genotoxrc effec ot f certain steroidal drugs · qadri for his ph.d work. . i am fully satisfied...

GENOTOXrC EFFECT OF CERTAIN STEROIDAL DRUGS

T H E S I S SUBMITTED FOR THE DEGREE OF

B o t t o r of $l|ilo£(optip

BIOCHEMISTRY

SHAFA7 A QADRI

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES

ALIGARH MUSLIM UNIVERSITY ALIQARH (INDIA)

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES

ALIGARH MUSLIM UNIVERSITY

CERTIFICATE

I certify that the work embodied in this thesis entitled "Genotoxic Effect of Certain Steroidal Drugs" has been carried by Shafat A. Qadri under my supervision. It is original in nature and has not been submitted for any other degree.

G (Prof. A.M. Sid|^iqui)

ALIGARH-202002 (U. P.) INDIA TELEPHONE : 2 5 7 4 1 TELEX ; 564-230 A.ViU IN

CONTENTS

PAGE NO

Certificate Acknowledgments List of Abbreviations ... i List of Tables iii List of Illustrations — vi Preface ... ix

CHAPTER-I: Review of Literature — 1

CHAPTER-II: General Materials and Methods — 29

CHAPTER-III: Ames Testing of Steroids .•• 41

CHAPTER-IV: Role of Active Oxygen Species ... 55 in Steroid Induced Mutagenesis

CHAPTER-V: Steroids as the Inducers of SOS ... 82 Response in E. coli and -phag0 systems

CHAPTER-VI: Steroid Induced DNA Lesions (In — 118 Vitro and In Vivo Studies).

CHAPTER-VIIi General Discussion ... 144

Bibliography ... 152

List of Publications ... 171

Summary

United Nations Industrial Development Organization

International Centre Shaheed Jeet Singh Marg

for Genetic Engineering TresMiaV/SOT M ^ Lli^H.-X'-v , . , , 6867358/6867360/6867361

a n d Biotechnology leiex 31-73286icgbin Telefax 91-11-6862316 - '

To whom so ever it mav concern

I feel pleasure to certify that the research problem entitled "Genotoxic Effect of Certain Steroidal Drugs" was assigned by me to Mr. Shcifat A. Qadri for his Ph.D. work. I am fully satisfied with the data embodied in the dissertation, in view of my direct research guidence. The present work is suitable for the award of Ph.D degree in Life Sciences.

4 = 1 New Delhi: (MASOOD AHMAD) 12^ February, 1993. Reader in Biochemistry

F/o Life Sciences Aligarh Muslim University ALIGARH.

T I' , 1 .

^ Tr n iiiie i i o v r n g m e m o r y o i

m y y o u n g e r b r o t h e r s

Late Nasir Iqbal and Late Nisar Iqbal

DKRICATED TO MY

DEAR PARENTS

ACKNOWLEDGEMENTS

I wish to express my most sincere and profound gratitude to Dr. Masood Ahmad who suggested this problem, and gave me full attention, indispensable guidance with constructive criticism and trust during this investigation. He painstakingly initiated me into the field of molecular genetics and nurtured in me the ability of independent work.

I am humbly grateful to Prof. A.M. Siddiqui for his unceasing encouragement and constant support. His magnanimous generosity and unflagging interest helped me in all my endeavours.

My sincere thanks are due to Prof. S.M. Hadi, Chairman, Department of B i ochemi stry and other teachers of this department.

I am also thankful to all members of Virology lab ICGEB, New Delhi especially to Dr. S. Jameel for fruitful suggestions, Dr.K. Rao for helping in performing H.P.L.C. and Dr. N. Jaysuryan for moral encouragement and interest in my work.

A token of deep appreciation to Dr(s) J. Musarrat, S. Tayyab and J.H. Wani for constructive suggestions. My thanks are extended to all my lab col leagues, other friends and well wishers.

I feel short of words to express my heartful gratitude to my younger brother late Nasir Iqbal for his encouragement and support from every corner to continue my higher studies. I also express the infinite gratitude to my dear parents for their benedictious and constant inspiration throughout my studies. I warmly acknowledge Bhai Sahib, Riaz Sahib, Shah Faisal, Ali Jan and Rauf who contributed with their affection and love.

Last but not least Mr.Shoaib Ahmad and Mr Suhail Ahmad deserved word of praise for expert word-processing.

UGC, New Delhi is acknowledged for the award of Research Fel lowship, as a NET fellow.

(Shafat A. Qadri)

LIST OF ABBREVIATIONS

Ap Ampicillin

arg Arginine

bp Base pairs

bio Biotin

CFU Colony formig units

^ uvrB Deletion in excision repair gene

dsb Double strand breaks

F~ Female

g/1 Grams litre"^

h Hour

his Histidine

lac Lactose

Lambda sensitive

leu Leucine

lex A regulatory locus for coding for repressor of SOS genes

met Methionine

ug Microgram

ul Microlitre

mA Milliampere

ml Millilitre

min Minutes

MM Minimal medium

M Molar

NBT Nitro blue tetrazolium

nm Nanometer

0/N Overnight

PFU Plague forming units

PRNO N, N dimethyl-p-nitrosoani1ine

pro Pro!ine

rec Recombination

rfa Deep rough mutation

sec Seconds

ssb Single strand breaks

ssDNA Single stranded DNA

Str*" Streptomycin resistant

TA7 Soft agar

thi Thiamine

thr Threonine

thy Thymine

VB salts Vogel - Bonner sallts

w/v Wight by volume

w. t. Wi Id-type

LIST OF TABLES

CHAPTER TABLE PAGE NO.

II 1. The Salmonella typhimurium and 29-30 Escherichia coli strains used under study.

2. The strains of phage A used in this 31 study.

3. The steroids used under study. 32-33

III 1. Reversion of Ames tester strains in 51 the presence of steroids without metabolic activation.

2. Reversion of Ames tester strains in 52 the presence of steroid after metabolic activation by post-mi tochondrial supernatant,

3. Relevant genetic and biochemical 53 markers of Ames tester strains.

IV 1. Generation of superoxide anion (Og'") 69 by steroid I in terms of the conversion of nitroblue tetrazolium to a formazan at different incubation times and varying concentration of Steroid I.

2. Generation of superoxide (Og"") in 70 terms of conversion of nitroblue tetrazolium to a formazan at different incubation timings and varying concentration of steroid VIII.

3. Production of the hydroxyl radicals 71 with varying concentrations of the steroid I in light.

4. Effect of quenchers on the formation 71 of hydroxyl radical by the steroid I.

CHAPTER LIST OF TABLES PAGE NO.

5. Production of hydroxy! radicals with 72 varying concentrations of the steroid VIII in light.

6. Effect of quenchers on the production 72 of hydroxyl radicals by steroid VIII.

7. H2O2 production by steroid VIII 73

8. Effect of scavengers of reactive 74 oxygen species on the mutagenic activity of the steroid I in S. typhimurium TA102 strain.

9. Effect of scavengers of reactive 75 oxygen species on the mutagenic activity of the steroid I in S. typhimurium TA104 strain.

V 1. Induction of prophage in XcISdl B. 100 CO77 (AB1157) lysogen by steroid I.

I 2. Effect of steroid treatment on the 101

transformation frequency of E, coli cells with pBR322 plasmid DNA.

3. Steroid induced mutagenesis in pBR322 102 plasmid after the uptake by E. coli cells.

4. Effect of oxygen radical scavengers 103 on the survival of X phage within the steroid I treated A red - recA E. coli complexes.

5. Effect of oxygen radical scavengers 103 on the survival of A phage within the steroid VIII treated A red - recA E. coli complexes.

6. Frequency of c mutation in steroid 104 treated E. coli A c lysogen.

7. Steroid I induced c mutagenesis in 105 Ac E. coli complexes.

CHAPTER LIST OF TABLES PAGE NO.

8. Steroid VIII induced c mutagenesis on 106 A c^ E. CO 7 7 comp1 exes.

VI 1. Effect of transition metal ions (0.4 132 mM) on the S^ hydrolysis of calf thymus DNA by steroid I treatment (0.2 mM) in presence of light and dark.

2. Inhibition of S^ nuclease hydrolysis 132 of DNA after treatment with steroid I (0.2 mM) and Cu(II) (0.4mM) in the presence of scavengers.

3. Degradation of calf thymus DNA 133 treated with steroid I in the absence of S^ nuclease.

4. S^ nuclease independent degradation 133 of calf thymus DNA treated with varying concentration of steroid I in presence of Cu(II).

LIST OF ILLUSTRATIONS

FIGURE PAGE NO.

Ill 1. Dose-response effect of test steroid 54 I-IV with Ames tester strains (TA102 and TA104),

2. Dose-response effect of test steroid 55 I-IV with Ames tester strains (TA102 and TA104) in the presence of post-mi tochondrial supernatant.

IV 1. Generation of superoxide anion (02*") 76 by steroid I in light. Effect of incubation in dark and in presence of SOD.

2. Generation of superoxide anion (Oo'") 77 by steroid VIII in light. Effect of incubation in dark and in presence of SOD.

3. Production of HgOg as a function of 78 steroid I concentration.

4. Formation of singlet oxygen at 79 various concentrations of steroid I.

5. Formation of singlet oxygen at 80 various concentrations of steroid VIII.

6. Possible mechanism of mutagenic action 81 of test steroid I

V 1. Survival of E. col i K-12 cells 107 exposed to steroid I.

