-disulfonyl-

5 06 -cholestane

3 ^-Brorao-5, 6-ethylene d i t h io -

5pt -cholestane

3 P-Brorao-6, 6-ethylene- oc -sulfonyl-

p -sulfinyl-ScC.-cholestane

3 ^-Chloro-5o<.-cholestano (5, 6-b)-

N-Phthaliraidoaziridine

5cC-cholestane-3, 6-dione bis ethylene

di thiolane

3 p -Chloro-5, 6-ethylenedithio-

5 06-cholestane

3 ^-Chloro-6, 6-ethylene-o(-sulf inyl-

^ - th io -5 ot-choles tane

3^- Iodo-6 , 6-ethylene d i th io-5 -cholestane

3 p -Iodo-6, 6-ethylene- oC-sulf inyl - p> -

thio-5 oL-choles tane

3p -Iodo-6, 6-ethylene-ot-sulfonyl- /J- thio-

5 c<-choles tane

Source

Chemistry Department,

A.M.U.; India

- do -

- do -

- do -

- do -

- do -

- do -

- do -

- do -

- do -

- do -

CHAPTER III ; AMES TESTING OF STEROIDS

40

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.

Ihe Salmonella/microsome mutagenicity test (Ames, 1971; Ames et al.,

1973; McCann et al., 1975b) has been sufficiently developed and

validated to be seriously considered for widespread use in this way.

Ihe considerable evidence accumulated so far indicates that with few

exceptions carcinogens are mutagens (Ames et al., 1973; Flessel et

al., 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).

Ihe work embodied in this chapter was, therefore, designed to

screen the available cholesterol derivatives of steroids for their

potential mutagenic and carcinogenic behavior employing Ames-testing

system.

Materials and Methods

Bacteria, steroids and media are listed in Chapter II. All the

media were freshly prepared. Two tests were routinely performed for

testing the mutagenicity of the steroids : spot test and plate

incorporation assay. Except otherwise stated, the cells were treated

41

for 20 minutes following the preincubation procedure in case of plate

incorporation assay.

Spot test : This test is the simplest way to test compounds for

mutagenicity and is useful for the initial rapid screening of large

number of compounds. This is a variation of the plate incorporation

test in which the test chemical (steroid) was left out of the soft

agar overlay and was applied directly to the surface of the minimal

agar plate after it had been seeded with the bacterial tester strain.

Appropriate concentrations of the steroids were added to the agar

surface.

A positive result in a spot test was generally not considered to

be an adequate evidence for mutagenicity. Mutagenicity was therefore

confirmed by quantitative plate incorporation test.

Plate incorporation assay : This test was performed by combining the

steroid, 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 37 C for 48 hours, revertant colonies were counted and

mutation frequency was then calculated by subtracting the spontaneous

mutation from induced mutation.

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 chemical and in the

interpretation of results.

42

Results

Ames spot test of certain steroids : Table 2 depicts the characte­

ristic reversion patterns of the standard strains of some test

steroids. In view of the qualitative assessment of the sensitivity of

the tester strains, the number of revertants scored per spot for TA104

was found to be the maximum. All the steroids that were tested

exhibited some amount of mutagenic activity in the tester strains but

ACPIA, BEDSC, CCPIA, CESTC and lESiTC exhibited relatively greater

number of revertants as compared to other steroids.

Dose response relationship test of all the steroids that were

diagnosed in the spot test was performed. BEDSC and ACPIA showed the

highest number of revertants per plate as compared to other steroids.

Except the BEDSC and its parent compound, BEDTC, data of other

steroids are not shown. BEDSC was selected for further studies.

3p-Bromo-6, 6-ethylenedithio 5PC -cholestane (BEDTC) induced reversion

of Ames tester strains : Reversion pattern of BEDTC with TA98, TA102

and TA104 at various concentrations is shown in Fig. 3. This steroid

is more active in TA104 as compared to TA102 and TA98. It showed a

significant level of mutagenicity with TA104 at very low dose (0.25

Mg/plate). The reversion property declined very sharply beyond this

concentration. Similar declining trend was also observed with TA102.