2. Survival of E. col i K-12 cells 108 exposed to steroid II.

3. Survival of E. coli K-12 cells 109 exposed to steroid III.

4. Survival of E. coli K-12 cells 110 exposed to steroid IV.

CHAPTER FIGURE PAGE NO.

5. Propagation of extracel1u1arly 111 treated A c with steroid I in various E. coTi host strains

6. Plaque forming ability of Xvir 112 particles in various B. coli host strains.

7. Survival patterns of intrace- 113 llularly treated A c with steroid I in various E. coli host strains.

8. Survival patterns of Intrace- 114 llularly treated X c with steroid VIII in various E. coli host strains.

9. Survival patterns of intrace- 115 llularly treated "hred with steroid I in various E. coli host strains.

10. Survival patterns of intrace- 116 llularly treated "hred with steroid VIII in various E. coli host strains.

11. Survival patterns of intrace- 117 llularly treatedA^/or with steroid I in various E. coli host strains.

VI 1. Sj dependent hydrolysis of calf 134 thymus DNA after treatment with steroid I.

2. Thermal melting profile of native and 135 steroid I treated DNA.

3. Sedimentation profiles of solvent and 136 steroid I treated calf thymus DNA on alkaline sucrose gradients (5-20%).

4. Sedimentation profiles of solvent and 137 steroid VIII treated calf thymus DNA on alkaline sucrose gradients (5-20%).

5. Sedimentation profiles of solvent and 138 steroid I treated calf thymus DNA on neutral sucrose gradients (5-20%).

(viii)

CHAPTER FIGURE PAGE NO.

6. Sedimentation profiles of solvent and 139 steroid VIII treated calf thymus DNA on neutral sucrose gradients (5-20%).

7. Agarose gel electrophoresis of pBR322 140 plasmid DNA incubated with 200 uM Cu(II) and varying concentrations of steroid I.

8. Agarose gel electrophoresis of 141 steroid I treated single stranded M13 mp8 phage DNA.

9. Effect of the steroid I treatment on 142 the survival of S. coli K-12 strains (Po 7A and 7 ig~).

10. Effect of the steroid VIII treatment 143 on the survjval of E. coli K-12 strains {polA" and lig~).

PREFACE

Cancer is still eluding the grasp of scientists even

though tremendous amounts of researches have been directed

towards understanding the etiology of this dreadful syndrome.

In fact, neoplastic transformation ultimately signifies the

alteration in the genetic material vis-a-vis its altered

expression. This is brought about by both the endogenous and

exogenous factors. DNA damage and mutation, therefore, seem

to be crucial events in many, if not all, malignant

transformation processes.

Even though prevention is always better than cure, in

case of cancer it is the only option available so far. There

is increasing evidence that environmental chemicals, both man

made and naturally occuring, play important role in causing

human cancers (Ames and Gold, 1990). Thus the appropriate

identification of the environmental chemicals responsible for

carcinogenesis could be a significant measure towards its

prevention (Sobels, 1984).

Knowledge of the mechanism of carcinogenesis is in fact

necessary for .prediction of human cancers. Results from

animal cancer tests, which are conducted at near toxic doses

of a test chemical can not predict the cancer risk at the

usually low levels of human exposure. Moreover, nothing can

be predicted regarding positive association of human

carcinogenesis and tumor initiating potency in some

experimental animal studies (Wogen and Gorlick, 1985).

Therefore, lifetime carcinogenicity studies will be needed

for those nongen<toxic carcinogens that are tissue, sex,

species specific in contrast to the genotoxic carcinogens

that cause tumors in different species at multiple sites

(Hay, 1988).

Innumerable number of hazardous chemicals are present

in the environment and thousands of new compounds are being

poured into the environment each year. That makes it

impossible to carry out the long term tests which are time

consuming and costly in addition to other short-comings

(Flessel et al., 1987 ). Several short-term tests have,

therefore, been developed during the last two decades (Ames,

1971; Ashby and Tennant, 1988). These tests are usually the

valid indicator of the genotoxic potential of the test agent,

at least when a combination of tests is performed (Maron and

Ames, 1983; Friedberg and Bridges, 1983). The beauty of the

tests is that one could also understand the mechanism of

action of the test compound and thus could gain insight into

the process of carcinogenesis. This further raises the hope

of investigating the role of active oxygen species in the /

chemical carcinogenesis which has recently attracted the

attention of biological scientists in understanding the life

processes in the imposing oxygen environment. (Smith and

Johnson, 1989).

Naturally occuring as well as synthetic steroids have

been constantly used in various industries (Fishbein, 1984)

and also have wide application in medicine (Barnes, 1987;

Henery et al. , 1988; Ryoji et al., 1988). Several steroids

and/or their metabolites have been reported to be mutagenic

and carcinogenic (Dunkel et al. , 1985; Ochs et a/., 1986;

Smith and Johnson, 1989). Therefore, it seems worthwhile to

assess the genotoxic behaviour of these chemicals, especially

of those having some biological activities.

The test steroids were synthesized and derivatized to

widen their chemotherapeutic use (Shafiullah et al., 1983;

1988; 1989; 1990). As we anticipate further developments in

this area of pharmaceutical chemistry, these steroids were

selected for the genotoxicity testing in the light of

available literature (Chapter-I) employing the Ames test

(Chapter-Ill) as well as the E. coTi and phage lambda

testing systems (Chapter-V). In vitro studies were also

carried out to study the interaction of the highly mutagenic

steroid I and VIII (Islam and Ahmad, 1991) with DNA and to

identify the lesions caused by them (Chapter-VI). Moreover,

an attempt was also made towards the structural requirement

for the mutagenicity of test steroids to elucidate the

mechanism of steroidal action (Chapter-Ill). Role of active

oxygen species in steroidal mutagenicity was also studied

(Chapter-IV). An overview of the findings has been presented

in the last two chapters.

CHAPTER I

Review of Literature

Cancer is fundamentally a degenerative disease of old I

age, although exogenous factors can increase cancer rates or

decrease them (Ames and Gold, 1990). The word cancer is

derived from the Greek word 'Karkinos' meaning crab. It is a

condition resulting from uncontrolled reproduction of cells

which escape controlling factors and eventually form a

cluster of cells called tumor. The tumor can be benign or

malignant. The later condition is called cancer, meaning

that the cells are radiating out like the 'arms of a crab'

(Alcamo, 1987).

Cancer may be malady of genes, arising from genetic

damage of diverse sorts - recessive and dominant

mutations, large re-arrangements of DNA and point mutations,

all leading to distortions of either the expression or

biochemical function of genes (Bishop, 1987; Ponder, 1988).

The belief that genetic damage might be responsible for

cancer grew from diverse roots: the recognition of hereditary

predisposition to cancer (Lynch, 1976; Schimke, 1978;

Ponder, 1980), the detection of damaged chromosomes in cancer

cells (Rowley, 1983, 1984), the apparent connection between

susceptibi1ity to cancer and impaired ability of cells to

repair damaged DNA (Lehmann, 1982; Hanawalt and Sarasin,

1986), evidence that relates mutagenic potential of

substances to their carcinogenicity (Ames, 1979) and the

discovery of oncogenes that can cause neoplastic growth and

transform a normal cell to a cancer cell (Cooper, 1982; Fink,

1984; Bishop, 1987). The pathogenesis of cancer involves

permanent alterations in gene structure i.e. mutations i

fFriedberg, 1985a). This theory is known as somatic mutation

theory (Florey 1962; Lloyd-Luke et al. , 1 978) and 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 fFlessel

et al. , 1987 ).

It has long been known that cancer can arise as a

result of exposure to a variety of agents both physical as

well as chemical. Genotoxicity due to environmental mutagens

may be the main cause of death and disability in advanced

societies (Cairns, 1975) initiating most human cancers and is

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

heart diseases as well (Pearson et al., 1975).

Since a large proportion of chemicals have been found

to be genotoxic and cause human cancer. The prevention of

cancer will depend to a large extent upon the recognition and

possible elimination of environmental and endogenous exposure

to genotoxic agents (Doll and Peto, 1981). Rapid and accurate

in vitro tests such as the SalmoneTTa microsome test, play a

crucial role in the identification of environmental mutagens

and minimizing human exposure (Rinkus and Legator, 1979;

McCann and Ames, 1976; Masayoshi, 1988).

Genotoxicants as Carcinogens and their mechanism of action

A majority of chemicals whether natural or synthetic

have been shown to interact with the genetic material.

Abundant evidence exists to support the assertion that

interference with DNA (genetic defects) in almost any

conceivable way, is likely to serve as a prelude to the

induction of cancer. A majority of these chemical

carcinogens are known to form covalent adducts with DNA

(Miller, 1978; Ames, 1979; O'Connor, 1981; Waring, 1981).

It is often assumed that DNA damaging agents are

carcinogenic because they induce mutations. However, another

strong possi bility is that the damage leads to heritable

changes in the methylation of cytosine in DNA. 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 specialized 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).

Most chemical carcinogens are alkylating or acylating,

or produce such compounds following metabolism in the body.

Such agents have electron deficient center and will combine

with electron-rich center within the cell. Reaction with DNA

is the most important process in the initiation of

mutagenesis and carcinogenesis, though reaction with some

amino-acids in proteins may also occur (Farmer, 1982; Saffhil

et al., 1985).

Since virtually every chemical known to cause cancer in

humans also causes cancer in animals flARC Monographs, 1972-'

75; Tomatis et a/., 1973; Epstein, 1974; Ames and Gold,

1990), 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 many uncertainties in

determining the risk to humans from animal data (Epstein,

1974; Mantel and Schneiderman, 1975; Ames et a?., 1987).

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

DNA damage can also be assessed by cytogenetic analysis. The

human disorders ataxia telangiectasia, Bloom's syndrome and

Fanconi's anemia are genetic diseases characterised by an

increased susceptibility to cancer (Walker, 1985).

Studies with Poecilia formosa showed an increased

incidence of tumors in recipients bearing cells maintained

under non-DNA repair conditions, thereby implicating DNA

damage as a specific etiological factors in the neoplastic

transformation (Hart and Setlow,1975; Woodhead and Scully,

1977; Woodhead et a1., 1977). Studies with syrian hamster

also suggested a causal relation between DNA damage and

neoplasia (Dipaolo and Donovan, 1978; Doniger et al., 1981).

DNA Repair

DNA is the primary carrier of genetic information and

the structural integrity of DNA is a prerequisite for gene

expression (Modak, 1972). It is a known fact that primary

structure of DNA 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. The living systems have

therefore, evolved repair processes to maintain structural

and functional fidelity of DNA against a large range of

insult CFriedberg, 1985b). The molecular mechanisms involved

in repair of damaged portions of DNA and their restriction

into functionally intact informational units is fundamental

to the maintenance of the genomic integrity (Modak, 1972).

Defective DNA repair could cause an accumulation of lesions

or mutations which might be either lethal, lead to an altered

phenotype, or neoplastic transformation. Repair at the

cellular and macromolecular level is multiple in its form and

varies as a function of 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 genetic studies

have identified, a large number of genes participating in the

repair of damaged DNA (Walker, 1985).

The following repair systems have been shown to be

existing in bacteria:

(i) Photoreactivation (ii) Excision repair (iii) Post-

recombinational repair (iv) SOS repair (v) Adaptive response,

Since SOS repair is error-prone in nature thus

involving mutations in the DNA of bacterial cell, we are

discussing some important features of SOS repair system in

the following 1ines:

The term 'SOS' (international distress signal) repair

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 postulated by Defais et al (1971) and was further

developed and amplified by Radman (1974, 1975) and Witkin

( 1976). The exposure of E. coTi to the agents that damage

DNA or interfere with DNA replication results in the

induction of a diverse set of physiological responses called

SOS response. It requires recA^ and lexA^ genotypes of host

(Miura and Tomizawa, 1968; Defais et al., 1971). 'SOS' repair

is a highly integrated and sophisticated regulatory network

that require de novo protein synthesis for expression (Koval,

1986). It is an inducible repair process and is believed to

be responsible for a common mutagenic pathways (Radman, 1974;

Witkin, 1976; Walker, 1985; Strauss, 1989). In response to

DNA damages calling for the SOS repair, DNA 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 which

coordinately function in 'SOS' repair. These are uvrA and

uvrB (DHA repair), umuC (mutagenesis), sfiA (filamentation),

himA (site-specific recombination) and several din genes with

unknown functions in addition to the recA and lexA genes

fWitkin, 1976; Kenyon, 1983). Role of recBC and recN genes

has also been suggested in 'SOS' induction (Chaudhury and

Smith, 1985; Finch et aT., 1985).

The recA and TexA genes are the regulators which

control 'SOS' response. Under normal conditions LexA protein

represses the subordinate genes of the system and RecA

protein derepresses these loci in response to DNA damage

(Kenyon, 1983). LexA protein is a self repressor and also

binds to similar operator sequences in each gene (Little et

al., 1981; Sancar et 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; Sancar et al., 1982). Salles et al. (1985) and

recently Fridrichova et al. (1992) reported that RecA

immunological assay can be used as a tool to analyse the

'SOS' response and can be used as an auxiliary test for DNA

damage detection.

The accumulation of certain deoxyribonu1eoside

monophosphates and a high level induction of recA gene

expression have been suggested to stimulate the 'SOS' repair

fGottesman, 1981). Moreover, Salles et al (1983) reported

the full amplification of RecA protein without any

modification 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' response. Induction of 'SOS' repair by

several physical and chemical agents that induce single

strand breaks (ssb) in DNA have been reported (Salles and

Defais, 1984; Chaudry and Essigman, 1991; Dale et al., 1992).

Moreover, the ssbs in DNA were able to restore the induction

of RecA protein.

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 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 DNA polymerases (Radman et al. , 1 977; Villani et a/.,

1978). Moreover, the lexA dependent conversion involving the

subunit of DNA polymerase III holoenzyme to 'SOS' polymerase

has also been postulated (Scheuermann et al., 1983; Piechocki

et al., 1986). Lu et al (1986), in fact, have demonstrated

the RecA mediated inhibition of 3' -> 5' nuclease activity of

isolated subunit of DNA polymerase III and was suggested to

be responsible for targeted mutagenesis.

Repair of bacteriophage A :

Extensive researches on the radiation induced damage

and its repair in bacteriophage identified several DNA repair'

systems (Walker, 1984). Three major repair systems are as

follows:

(i) Host cell reactivation, (ii) Prophage reactivation, and

(iii) Weigle reactivation

Weigle 'reactivation accompanies with high frequency of

mutation. Therefore, certain significant points relevant to

our work are given below:

It is defined as the increased survival of UV

irradiated phage when the host bacterium is also exposed to

UV prior to infection (Weigle, 1953). This phenomenon is

accompanied by high frequency of mutation. Devoret et al,

(1975) have suggested that W-reactivation of bacteriophage' « \

lambda is independent of both excision and recombinational

repair and that it depends on a new error—prone repair

activity induced in the host cell by UV radiation and other

•SOS' inducing treatments. W-reactivation does not occur if

the chloramphenicol is present in the preincubation medium

(Defais et a/., 1976). W-reactivation of UV irradiated

lambda does not require the function of uvr and pol genes but

it requires the functional rec*' and lex^ genes (Yamamoto and

Shinagawa, 1985).

Two genes of phage lambda have been shown to influence

radiation sensitivity. These are designated as red and gam

and have been assumed to act independently (Trgovcevic and

Rupp, 1975). Moreover, the red gene of lambda is considered

to play a role complementary to recA gene of E. coTi and is

involved in the repair of mild heat (52°C) and alkali induced

lesions in DNA (Ahmad and Srivastava, 1980; Musarrat and

Ahmad, 1991).

Role of free radicals and active oxygen species in

carcinogensis:

Reactive oxygen species (superoxide radicals, hydrogen

peroxide, hydroxyl radicals) are responsible for damage to

DNA (Sies, 1986; Imly and Linn, 1988; Pryor, 1988), causing

strand breaks and base alterations that can lead to cell

death or mutations (Ames, 1979; 1982). In addition, oxygen

and a variety of oxidants have shown to be mutagenic (Lewin

et al., 1982a; Storz et al., 1 987; Greenberg and Demple,

1988). One of the theories of etiology of cancer, which is

being widely accepted, holds that the major cause is damage

to DNA by oxygen radicals, free radicals and lipid

peroxidation (Totter, 1980; Copland, 1983; Ames, 1983).

There is an increasing amount of data which suggests that

chemical carinogens may cause both direct DNA damage i.e.,

carcinogen-DNA adducts, and indirect DNA damage by causing

formation of free radicals and active oxygen species that

react with DNA (Kadlubar et al., 1980; Cerutti, 1985; Michael

et al., 1989). It has been estimated that the number of

oxidative hits to DNA per cell per day is about 100,000 in

rats and 10,000 in humans (Ames, 1989; Shigenaga et al.,

1989; Fraga et al., 1990). In addition, active oxygens and

the ensuing lipid peroxidations could affect carcinogenic

processes in at least two ways: by causing chromosomal damage

and rearrangements, and by modulating cell growth and

differentiation through epigenetic mechanisms (Cerutti,

1985). For chemical carcinogens such as aminoazo dyes and

Naphthylamines, there is a good correlation between the

formation of active oxygen and corcinogenicity (Nakayama et

al., 1983). Moreover, oxygen radicals generated by enzymatic

reactions (Morgan et al. , 1 976 ) and directly from several

chemical compounds including drugs cause DNA strand scission

Halliwell and Gutteridge, 1990; Bhat and Hadi 1992).

The genotoxicity of 02*" and HgOg in vivo is largely

thought to arise from their metal ion catalysed conversion

into highly reactive 'OH radical (Brawn and Fridovich, 1981;

Halliwell, 1987; Nassi-Calo et a/., 1989). Evidence indicates

that iron ions can lead to formation of -OH from 02*" and

H2O2 (Halliwell, 1987). Ionizing radiations produce 'OH

which result in DNA base modification (Dizdaroglu et al. ,

1991). Formation of -OH in the reaction of copper ion with

H2O2 has been well documented (Masarwa et al., 1988; Reed and

Douglas, 1989; Sagripanti and Kreamer, 1989) H2O2 with copper

ions also produces extensive strand scission (Sagrapanti and

Kreamer, 1989; Yamamoto and Kawanishi, 1989). Moreover, DNA

strand breakage and formation of DNA base products by copper

ion/H202 has been shown to be more extensive than that by

iron ion/H202 under similar experimental conditions (Reed and

Douglas, 1989; Tachon, 1989). Kazakov and co-workers (1988)

have found only copper ion among several transition metals as

a cofactor in the ascorbate mediated site-specific cleavage

of DNA. Most of the "OH generated in vivo, except during

excessive exposure to ionizing radiations, comes from the

metal dependent breakdown of H2O2 according to general

equation.