However, it showed a sharp decline beyond the dose of 0.5 ;jg/plate.

The dose level of BEDTC was not at all lethal for TA98. It followed

about a linear dose response pattern. Ihe tester strains can be

sequenced in order of their responsiveness with the steroid as

43

follows : TA104>TA102>TA98.

3^-Bromo-6, 6-ethylenedisulfonyl 5oC-cholestane (BEDSC) induced rev­

ersion of Ames tester strains : Fig. 4 depicts the reversion pattern

of tester strains at various concentrations as a result of BEDSC

treatment. Again, the mutant TA 104, harboring a multicopy plasmid, pK

MlOl was the most responsive strain. This strain is reverted many

times more efficiently by BEDSC as compared to other steroids (data of

other steroids not shown). BEDSC also showed a significant level of

mutagenicity with TA104 at very low dose of 0.5 ;jg/plate but beyond

the concentration of IQug the reversion property showed a sharp

decline.

BEDSC also induced a positive response in an ochre mutant,

TA102 in being reverted at such low doses as 0.5 ;ug/plate v^ile it

showed a sharp decline beyond the dose of 15 jug/plate. This steroid

demonstrated a linear dose- response at levels that are not

excessively toxic to the frameshift mutants, TA97a and TA98 and a

base- substitution mutant, TAIOO. These tester strains could be

sequenced in order of BEDSC induced mutagenesis as follows :

TA104>TA102>TA97a>TA98>TA100.

Discussion

The Salmonella test was first validated in a study of 300

chemicals most of which were known carcinogens (McCann et al., 1975a;

McCann and Ames, 1976; McCann and Ames, 1977). It was subsequently

employed in other studies by the Imperial Chemical Industries

(Purchase £t al., 1976), and the International Agency for Research on

44

Cancer (Bartsch ^ al., 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 5 mg/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 etal., 1984; Flessel et al., 1987). Our results indicated

an invariable increase in number of revertant colonies with all the

test steroids. Moreover, all the steroids exhibited a remarkable

degree of mutagenicity with TA104 and TA102 strains (Table 2).

Table 1 shows the relevant genetic and biochemical markers of

the Ames tester strains. All the strains carry the pK MlOl plasmid

which is believed to enhance the error-prone repair process

(Shenabruch and Walker, 1980; Levin £t al., 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 TAIOO tester strains contain G-C base

pairs at the critical site for reversion (Isono and Yourno, 1974;

Barnes et_al., 1982; Levin £t al., 1982a). With regard to the

individual difference among the G-C specific mutants,TA97a, TA98 and

TAIOO, the first two strains are the frameshift type while TAIOO is

45

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 hisG428 gene on the

multicopy plasmid, pAQl. On the contrary, TA104 carries the same

mutation on the chromosome but carries a defective excision repair

gene, uvrB which obviously diverts the repair towards the errorprone

side and thus enhances the mutagenic activity of the compound (Levin

et al., 1982b). In view of our findings, it is noteworthy that the

test steroids preferentially act on the A-T base pair mutants, TA102

and TA104 as compared to those having G-C base pairs at the site of

mutation. However, the steroid, lESiTC seems to revert the frameshift

tester strain, TA97a, more efficiently as compared to base-pair and

transition mutants (Table 2).

We have then, concentrated our studies on two steroids only,

the parent steroid, BEDTC and its oxygenated derivative, BEDSC. Infact

the parent, BEDTC presumably contained the reactive S-CH,~CFL-S moiety

which was chemically converted into O^S-CH^-CH^-SOj^in the BEDSC (Figs.

1, 2). Quantitative Ames testing of the steroids revealed that the

tester strains, TA104 and TA102 happened to be the most responsive.

Moreover, a strong mutagenic activity was observed with the derivative

as compared to its parent compound (Figs. 3, 4). In view of the

present findings it is clear that the steroid treatment brought about

a transition mutation owing to the error-prone repair of the damaged

cell. This idea further gains support by the relatively strong

mutagenic response of the test compound on the uvrB defective, TA104

46

strain.

According to Tennant et al (1987) the carcinogenic compounds

containing the electrophiles only would be detected as mutagens by the

Salmonella test. The electrophilic portion of the molecule is an

electron-deficient region present either because of the particular

structure of a chemical, or as a result of metabolic activation. Ihis

electrophilic region accepts electrons from nucleic acids in WA,

allowing the chemical to react and combine with it, leading to

mutation. The test derivative of steroid being oxygenated, acts as

electrophile and can accept single electron to yield the sulfonyl

radical(Figs. 1, 2). The radical might, in turn, react directly with

DNA or generate oxygen radicals like'0„, 'OH and'O". With regard to the

mutagenic potency of the test steroids, both the criteria seem to have

been fulfilled as were laid down by Ames and Whitfield (1966), Ames et_

al (1972) and Creech et_al_(1972). According to the authors the

compound should have a ring system capable of stacking interaction

with WA as well as should also contain an electrophilic group that

can react with it.

47

Table 1. Relevant genetic and biochemical markers.

S.No. Markers TA97a TA98 TAIOO TA102 TA104

1.

2.

3.

Type of mutation

Location of mutation in histidine gene

Excision-repair

Frame- Frame-shift shift

his01242 hisD3052

4UvrB AUvrB

Base pair substi­tution

hisG46

<au\T:B

Transi­tion

hisG428 Uchre mutation on a multicopy plasmid, pAQl

uvrB Profici­ent

Transi­tion

hisG428 on the chromos­ome

extra AUvrB

4. Loss of polysacc- rfa haride layer (rfa)

rfa rfa rfa rfa

5. R-factor plasmid pk MlOl pK MlOl pK MlOl pK MlOl pK MlOl pAQl

6.

7.

8.

9.

Base-pairs at the GC site of reversion

Antibiotic Amp^ resistence

Spontaneous 90-180 reversion frequency

Replacement of TA1537 the strains

GC

Amp

30-50

TA1538

GC

Amp

120-200

TA1535

AT

AmpT, Tet^

240-320

—

AT

Amp

350+75

"

Symbols : ^ - deletion r - resistent

Fig. 1. 3^-Bromo-6, 6-ethylene dithio 5PC-cholestane (BEDTC)

M.P. 140 C.

Fig. 2. 3^-Bromo-6, 6-ethylene-disulfonyl 5oc-cholestane (BEDSC)

M.P. 167 -168 C.

Table 2. Ames spot test of certain steroids.

49

S.No. Steroids Amount S9 TA97a TA98 TAIOO TA102 TA104

spotted

(>ug)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

ACPIA

BEDSC

BEDTC

BESSC

CCPIA

CDBED

CEDTC

CESTC

lEDTC

lESiTC

lESoTC

5-15

-do-

-do-

-do-

-do-

-do-

-do-

-do-

-do-

-do-

-do-

+

+

N.D.

+

+

+

+

+

+

+

N.D.

+

+

N.D.

N.D.

+

N.D.

+

+

++

N.D

+

+

+

+

+

+

+

+

+

+

+

+

++

++

+

+

Sjonbols for the number of revertants/spot (Spontaneous subtracted) :

- = ^ Control; + = ^ 10 - 30; + = 31 - 50;

++ = 51 - 120; N.D. = not done.

All the steroids were dissolved in acetone/dimethylsulphoxide mixture.

5(D

Fig 3. Dose-respopse curves of BEDTC with Ames tester strains

TA97a : N.D.

TA98 : O O

TAIOO : N.D.

TA102 : if- i(:

TA104 : A •

5 10

BEDTC ( j u g / p l a t e )

51

Fig 4. Dose-response curves of BEDSC with Ames tester strains.