M"" + H2O2 — > + -OH + OH"

where M""*" is a metal ion like Fefll) (Halliwell and

Gutteridge, 1990). The Fe "*" dependent decomposition of H2O2

(the so-called Fenton reaction) is usually written as:

Fe^"^ + H2O2 — > Fe^"^ + -OH + 0H~

In addition to the causation of cancer through DNA

damage, these free radicals and oxidants are involved in many

other human diseases like aging, arthritis, infection with'

malaria and other parasites, neurological damage, diabetic

cataract, dysbaric osteonecrosis, silicosis, asbestosis.

Downs syndrome and immune injury to kidney, liver and lung,

as well as atherose 1erosis and Ischemia (Marx, 1 987;

Halliwell and Gutteridge, 1990).

Certain promoters of carcinogensis act by generating

oxygen radicals. The mechanism of action of promoters

involves the expression of recessive genes and increase in

gene copy number through chromosome breaks and hemizygosity

(Varshavsky, 1981; Kinsella, 1982).

Correlation of carcinogenasis 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 95^ fMcCann et al. , 1975b; Rinkus

and Legator, 1979; Levin et al., 1984; Tennant et a/., 1987;

Zeiger 1987). 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 ( 1 987 ), there appears a

continuous decrease in the degree of sensitivity by Ames

testing. McCann et al f1976a) reported a correlation of

around 90% which was further supported by many reports

(McCann and Ames, 1976; Ames and McCann, 1981; Hartman and

Auckerman, 1986). But an analysis of 465 compounds with

known or suspected carcinogenic activity indicates that about

58% have mutagenic activity in Solmonella test (Rinkus and

Legator, 1979). Zieger (1987) reported that the Ames test

has a sensitivity (percentage of carcinogens identified as

mutagens) of only 54% and sepcificity (percentage of non-

carcinogens identified as non-mutagens) of only 70%. Fven

worse, Tennant et al (1987) found that the Salmonella assay

would only identify about 46% of carcino'^^ns which is at a

hi gh vari ance to the original report of 90% correlation

(McCann et al. , 1975a). This looks disastrous for

carcinogenicity sc-eening that animal carcinogenicity studies

were the only way to identify carcinogens. But the later

analysis of Ashby and Tennant (1988) lifts this gloom. They

still conclude that there is about 90% correlation between

structural analysis and Salmonella assay results for a

variety of chemicals including 115 carcinogens, 24 equivocal

carcinogens and 83 non-carcinogens. Salmonel 1 a test, the

authors argue, is a sensitive method for picking out

chemicals that are genotoxic and could attack DNA (Ashby and

Tennant, 1988).

Mutagenicity and 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 systems and over the last few decades a

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

Ingvardsen, 19Si3).

Long-term tests are expensive and time consuming,

although they have given considerable evidence in estimating

the potency of a carcinogen. But still there is a problem of

predicting whether human 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 to

extrapolate the risk from rodents to humans, a very

dissimilar long-lived species. Opposite was also found in

Which a chemical was found to be c a r c i n o g e n i c 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 mechanism 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 (SCE),

mutagenicity assay using mouse lymphoma or hepatic cells and

the Ames test. No combination of the four STTs is any better

than Salmonella on its own for flagging probable carcinogens

fWolff, 1984; Renata et al., 1989). However, it is always

desirable to carry out a battery of tests for a better

understanding of the actual behaviour of the test compounds

(Maron and Ames, 1983). In addition, the role of these tests

has increased 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 in vitro are genotoxic in short-

term tests (Ames, 1979). The in vitro STTs have advantages

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 concordance between results from in vitro STTs and

rodent carcinogenicity tests were highly encouraging fDurston

et al., 1973; Sugimura et al., 1976; Devort, 1979).

Wide use of short-term tests for detecting mutagens

(Ames, 1979) and a number of animal cancer tests on plant

substances have contributed to the identifications of many

natural mutagens and carcinogens in human diet fKapadia,

1982).

On the basis of literature - derived study of

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 non-carcinogens (Shelby and Stasieweiz, 1984;

Kier et al., 1986; Ray et a/., 1987) and too few chemicals

had been tested in multiple STTs 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 a/., 1987).

In addition to the aforementioned STTs, there are few

other short term tests in which f. co7i and phage lambda have

been used as target cells (Kondo, 1974). The 'Inductest,

Mudtest and Chromotest' are the different parameters to

estimate the extent of DNA damage to determine the mutagenic

potential of a chemical based on the expression of SOS-genes

in treated E. col 1 cells fMoreau et a/., 1976; Bagg et a1.,

1981; Kenyon and Walker, 1981).

Ames Testing System

Mutagens cause cancer by mutating the DNA of cells in

ways that cause cells to proliferate in an uncontrolled

manner. It is generally agreed that several mutations are

necessary to convert a normal cell into a cancer cell capable

of uncontrolled growth (Weinberg, 1989; 1990). Among 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

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

other short-term assays (Hollstein et al., 1979), is being

extensively used to survey a variety of substances in our

environment for mutagenic activities (Ames, 1984). The test

measures back mutations in several specially constructed

mutants of Solmonella.

The test has been adopted for use in detecting

chemicals which are potential human carcinogens or mutagens,

by adding homogenates of rat liver (or other mammalian

tissues) directly to the petri plates as an approximation of

mammalian metabolism into the in vitro test (Ames et a1.,

1973b).

Several histidine - requiring mutants in the standard

set of SolmoneTIa tester strains have GC base pairs

at the critical site for reversion e.g.; -C-C-C- in the

base pair substitution strain, TA100 (Barnes et aT.,

1982), -C-C-C-C-C-C- in the frame shift tester strain, TA97a

(Levin et a/., 1982b) and -C-G-C-G-C-G-C-G- in the frame

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

TA102 and TA104 strains have AT base pairs at the critical

site for reversion. TA102 and TA104 strains detect a variety

of oxidants and other agents as mutagens which were not

detected in the standard tester strains (Levin et al., 1984;

De Flora et al. , 1989).

Steroids: Uses and Applications

Naturally occurring as well as synthetic steroids have

been commonly used in various industries (Fishbein, 1984).

Several steroids have been reported to possess anti-

inflammatory (Boltralik, 1988; Henry et a/., 1989),

anesthetic (Evelyne et al., 1988), anabolic (Fennessey et

al., 1988), angiostatic (Larrian, 1989), antiangiogenic

(Roger et al., 1988; Judah et al., 1989), cardioactive (Karel

and Thomans, 1987), antidiabetic (Ryoji et al., 1988) and

anti AIDS (Barnes, 1987) activities.

Certain steroids have also been shown to control e'nergy

metabolism throughout pregnancy (Baird et a1., 1985).

Steroid-alkaloid formulations have been used for skin

disorder treatment (Chain et al., 1984). Also, there are

some steroidal compounds which increase resistance against

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

found that medroxy progesterone acetate inhibits growth of

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

1986). Fluorouracil estradiol conjugate (Asano et al., 1978)

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

1983) as well as gestagens (Nishida et al., 1986) have been

reported to possess antitumor and antimutator activities

respectively.

Steroids as Potential Carcinogens

There is a high risk of development of cancer with the

use of steroids too, since several steroids have been

reported to possess tumorigenic and carcinogenic activities

(Allen et al., 1981; Kay, 1981; Candrian, 1984).

Several steroids were mutagenic and carcinogenic on

the basis of short-term tests (Dunkel et al., 1985).

Moreover, 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 fKay,

1981; McKillop et al., 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

fKatzenellenbogen, 1 986; Ochs, et al., 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 homogenate or

cytosol fractions fPrakash et al., 1986). Tissue steroids

have been found to play a role in regulating aromatase

activities in breast and endometrail cancer (James et al.,

1986). Steroid alcohol sulfotransferases have been isolated

from the cytosol of a human breast carcinoma cell line MCF-7

fRohzim et al., 1986). Moreover, the stereochemical

complementarity of DNA and reproductive steroid hormones have

been found to correlate with biological activity (Lawrence et

a/., 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 and reproductive glands (Heywood,

1986). They have also been shown to enhance liver tumor in

the Egyptian toad (Sadek and Abdelmeguid, 1986). Moreover,

contraceptive steroids have been shown to enhance mutagenic

activity in Salmonella (Rao et a1., 1982) and were also found

to be the promoter of tumorous growth in rat liver (Heike et

aT. , 1986; Reinhard et a/., 1989). Certain steroidal

derivatives were reported to have remarkable mutagenic

activity with Ames tester strains TA102 and TA104 which

detect oxidative mutagens (Islam et a1., 1991; Islam and

Ahmad, 1991).

Autoxidation of steroids like cholesterol have resulted

in the production of some active radicals like carbon

centered and peroxyl radicals as well as H2O2 (Smith, 1981;

Sevilla, et al., 1986), Moreover, some biologically active

oxysterols have been produced by the autoxidation of

cholesterol with active oxygen radicals (Assmann et al.,

1975; Painter et al., 1982; Smith and Johnson, 1989). These

oxysterols have been reported to be toxic, mutagenic and

carcinogenic (Smith, 1981; Ansari et aJ., 1982; Smith et a1.,

1986; Smith and Johnson, 1989).