TA97a : • •

TA98 : 0 0

TAIOO : ^ <>

TA102 : * ^

TA104 : • A

1000

9 0 0 h

8 0 0 h

700h UJ t -< a. cr UJ Q.

lO h-Z

< 1 -Q: Hi

> UJ Q:

600

500

AOO

300

200

100

BEDSC (^ig / p l a t e )

CHAPTER IV : ROLE OF SOS REPAIR AND MUTAGENESIS IN BEDSC - INDUCED

INJURY : IN VIVO AND IN VITRO STUDIES

52

Introduction

Various physical and chemical environmental agents have been

shown to damage cellular EWA 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 of microorganisms, raaimialian 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

Makinodan, 1973; Lohraan and Bootsma, 1974).

E.coli responds to DNA damage with the expression of a set of

functions usually termed as SOS response. Ihis includes the induction

of a transitory mutagenic DNA repair system, the activation of

inducible propiiage and of several other functions involved in cell

division and DNA metabolism (Radman, 1974; Witkin, 1976).

The in vitro studies on the minor distortions or changes in the

secondary structure of DNA have been studied employing several

techniques. The BND-cellulose chromatography has been used to measure

the growing points in a replicating CNA (Scudiero and Strauss, 1974)

and to detect in vitro post-replication repair. The method of

detection of post-replication repair relies on the ability of the

column to absorb DNA containing single stranded regions (Brash and

Hart, 1978).

The work embodied in this chapter was, therefore, planned to

53

estimate the extent of injury produced, to understand the mutagenic

behavior and the mode of interaction of steroid, BEDSC with DNA.

Our contention was that SOS defective strains of E.coli K-12 as

well as lysogen might serve as a convenient model for this purpose

because even a slight change in environment could be reflected in its

survival and plague forming capacity. Also, in vitro studies with

BND-cellulose chromatography might serve as a good tool in the

understanding of the mechanism of DNA destabilization induced by the

test steroid at different molar ratios.

Materials and Methods

Bacteria, the lysogenic strain, media, test steroid, BEDSC and

buffer are listed in Chapter II. Relevant genetic markers associated

with the strains is also given in Chapterll. Survival patterns of

E.coli strains, induction of prophage X and in vitro study employing

BND-cellulose chromatography have been studied in the presence of

BEDSC.

The SOS defective E.coli strains and lysogen were treated with

that concentration of BEDSC at which the highest peak was observed in

the Ames test as well as with the concentration three times that to it

with lysogen only to identify the genes involved in this system.

Except otherwise stated, the E.coli cells were treated for 6 hours.

All the media were freshly prepared.

BEDSC treatment to bacteria : The SOS defective recA and lexA

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

harvested by centrifugation from exponentially growing culture ( 1 - 3

54

X 10 viable counts ml" ). The pellets so obtained were suspended

directly in 0.01 M MgSO solution and treated with the steroid.

Samples were withdrawn at regular intervals, suitably diluted and

plated to assay the colony forming ability. Plates were incubated 0/N

at 37 C. Treated controls were also run simultaneously.

Prophage induction : Exponentially grown lysogen Acl857/HB101 (1-4 x

10 cells ml^ ) was centrifuged, suspended in 0.01 M MgSO buffer,

treated with steroids separately and incubated at 32 C for 3 hours.

Ihe cells were again centrifuged, washed and resuspended in nutrient

broth and incubated for the next 3 hours at 32 C. Aliquots were taken

out at regular intervals, suitably diluted and plated with C600 cells.

Ihe plaques were scored after 0/N incubation of plates at 42 C.

Treated controls were also run simultaneously.