Recent Advances in Carcinogenicity Testing Systems

Ashby and Tennant (1988) concluded from their survey of

around 222 carcinogens and non-carcinogens that the

SalmoneJ 7a assay (the so-called Ames test) does not identify

all carcinogens. It does work for certain chemicals

particularly those that become reactive electrophiles and

attach to DNA (Miller and Miller, 1977), but it certainly

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

Tennant et al. (1987) observed that three of the better known

short-term tests: (the chromosome aberrations, sister

chromatid exchange and mutagenicity assay using mouse

lymphoma cells) would falsely label more non-carcinogens as

carcinogens owing to their high sensitivity and less

specificity than the Ames test. 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 suggested by Ashby and

Tennant (1988). The test is the mouse micronucleus assay

which works 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

molecular markers for human exposure to chemical carcignogens

is no longer barrier to further study, as several

sophisticated analytical techniques such as tandem mass

spectrometry and fluorescence line-width narrowing

spectrophotmetry are now available (Shuller, 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).

The Salmonena TA1535/psk 1002 strain is used to detect

environmental mutagens in which the expression of the umuC

gene for (3-D- galactosidase is used as a parameter. The

sensitivity of detecting this enzyme in this method is

amplified by 10-1000 fold by a chemiluminescence spectrometry

method (Masayoshi, 1988).

Ames et a1 f1987) and Gold et a1. (1992) have invited a

great deal of controversy over the popular view that

synthetic carcinogens are posing a higher risk than the

natural ones. According to these reviews 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. Gold et a1. f1992)

have suggested that natural substances like plant pesticides

and products of cooking are more potent rodent carcinogens

than synthetic pesticides and environmental pollutants,

according to HERP (human exposure/rodent potency) index.

Animal models provide invaluable information in studies

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

of this information from experimental animals to humans

remains, however, a problematic endeavour. 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 fWogen and Gorlick, 1985) it is not known whether 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 objectives of the Ph.D. work

Natural as well as synthetic steroids both have been

widely used for chemotherapy against several diseases

including tumors fMoreau et al., 1979), inflammation (Verma,

1981; Henry et al., 1989), cancer (Chang et al., 1955;

Blickenstaff and Foster, 1961) and even AIDS (Barnes, 1987).

Moreover, some oxazoles and their derivatives have been

reported to possess anti-inf1ammatory , analgesic,

antibacterial, antimicrobial and antiviral activities (Turchi

and Dewar, 1975). As pharmaceuticals, oxazolines are useful

as anti-hypertensive as well as central nervous system

regulators (Levitt, 1970a,b). Azridines have also been shown

to possess diversified types of biological activities

including their contraceptive, antitumor, antineoplastic,

cytotoxic, antibacterial and antiviral activities (Borokovec

and Woods, 1963; Hassner and Healthcock, 1965; Derner and

Hans, 1969; Lusthof et al., 1988).

Some steroids have been derivatized in order to widen

their spectrum in pharmacological use (Shafiullah et al.,

1988; 1989; 1990), Before expecting any further development

in this field of pharmaceutical chemistry, the long term

hazardous effect of such pharmaceuticals should be tested.

The genotoxic effect of some of these steroids and their

interaction with the DNA have been already reported by our

group (Islam et al., 1991; Islam and Ahmad, 1991). Several

authors have also reported the genotoxic effect of ceVtain

other steroids (Rao et a7., 1983; Heywood, 1986; Sadek and

Abdelmeguid, 1986). The mutagenic potential of sulfonyl and

aziridinyl derivatives have been attributed to the active

oxygen radicals (Islam et a1., 1991; Islam and Ahmad, 1991).

In view of the available literature and in the light of

our earlier findings, we initiated the work on the screening

of some oxathiolane and aziridinyl derivates of steroids for

mutagenicity and DNA damaging activity employing a battery of

tests. The major objectives were as follows:

(i) Quantitative mutagenicity screening of test oxathiolane

series and disulfonyl steroids to construct the

complete dose response curves.

(ii) Studies on the role of SOS response in the mutagenic

activity of the potent oxathiolane and aziridinyl

steroidal derivatives employing E. coTi and lambda

systems.

(iii) Investigation for the involvement of active oxygen

species (02'~. * OH, ^02) by the potent

oxathiolane and aziridinyl steroids and their role in

genotoxicity.

(iv) Identification of steroid induced DNA lesions in

plasmid, phage E. coTi and calf thymus DNA by a variety

of biochemical and genetic methods.

CHAPTER II

General Materials and Methods

TABLE-1: The Salmonella typhimurium and Escherichia coli strains used in this study have been tabulated as under:

Strain Relevant genetic markers designation

Source

Ames Strains

uvrB, hisD6610, bio, rfa, R-factor plasmid-pKMIOI

uvrB, hisD3052, bio, rfa, R-factor plasmid-pKMIOI

uvrB, hisG46, bio, rfa, R-factor plasmid-pKMIOI

Ames, B.N.

rfa, R-factor p^asm^6-pKMI01, Ames, B.N. multicopy plasmid-pAOl containing hisG428 auxotrophic marker and tet""

uvrB, hisD428, rfa, R-factor plasmid-pKMIO!

Ames, B.N,

AB1157

AB2463

AB2470

AB1886

AB2494

E. coli K'12 strains

thi-1, argE3, thr-1, 1euB6 proA2, hisG4, 1acY1,F~, St^^ X®

recA13, thi-1, argES, thr-1 leuB6, ^roA2, hisG4, F~, S t r^, A

recB21, thi-1, argE3, thr-1 TeuB6, proA2, hisG4, F . St^^ X^

uvrA6, thi-1, argE3, thr-1 1euB6, proA2, hisG4, lacYI, P- Str^, X®

lexA, thi-1, thr-1 leuB6, proA2, hisG4, metB, lacYI, F" 5tr^ A®

Bachman, B.J

Howard-Flanders, P

Bachman, B.J

Howard-Flanders, P

JC9239

rerr arg, thr-1, leu, pro, his, F , Str^,

thr, leu, thi, lac,

Srivastava, B.S.

Thomas, R.

recF143, thr-1, TeuB6, proA2, Bachmann, B.J. hisG4, thi-1, lacY1,

thyA54, ma1A38, lacZ36, Bachmann, B.J. thi-1 1euB6, proC32, hisF860 ara-14, mt1-1, xyl-S, strA109, spc-15,

polAI, thyA54, ma1A38, lacZ36, Bachmann, B.J. thi-1 leuB6, proC32, hisF860 ara-14, mtT-1, xyl-S, rpsE2115, rpsL109,

lig-T, relAI, spoTI, bglRd rpsL150, A®

Bachmann, B.J.

TABLE-2: The strains of phage A tabulated as under:

used in this study are

Strain Designation

Description Source

or A""

Tibial

hcI857

Wild-type strain, It forms turbid plaques on all E. coTi K-12 strains described in Table-1.

Recombination defective mutant. It is defective in Exonuclease V and B-protein. It is also having a temperature sensitive cl mutation and does not form detectable plaques on polA mutant of E. coTi K-12.

Recombination deficient mutant (i.e. reef gam~). It does not multiply in recA mutant of E. col i K-12.

Virulent strain. It contains an absolute defective mutation in the immunity region and, therefore, forms clear plaques.

Conditionally defective cl mutant. The strain codes for a temperature sensitive immunity repressor. Lytic cycle is operative at 42°C and lysogenic at 32°C.

Caillet-Fauquet,

Thomas, R

Maenhaut-Michel, G

Srivastava, B.S.

TABLE-3: Ttw steroids uswi urxter study

S Steroids No.

1. 38-Acetoxv-5 oO-cholestano I I6oC.5-dl-1' .3'-oxathiolan®-2' -thiofw

Symbol Melting Point (°C)

m structure

2. 3B-Ch1oro-5aC-cho1e8tano II .5-dl-1'.3'-oxathiolane-2'

-thione

3. 3B-Hydroxy-5 oC-chole«tano III t8«c.5-dl-1',3'-oxath1olane-2' -thione

181 520

4. SoC-Cholestano [6«C.5-d]-1'-, IV 87®-88° 3'-oxathio1ane-2' thione

V 11 S

478 CsHn

0 S \ / c II s

5. 5 oC-Cho1©8tano 3. 6-<Hon« bis ethylene dithlolane

165' 570 C8H17

0 2 ^ 5 0 2

6. 3B-Acetoxy-6-n1tro. chole8t-5-en©

VI 102" 98^17

7. Cho1e8t-5-ene (Parent coapd.)

VII 94-95"

8. 3B-Acetoxy-5«ccho1e«t!mo VIII [5-6b] N-phthaiiildoazlrlcHn®

18 C8H17

Methods

Maintenance and growth of bacteria:

Each strain of Salmonella typhimurium >fias 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. col 1 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 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 and 32®C respectively. The culture was diluted fifty times in fresh broth followed by shaking at 37^C and 32^C till the cell density reached to about 2x10® viable counts ml Such exponential cultures were used in all he experiments.

Preparation of phage stock:

Stocks were prepared on plates by confluent lysis method. Phage ^ was obtained from isolated plaques streaked on agar plates. Bacteria from exponential culture were harvested and resuspended in Mg^ solution. 0.3 ml of C600 cells were infected with phage ^ . Adsorption was allowed for 20 min at 37^0 and plated with 3.0 ml of molten TAy. Plates were then incubated at 37°C or 42*^0 (in case of }\cl&57 phage) till confluent lysis was visible to naked eyes. Soft agar containing A was scraped with the help of MgSO^ solution, 15*; chloroform was then added to it and the agar was beaten by gentle vortexing. Phage A was obtained in the supernatant by centrifugation of the lysate. The phage stock thus obtained was stored over few drops of chloroform at 4* 0.

Construction of lysogen:

The overnight grown cells of AB1157 were centrifuged and suspended in equal volume of Tris-Mg buffer (pH 8.0). To 1 ml of cell suspension the phage cI857 particles in the

ratio of 5:1 were added and allowed to adsorb for 20 min at 32^C. These complexes were then transferred to the baiTing tubes containing sterile Luria broth and incubated at 32' C till the cell density reached to 1-3x10® CFU/ml. This culture was then streaked on agar plates to get isolated colonies. Several bacterial colonies were picked up and tested for prophage induction at 42^C. The clones growing at 32®C and undergoing lysis at 42®C were stored.

Materials

Media for Ames Strains

Medium for master pTates and slants:

The composition of the medium for Ames tester strains to prepare master plates and slants is as under:

Sterile 50 x VB Salts* 20 ml Sterile agar 15 g/ 910 ml Sterile 40% glucose 50 ml Sterile histidine. HCl.HpO 10 ml (2 g per 400 ml HgO) Sterile 0.5 mM biotin 6 ml Sterile ampicillin solution 3.15 ml (8 mg/ml 0.02 N NaOH) Sterile tetracycline solution** (8 mg/ml 0.02 N HCl)

For the preparation of plates, the above components were mixed with the molten agar.

** Tetracycline was added only for use with TA102 which is tetracycline resistant.

* Stock solution of VB salts (IX) was prepared using the following ingredients,

MgS04.7H20 0.2 g/1 Citnc acid monohydrate 2.0 g/1 KpHPO^ (anhydrous) 10.0 g/1 NaHNH4P04.4H20 3.4 g/1

The salts were added in the order indicated to warm distilled water and each salt was allowed to dissolve completely before adding the next. The solution was then autoclaved for 20 min at 12l"^C.

Minimal glucose plates for mutagenicity assay:

Sterile VB salts 20 ml Sterile 40^ glucose 50 ml Sterile agar 15 g/930 ml

disti1 led 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% agar powder and 0.5% NaCl. 10 ml of sterile solution of 0.5mM histidine. HCl/0.5mM biotin was added to the molten agar and mixed thoroughly by swi rling.

0.5 mM histidine/biotin solution for mutagenicity assay:

D-Biotin 30.9 mg L-Histidine, HCl 24.0 mg Distilled water 250.0 ml

First biotin was dissolved by heating the water to the boiling point. Then histidine was mixed to it and finally the solution was sterilized for 20min at 121*^C.

0.2M Sodium phosphate buffer, pH 7.4:

NaHpPO^.HpO 13.8g/500 ml Na2HP04 14.2g/500 ml

The pH was adjusted to 7,4 and sterilized at 121®C for 20 min.

Preparation of liver microsomal fraction (Sg):

Sg fraction was prepared from the livers of Aroclor-'1254 induced Balb C mice according to Maron and Ames (1983).

Sg mix for mutagenicity assay

Sg mix was prepared by mixing (per 3.0 ml); sterile distilled water, 1.185 ml; 0.2M NADP, 0.120 ml; 1M glucose-5-phosphate, 0.015ml; sterile 0.4M MgCl2-1.65M KCl salts, 0.060

ml; Rat liver Sg fraction, 0.120 ml (4%). Sg mix was fr.eshly prepared for each experiment.

Oxygen radical scavengers:

Stock solutions of superoxide dismutase (2 mg/ml), catalase (4 mg/ml), mannitol f0.1M), formate fO.IM), thiourea (0.1M), benzoate (0.1M), iodide fO.IM) and azide (6.1M) were prepared in sterile 0.1M phosphate buffer (pH 7.8).

Media for E.coli K-12 strains and lysogen

Nutrient borth (13g/1):

Nutrient broth obtained from Hi-media fIndia) had the following composition:

Peptone 5.0 g/1 NaCl 5.0 g/1 Beef extract 1.5 g/1 Yeast extract 1.5 g/1 pH (approx.) 7.4 ± 0.2

Nutrient agar (Hard agar):

Nutrient broth 13 g/1 Agar powder 15 g/1

Soft agar:

The composition of soft agar used for lysogen work was as under:

Nutrient broth 13 g/1 Agar powder 7 g/1

MgS04.7H20 Solution (0.01M) : For all dilutions, 0.01 M MgS04 solution was used.

Buffers and solutions for in vitro tests

0.01M TNE buffer: For preparing DNA solution 2.5 mg/ ml of calf-thymus DNA was prepared in 0.01M TNE (O.OIM Tris-HCl. pH 7.5, 0.01M NaCl and 10"" M EDTA) buffer.

Solutions for S^ nuclease hydrolysis:

S^ nuclease dilution (2 units/ml):

Sterilized Sj nuclease buffer (pH 4.5) 200 ul Sterilized glycerol 50 ul Sterilized distilled water 738 ul S^ nuclease 12 ul

(2000 units)

S^ nuclease buffer (pH 4.5)

0.5 M Sod i urn acetate (pH 4.5) 1.0 mM ZnSO^

Perchloric acid was 14* and bovine serum albumin 10 mg/ml in sterilized distilled water.

Diphenyl amine reagent for estimation of DNA:

Diphenylamine 250 mg CHoCOOH (glacial) 25 ml HgSO^ (Cone.) 675 ml

Preparation of sucrose gradients (5-20%)

5 and 20% sucrose was dissolved separately in 0.1N NaOH and 0.01M EDTA (for alkaline gradient) and 0.01M TNE buffer, pH 8.0 (for neutral gradient). 3.8 ml 5-205« gradient was prepared in polyalomer centrifuge tubes by a gradient mixer (Auto Japan).

Buffers and solutions for transformation

Tris-CaClg buffer (pH 8.0)

0.05 M CaClp 0.01 M Tris

Treatment buffer (pH 7.0)

0.01 M Tris 0.05 M MgS04.7H20

Chemicals

The following chemicals were used:

Chemicals Source

Acetone Agar powder Agarose

Ammonium chloride Ampici11 in Arochlor 1254

3,4-Benzo Biotin

'a' pyrene

Bovine serum albumin Calcium chloride

Calf thymus DNA (Type I) Catalase Chloroform Citric acid monohydrate Cobalt chloride Copper sulphate Cupric chloride Deoxy ribo nucleic acid (calf thymus) Dimethyl sulphoxide Deoxy ribo nucleic acid (Ml3 and ?^-phage^ Diphenylamine Dipotassium hydrogen phosphate (dibasic) Disodium hydrogen phosphate (di basic) Ethylene diamine tetraacetic acid Ferric chloride Ferrous sulphate

Formamide G1ycerol D-Glucose Histidine monohydrochloride Hydrochloric acid Hydrogen peroxide

B.D.H., India Hi-media, India Sigma, U.S.A. Gift from ICGEB, New Delhi B.D.H., India Ranbaxy, India Gift from Dr J.H.Parish Univ. of Leeds, U.K. Koch-Light Ltd., England Nutritional Biochemical Corporation, U.S.A. Sigma, U.S.A. Sarabhai, M. Chemicals, India Sigma, U.S.A. Sigma, U.S.A. B.D.H., India B.D.H., India B.D.H., India E. Merck, India B.D.H., India Sigma, U.S.A.

B.D.H., India Sigma, U.S.A.; Gift from ICGEB, New Delhi Glaxo, India B.D.H., India

Sarabhai, M. Chemicals, India S.d. Fine Chem., India

Loba, Chem. India Sarabhai, M. Chemicals, India. E.Merck, India Hi-Media, India B.D.H., India E.Merck, Germany B.D.H., India S.d. Fine Chem., India

Chemicals Source

Magnesium sulfate E.Merck, India Manganese chloride E.Merck, India Manni to! Difco, U.S.A. f3-Mercaptoethanol E.Merck, Germany Nickel sulphate B.D.H., India Nicotinamide dinucleotide S i gma, U.S.A. phosphate Nitroblue tetrazolium Si SCO Research Labs,

India Bombay,

Nutrient agar Hi-media, India Nutrient broth Hi-media, India;

Difco U.S.A. Perchloric acid E. Merck India Plasmid DNA (pBR322) Gift from ICGEB, New Delhi Potassium chloride B.D.H., India Potassium dihydrogen phosphate S.d. Fine. Chem. India (monobasic) S^ nuclease Sg Fraction

Sigma, U.S.A. S^ nuclease Sg Fraction Prepared in our lab Sodium acetate B.D.H., India Sodium ammonium phosphate E.Merck, Germany Sodium carbonate Glaxo, India Sodium chloride Sarabhai, M. Chemicals,

India. Sodium dihydrogen phosphate B.D.H., India (monobasic) Sodium dodecyl sulfate Sisco, India Sodium formate B.D.H., India Sodium hydroxide Hi-Medica, India Sucrose Glaxo, India Superoxide dismutase Sigma, U.S.A. Tetracycline IDPL, India Thiourea E.Merck, India Trichloroacetic acid Glaxo, India Tris-HCL Sigma, U.S.A. Triton X-100 B.D.H., India Zinc sulphate E.Merck, India

Note: All other chemicals were the commercial products of analytical grade.