BND-Cellulose Chromatography : A slurry of BND-cellulose was prepared

in 0.3 M NET buffer and the fines were removed by decantation. The

resin was regenerated following the standard procedure which

constitutes the stepwise washing with water (50ml per 6 gm of resin),

0.3 M NET buffer, 50% formamide in 1 M NET buffer and finally with 0.3

M NET. Ihe washed resin resuspended in 0.3 M NET was poured into the

column of 1 cm diameter containing glass-wool at the bottom so as to

achieve a column length (bed height) of 4 cm. Ihe column was

equilibrated 0/N with 0.3 M NET buffer. Calf thymus DNA was purified

over BND-cellulose column of the commercial sample to complete native

DNA. Ihe 1 M NET eluate was dialyzed against 0.01 M TNE and used to

study the effect of BEDSC on dsEWA. After regeneration and 0/N

55

equilibration of BND-cellulose with 0.3 M NET, 250^g DNA was treated

0/N at 37 C with 1:0.5 and 1:1 molar ratios of the steroid and then

subjected to binding to BND-cellulose. A control was also run

simultaneously. Stepwise elution of DNA with the following buffers in

the given order was carried out i.e. 0.3 M NET, 1 M NET and 1 M

NET+50% formamide buffers. 7-10 fractions of each buffer were

collected at the rate of 10-12 ml/hr. DNA eluted in various fractions

was determined spectrophotometrically at 260 nra.

Results

Effect of BEDSC on the SOS defective E.coll strains : The survival

patterns of recA and lexA mutants of E.coli are shown in Fig. 1. The

damage brought about in the cell in the presence of the steroid is

much pronounced. Both the mutants invariably exhibited a significant

decline in their colony forming ability as compared to their wild-type

counterpart. LexA mutant was found to be more sensitive as compared to

recA mutant.

Prophage induction in the presence of BEDSC : Table 1 shows the

induction of prophage as a result of BEDSC treatment to lysogen during

the post treatment liquid holding in the nutrient broth. A fraction of

the lysogenic population exhibited induction of lytic cycle during the

liquid holding at 32 C. Moreover, on increasing the concentration of

the steroid, the fold induction was also increased. There was no

significant increase in the prophage induction over and above the

background level in the control as well as in the chloramphenicol

supplemented samples.

56

BND-cellulose chromatography : The elution patterns of untreated and

BEDSC treated WA in the BND-cellulose column are shown in Fig. 2. O/N

incubation of steroid with the EWA at 37 C caused adherence to

BND-cellulose indicating the production of single-stranded regions or

breaks in the native dsDNA molecules. An increasing degree of EWA

hydrolysis was observed with the enhancement in the steroid : DNA base

pair ratio. The percent of DNA adhered on the BND column in case of

0.0:1.0, 0.5:1.0 and 1.0:1.0 molar ratios were 55.29%, 55.49% and

60.71% respectively.

The untreated and steroid treated EWA at different molar ratios

were eluted out in two major peaks but the area covered in the two

peaks are remarkably different under the treated and control

conditions. The amount of dsDNA eluted out at 1 M NET buffer in terms

of decreasing order of area were as follows : 0.0:1.0 0.5:1.0

1.0:1.0 whereas the peak-area eluted out at 1 M NET+50% formamide

buffer presumably containing the ssDNA followed the reversed order.

Discussion

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 LWA damage, and several

classes of EWA repair enzjmies have been identified (Friedberg and

Bridges, 1983).

The steroid (BEDSC) which was detected to be the most potent

57

mutagen in our case in the Ames testing system was also tested with

SOS-repair defective recA and lexA 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 EWA whether produced by

radiation or by other agents (Walker, 1985). These mutants were found

to be highly sensitive to the test steroid, BEDSC (Fig. 1) suggesting

thereby the damage to the WA of the exposed cells as well as the role

of recA and lexA genes to cope with the hazardous effect of the test

steroid. The role of recA and lexA gene in initiating the error-prone

repair in E.coli is well documented (Witkin, 1976; Walker, 1985). Ihis

idea gains further support from the prophage induction (Table 1) and

by Ames testing studies (Figs. 3, 4) with the Salmonella strains

triggering the error-prone SOS response. Propiiage induction infact,

has also been used to detect the mutagenic activity of chemicals

(Moreau et al., 1976).

Our BND-cellulose chromatograj^ic studies suggested the enhance­

ment in the single-strandedness in the steroid-treated WA (Fig. 2).