CHAPTER III

Ames Testing of Steroids

Introduction

Since human exposure to toxic chemicals both natural as

well as synthetic is an established fact. There is

considerable evidence that a large proportion of human

cancer may be caused by exposure to chemicals in the

environment. Thus it seems worthwhile to evaluate the

genotoxic effects of these noxious chemicals. Moreover,

there is an increasing acceptance of mutagenicity testing as

an integral part of premarketing toxicological evaluation of

chemicals. Animal bioassays are too expensive and time-

consuming for routine screening of these toxic chemicals

(Flessel et al., 1987). So in vitro short term testing has

been widely accepted as a quick and relatively inexpensive

means to assess the mutagenic potential of chemicals (Rinkus

and Legator, 1979). The generally recognised Salmonella/

mammalian microsome test, developed by Ames and colleagues

(Ames, 1971; Ames et al., 1975; McCann and Ames, 1976; Rinkus

and Legator, 1979) measure the ability of chemicals to

produce mutations in sensitive strains of bacteria. The

results with the test strongly support the somatic cell

mutation theory of cancer. In a number of validation studies

involving several hundred chemicals, nearly four out of every

five (805^) animal carcinogens assayed were found to be

mutagenic (Ames and McCann, 1981; Hartman and Auckerman,

1986). This test has, therefore, utility in cancer risk

evaluation. Moreover, 5aZmone7/^/microsome mutagenicity'test

(Ames, 1971; McCann et al., 1975b) was sufficiently developed

and validated to be seriously considered the risk assessment.

A set of histidine - requiring Salmonella typhimurium

strains has been recommended for the Ames mutagenicity

testing. Each tester strain contains a different type of

mutation in the histidine operon (Table-3). In addition the

standard tester strains contain other mutations like rfa

which causes partial loss of the 1ipopolysaccharide barrier

that coats the surface of bacteria and, l^uvrB which is a

deletion of gene coding for the DNA excision repair system

(Ames, 1971, 1973a). The standard tester strains TA97a,

TA98, TA100, TA102 and TA104 contain the R-factor plasmid

pKMIOI. This plasmid further enhances the mutagenic response

in the harbouring Ames tester strains (McCann et al., 1975b).

Among these Salmons!la strains TA97a and TA98 detect various

frame shift mutagens and TA100 detects mutagens that cause

base-pair substitution.

Oxygen radicals have been reported to be the most

important class of mutagens contributing to aging and cancer

(Ames, 1982). Levin and coworkers have recommended the

standard tester strain TA102 for the detection of mutagens

that produce active oxygen species (Levin et al., 1982a;

1 984). They have used cumene hydroperoxide , a strong

oxidant, as a model for the numerous lipids and cholesterol

hydroperoxides (Ames, 1982). In addition to TA102, TA104 has

also been used and recommended for detecting the oxidative

mutagens (Levin et al., 1982a; De-Flora et al., 1989). TA102

and TA104 carry the ochre mutations at his G428 locus and

detect a variety of oxidative mutagens like H2O2 and other

hydroperoxides as well as those reduced to 02'" (Levin

et al. , 1982a).

Considerable evidence accumulated so far supports the

desirability of using this type of rapid and economical test

system as a screening technique (Ames,1971; Ames et al.,

1975a, b; Ashby and Tennant, 1988).

The work presented in this chapter was, therefore,

designed to screen the available steroidal derivatives for

their mutagenic and carcinogenic behaviour employing Ames-

testing system.

Materials and Methods

Bacteria, steroids and media are listed in chapter II. All the media were freshly prepared. The steroidal mutagenicity was detected by preincubation test according to Maron and Ames ( 1 983 ). For the metabolic activation of oxathiolane series of steroids, 20ul of Sg liver homogenate mix per plate was added.

Preincubation test: To 0.5ml phosphate buffer pH 7.5 (or 20 ul Sg mix) in sterile capped tube placed in an ice bath appropriate doses of steroid and 0.1ml of fresh overnight (0/N) culture of the tester strains were added and incubated

at 20-30 min at 37^C. Then 3.0ml molten top agar (held at 45*^C) supplemented with 0.5ml His/Bio solution was added to each of the tubes containing treated culture. The contents were mixed and poured immediately over minimal glucose agar plates with swirling action to achieve uniform distribution of the top agar and allowed to harden on a level surface. Plates were inverted and placed in a dark vented incubator maintained at 37* 0 for 48h. Revertant colonies were counted and mutation frequency was calculated by substracting the spontaneous mutation from induced mutation.

Solvent as control was also run simultaneously.

Results

Reversion of Ames tester strains in the presence of steroid

I-VII:

Table-1 shows the number of histidine revertants upon

treatment of Ames strains with steroids I-VII in the absence

of Sg fraction. The steroids exhibited a significant level

of mutagenic activity with strain TA104 followed by TA102.

TA97a and TA98 strains usual 1 y did not respond to test

steroids except for steroid VI which displayed a significant

mutagenic activity with these strains also. TA100 responded

comparatively weakly to all the steroids. The test steroids

exhibited relatively linear mutagenic response at lower doses

like 0.25 to 1.0 ug/plate, but beyond 5.0 ug/plate the

reversion property showed a sharp decline.

The tester strains could usually be listed in the order

of their sensitivity to steroids as follows:

TA104 > TA102 > TA100 ^ TA97a ^ TA98

Among the oxathiolane series of steroids fl-IV), steroid lil

exhibited the maximum mutagenic activity followed by steroid

I, II and IV. These could be listed in the following order

of preference

III > I > II > IV

The parent compound steroid VII did not show any

significant mutagenic activity fTable-1).

Reversion of Ames tester strains brought about by

steroids I-IV in presence of Sg fraction is shown in Table-2.

The results indicate that the number of revertants were

significantly increased by the metabolic activation at low

steroid concentrations fO.25 to 5.Oug/p1 ate ) . Beyond

5.0ug/plate dose, the steroids became lethal after attaining

a peak value. The maximum mutagenic activity was again

observed with tester strains TA104 and TA102.

The mutagenic activity in presence of Sg fraction was

higher with the steroid I followed by steroids III, II and IV

respectively.

Fig.1 shows the reversion frequency of tester strain

TA102 by the oxathiolane steroids I-IV in the absence

(Fig.1a) and presence (Fig.lb) of Sg faction. The mutagenic

activity was significantly enhanced by metabolic activation.

Fig.2 shows the reversion of tester strain TA104 by the

steroids I-IV before (Fig.2a) and after (Fig.2b) metabolic

activation by Sg fraction. Steroid I exhibited a remarkably

high mutagenic activity ( 1250 his"*" revertants/ plate) at the

steroid dose of 1.0 ug/plate.

Discussion

Although several short-term mutagenicity testing

systems have been developed during past 15 years, the Ames

Salmonena system is recognised as a valid indicator of

mutagenicity fMaron and Ames, 1983). This test was first

validated in a study of 300 chemicals most of which were

known carcinogens (McCann et al. , 1975a; McCann and Ames,

1976, 1977). It was subsequently employed in other studies

by the Imperial chemical industries (Purchase et al., 1976)

and the International Agency for research in cancer (Bartsch

et a/., 1980). Nearly 90% of the carcinogenic compounds

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

5mg/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; Lev'in et

al., 1984, Flessel etal., 1987).

Our results indicate an increase in the number of

histidine"*" revertants with all the test steroids. Moreover,

all the test steroids exhibited a remarkable degree of

mutagenicity with TA104 and TA102 strains (Table- 1 and

Figs.1,2). These strains carry A:T base pairs at the

critical site of mutation (Levin et a/., 1982, Table-3). It

is also noteworthy that even in the absence of Sg microsomal

fraction the oxathiolane series of steroids (I-IV) responded

significantly, however, the addition of liver microsomal

fraction, further enhanced the mutagenic activity of these

steroids (Table-2 and Figs. IB and 2B). These results are in

agreement with earlier findings (Islam and Ahmad, 1991; Islam

et al. , 1991 ).

We found that among the closely related steroids, those

which contained substituted groups at position 3 were more

mutagenic than the one devoid of any group at that position

(Table-1). It was demonstrated earlier that the moiety after

getting attached to the steroidal nucleus became more

mutagenic compared with the parent steroid and individual

moiety (Islam and Ahmad, 1991). Now we have obtained further

enhancement of mutagenic activity by increasing the number of

•individual moiety attached to the steroidal nucleus

fTable-1). Thus, attachment of disulfonyl moiety to the 3rd

position in addition to the 6th position further enhanced the

mutagenic activity fTable-1; Islam et a/., 1991). Our group

has recently reported that dithiolanes and sulfonyl

derivatives of steroids displayed mutagenic activity in

proportion to the electro-negativety of the halogen atom at

the 3rd position of the steroidal nucleus (Islam et a/.,

1991). In addition, it was also found that as the number of

oxygen atoms increased in the substituted groups, the

mutagenic activity of the steroid was also enhanced. Our

results obtained with steroid V (Table-1) are consistent with

the previous findings (Islam et a/., 1991).