This finding alongwith the elevated mutagenic response of the compound

with uvrB defective Salmonella strains (Chapter III) further supports

the idea of physical distortion brought about by the test steroid,

uvr endonuclease has been shown to act upon any sort of physical

distortion in the WA rather than the pyrimidine dimers only (Braun

and Grossman, 1974; Seeberg, 1981; Miller and Heflich, 1982).

58

Fig. 1. Survival of SOS defective E.coli K-12 strains exposed to

BEDSC.

Wild-tj^e

recA

lexA

0-

1.

o

.A

O

< > > a. Z3

UJ o cr.

0.

TIME ( h r s )

59

Table 1. Induction of A prophage on BEDSC treatment

8

Time of incubation Prophage induction per 10 lysogens

in nutrient broth (hr) •

Control After BEDSC treatment for 3 hrs

0.5;jg/ml 1.5;ag/ral In presence

of chloram­

phenicol

(lOOjug/ml)

0 1.0 1.0 1.0 1.0

1 0.9 1.6 1.7 1.0

2 1.2 1.8 3.3 1.0

3 1.3 2.5 3.9 1.0

60

Fig. 2. BND-cellulose chromatography of double stranded calf

thymus ENA treated with BEDSC with respect to DNA

base-pair : compound molar ratio.

Panel A :

Panel B :

Panel C :

Control

1 : 0.5

1 : 1

e c o

UJ U z < m tr o IT) CO <

20 40 60

ELUTION VOLUME { ml )

CHAPTER V : GENERAL DISCUSSION

61

Ames testing system has gained a world-wide recognition to

assess the mutagenic potential of great variety of synthetic and

natural chemicals present in our environment (Ames, 1984). Although

the validity of the test has been questioned in terms of the actual

carcinogenic behavior of the test compound yet a remarkable amount of

correlation was obtained with several mutagenic compounds tested by

the Salmonella system (McCann et al., 1975; Purchase et al., 1976;

Flessel et al., 1987).

Several steroids were found to have a great potential in

pharmacology, medicine and other biochemical industries (Wilpart,

1986). On the other hand these chemicals have also been reported to be

mutagenic, carcinogenic and teratogenic on the basis of short-term and

long-term testing systems (Lucier and Rambaush, 1983; Metzler, 1984;

Dunkel et al.,1985). Interestingly enough, the compounds which we have

selected for our studies have also exhibited a significant level of

mutagenic activity with the Ames tester strains (Table 2, Figs. 3, 4;

Chapter III). It is noteworthy that even in the absence of S^ 9

microsomal fraction, usually all the tester strains responded to a

remarkable extent. It does not, however, essentially imply that these

steroids would display a higher degree of mutagenicity after being

metabolized in the higher system.

It is always emphasized that a single testing system does not

reflect the actual behavior of the test compound and a battery of

various types of tests is recommended (Maron and Ames, 1983; Tennant

et al., 1987). We have, therefore, employed other systems like the

62

E.coli and lambda for the steroid 3p-bronio-6, 6-ethylene disulfonyl-5t>c

-cholestane. This steroid exhibited the toxicity towards the

SOS-defective E.coli K-12 recA and lexA strains. The sensitivity of

these radiation sensitive mutants also supports the notion that

DNA-damage is induced by the test compound (Fig. 1; Chapter IV). The

potential of test compound for inducing the SOS response was also

confirmed by the significant degree of>.-prophage induction in the

iysogenic E.coli strain (Table 1; Chapter IV) as well as the intense

mutagenic response with the TA104 Salmonella tester strain.

The test compound, BEDSC which is the sulfonyl derivative of

BEDIC Figs. 1, 2; Chapter III) seems to have initiated the

SOS-response with the concomittent induction of transition mutation

because of the presence of a multicopy plasmid(s) in the TA104 and

TA102 strains. TA104 strain carries a defective uvrB gene in addition

to the pKMlOl plasmid which is believed to enhance the error-prone

DNA-repair process (Shanabruch and Walker, 1980; Levin et al., 1982b).

Ihe uvrB gene is involved in the error-proof machinery. The defect in

this error-proof machinery would further lead to the enhancement of

the error-prone repair response.

We were also interested in the role of several chemical groups

and positions in the steroid nucleus to have an idea about the

contribution of different loci and moieties within the molecule

towards the mutagenic potential of the compound to put forward a

plausible mechanism of DNA-mutagen interaction. It was found that the

replacement of sulphur at the 6th position of the steroid nucleus by

63

the sulfonyl group (Figs. 1, 2j Chapter III) resulted in the

tremendous increase in the mutagenic activity of the compound (Figs.

3, 4; Chapter III).

One of the theories on the etiology of cancer holds that the

major cause induced by the carcinogen is the damage to HJA by oxygen

radicals and lipid peroxidation (Totter, 1980; Ames, 1983). Several

enzjmies in the living system like xanthine oxidase and peroxidase, are

thought to produce superoxide anion (' 0") during the oxidation of

their substrates (Faria et al., 1977; Buttner et al., 1978). Numerous

substances such as reduced flavins and ascorbic acid on auto-oxidation

produce superoxide anion. This radical further accepts an electron

from a reducing agent such as thiol to yield peroxide (H„0 ). There is

in vitro evidence that H^O^then reacts with certain chelates of copper

and iron to form the highly reactive free radical ("OH) (Wolff et al.,

1986). Actual appearance of superoxide anion in metabolism is

confirmed by the ubiquitous occurence of superoxide disrautase. Our

test compound 3^bromo-6, 6-ethylene disulfonyl-5o(.-cholestane (BEDSC),

owing to have a disulfonyl moiety has a great potential to form the

oxygen radical and/or reactive free radicals ('(M) and (' 0^) under the

experimental conditions.

With regard to the overall damage on the DNA molecule brought

about by the test compound, the BND-cellulose chromatographic studies

suggested the enhancement in the single strandedness in the DNA

molecule (Fig. 2; Chapter IV). It is an established fact that recA

protein binds to the single-stranded regions formed by any means and

64

thus is thought to initiate the SOS response (Salles and Defais,

198A).

In view of the high sensitivity of recA and lexA strains of

E.coli as well as significantly high mutagenic activity of the test

steroid with Ames testing system it is clear that :

(i) The damage is primarily at the level of DNA.

(ii) The injured cells cope with the situation employing both the

constitutive as well as the inducible DNA repair machinery.

(iii) Under the drastic conditions e.g. at relatively high dose,

mutation is brought about by the genetic defect in the error-

proof repair system,

(iv) With regard to the plausible nature of lesion, it seems that

distortion in DNA occurs as a result of steroid interaction

which is supported by genetic studies as well as with BND-

cellulose chromatography. The cell,owing to the multiple genetic

defects and relatively lethal action of the steroid under the

experimental conditions has no option but to initiate the SOS

response.

65

Future Plan

We would like to extend our studies on the following lines :

1. Quantitative screening of available steroids to construct the

complete dose-response curves.

2. Mutagenicity testing of the potent compound(s) employing the

E.coli and A-systems as well as the Mud systems.

3. Genotoxicity testing employing sister chromatid exchange (SCE).

4. Studies on the possible nature of potentially mutagenic lesion in

DNA employing (i) BND-cellulose chromatography (ii) hydroxyapatite

chromatography (iii) melting profile and (iv) alkaline hydrolysis.

5. (i) QSAR studies on the possible mode of interaction with EWA of

the steroid(s) exhibiting the transition mutation,

(ii) QSAR studies on the possible mode of interaction with ENA of

the steroid(s) exhibiting the frameshift mutation.

6. Identification of the region/sequences, if any, with the DNA for

preferential binding of the test steroids.

7. Studies on the role of several light and heavy chemical moeities

and of different positions of the parent nuclei to ascertain the

contribution towards mutagenic potential of the compound.

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