Table- 3 shows the relevant genetic and biochemical

markers of the Ames teser strains. In fact, the Salmonella

typhimuriurn strains per se lack error-prone repair (Walker,

1984) due to the absence of a functional umijD gene (Herrera

et a7.. 1988). However, this error-prone repair in Ames

tester strains is regained in the presence of plasmid pKMIOI

(McCann et al. , 1975b) which contains analogs of u/niX) and

umdD genes (Walker and Dobson, 1979; Walker, 1984).

The TA97a, TA98 and TA100 tester strains contain G-C

base pairs at the critical site for reversion (Isono and

Yourno, 1974; Barnes et al., 1982; Levin et al., 1982b).

With regard to the individual difference among the

aforementioned G-C specific mutants, the first two strains

are the frameshift type while TA100 is the base-pair

substitution mutant (Isono and Yourno, 1974; Barnes et a1.,

1982). The SaTmoneT la tester strains TA102 and TA104 detect

a variety of oxidants and other agents as mutagens (Levin et

a/., 1982b; DeFlora et al. , 1989). Our test steroids

exhibited a marked degree of mutagenicity with strains TA102

and TA104, inducing a transition mutation in the DNA of

tester strains (Table-1). With regard to the mechanism of

mutagenic action of the substituted group the test steroids,

present at position 3 is thought to be responsible for the

interaction with any reactive species in the system to form

oxygen radicals (Islam et al., 1991; Islam and Ahmad, 1991).

Water molecules may be one of the easily available reactive

species as it is the major component of most biosystems

(Simic et al., 1989).

Comparison of closely related structures permit

identification of a number of features essential for the

mutagenic activity. Among the test steroids, acetoxy

derivative of oxathiolane series (steroid I Table-3 of

Chapter II) was found to be relatively more mutagenic. This

may be due to the bulky nature of acetoxy group and its

cleavage from the steroidal nucleus in the form of acetoxyl

radical is more likely which can further interact with any

reactive species in the system to form 'OH radical and

hydrogen peroxide (Islam et a/., 1991; Islam and Ahmad 1991).

According to Tennant et a7 ( 1987) only the compounds

containing the electrophiles would be detected as mutagens by

the Salmonella test. This electron deficient region either

already exist in these steroids, or seems to have been

generated as a result of metabolic activation.

TA8LE-1 : Reversion of Aies tester strains in the presence of steroids without letabolic actiyatiw.

Steroid Ames Strains

Histidine revertants/olate'

Steroid (ug/plte)*'

Control 0.25 0.5 1.0 5.0 10.0 15.0 20.0

I TA 97a 178 + 16 151 + 15 168 + 16 188 + 16 305 + 15 258 + 15 262 + 10 105 + 14 TA 98 32 + 9 27 + 14 30 + 12 33 + 12 54 + 90 51 + 4 20 + 8 0 + 0 TA too 198 + 29 446 + 64 511 + 75 382 + 84 326 + 22 308 + 14 258 + 14 161 i 15 TA 102 269 + 21 601 +. 28 670 + 18 754 + 27 640 + 12 528 + 24 369 + 15 210 + 97 7A W4 42? t 75 943 t 57 mi t 28 t t20 87? t 8?} i m 45S t Its 38? i 2!

II TA 97a 15t + 18 120 + 12 92 + 13 118 + 13 146 + 18 133 • 15 53 + 21 0 + 0 TA 98 51 i 21 21 + 8 49 + 12 33 + 4 54 + 14 71 + 8 46 + 30 0 + 0 TA 100 128 + 14 138 + 9 178 1 29 352 + 15 258 t 8 243 + 13 158 + 12 93 + 12 TA 102 240 i 18 345 + 32 580 + 18 472 + 4 506 + 8 570 + 24 340 + 15 135 + 29 TA 104 442 i 21 1037 i 30 819 i 86 677 + 108 657 t 18 650 + 12 527 t 18 296 i 30

III TA 97a 180 + 18 186 i 14 212 + 14 236 + 66 196 + 44 42 + 12 20 + 21 0 1 0 TA 98 37 • 8 14 + 4 25 + 8 18 + 5 31 + 4 19 + 6 16 • 2 13 + 4 TA 100 212 + 14 405 + 18 428 + 18 418 + 18 355 + 8 330 + 14 170 + 30 58 + 9 TA 102 245 + 32 319 + 15 353 + 14 928 + 15 1238 + 21 583 + 21 423 t 45 195 + 10 TA 104 399 + 44 700 i 86 807 1 15 1277 1 115 1060 + 63 751 i 318 441 + 28 200 1 12

IV TA 97a 163 • 2 188 + 5 193 + 7 201 • 66 255 + 1 217 + 2 196 + 4 47 + 5 TA 98 39 + 2 11 • 3 18 + 2 31 + 2 55 + 3 47 + 4 10 • 3 0 i 0 TA 100 174 i 4 255 + 5 274 + 7 294 + 5 296 4 35 238 + 9 253 + 2 113 • 7 TA 102 252 + 14 360 + 14 505 + 16 530 + 23 510 + 8 394 + 5 310 + 8 208 + 3 TA 104 412 i 85 501 i 110 652 t 106 809 1 17 782 i 37 772 i 28 552 i 5 3 1 0 + 9

V TA 97a 175 + 2 228 + 8 282 i 6 296 + 3 280 + 6 272 + 5 254 • 3 98 + 12 TA 98 27 + 10 52 + 27 84 + 16 53 + 11 46 + 4 36 + 2 36 + 2 30 i 2 TA 100 220 + 20 282 i 33 284 + 29 300 + 7 226 + 14 306 • 4 386 + 8 191 + 13 TA 102 263 + 7 383 + 14 401 + 47 506 + 24 727 + 18 388 + 13 311 + 7 273 i 18 TA 104 382 + 7 1093 i 270 1162 i 81 1200 i 210 1098 + 154 992 + 53 763 + 71 410 + 29

VI TA 97a 183 t 6 349 t 10 443 + 7 414 t 8 200 + 5 193 • 6 20 + 2 0 + 0 TA 98 29 + 3 109 + 47 139 + 16 114 i 6 79 i 12 20 + 2 0 + 0 0 + 0 TA 100 212 + 14 225 + 5 231 + 4 262 + 8 322 + 19 362 + 12 292 + 8 72 + 2 TA 102 245 + 27 325 i 27 411 + 22 460 i 70 405 i 112 335 • 77 110 i 7 23 + 5 TA 104 410 + 18 490 + 72 590 + 30 443 1 180 410 + 90 309 + 9 120 + 4 0 + 0

VII TA 97a 314 + 28 307 5 328 7 340 + 10 350 + 3 339 + 20 347 7 319 + 6 TA 98 33 + 5 48 + 6 51 + 5 57 7 53 + 4 31 + 3 10 t 3 0 + 0 TA 100 263 + 15 265 + 10 268 4 260 6 251 + 9 256 + 43 164 + 5 64 + 6 TA 102 254 + 21 255 + 10 239 + 20 241 + 12 221 + U 231 + 28 282 + 9 230 + 9 TA 104 218 + 8 260 + 7 214 1 6 240 + 10 283 + 53 100 + 5 48 7 0 + 0

Ihs mbsr rmmnts 3civ3) itistiiiins rs}/srtM$ in tem of iim t 5.D.: 0*0, not 3 single colonji.

^Ttie molar concentration of the steroid for 1 uq per plate was equivalent to 3.7. 3.8. 4.0. 4.?. 3.4 » 5. nano nolar (nM) for steroid I to VI respectively.

TABLE-2; Reversion of Aies tester strains in the presence of steroid after wtabolic activation l>y Post-Bitochondrial supernatant (Sg fraction)

Steroid Ames Strains

Histidine^ revertants/olate^

Steroid (ug/olte)''

Control 0.25 0.5 1.0 5.0 10.0 15.0 20.0

I TA 97a 180 + 20 205 + 14 256 + 19 321 + 15 380 + 24 280 i 12 852 + 18 0 + 0 TA 98 59 + 9 135 + 18 180 + 29 187 1 25 104 + 15 89 + 12 50 + 13 11 + 11 TA 100 210 i 18 325 + 21 536 + 124 515 i 14 410 + 14 385 • 15 361 + 15 108 + 9 TA 102. 298 + 21 709 + 29 777 + 95 907 + 60 889 + 275 723 + 36 499 + 28 320 + 24 TA 104 460 i 43 1590 i ' 166 1670 i 82 1715 1 156 1671 + 540 950 i 30 835 + 129 390 i 27

II TA 97a 198 + 40 223 1 3 234 + 14 288 1 28 318 t 18 248 + 13 140 + 12 98 + 21 TA 98 55 + 15 85 + 9 129 + 13 137 + 16 145 + 15 87 + 18 68 + 14 35 + 12 TA 100 205 + 21 410 + 32 430 + 29 455 + 21 478 + 33 375 + 18 295 • 15 175 t 48 TA 102 275 + 14 403 + 42 501 + 72 735 i 36 775 i 24 475 + 24 398 + 24 295 + 21 TA 104 431 i 22 741 i 92 822 i 128 886 i 128 964 i 136 740 + 127 630 1 ^ 4 421 i 15

III TA 97a 205 i 13 267 + 15 357 + 34 492 + 9 437 + 21 320 + 18 53 + 6 0 + 0 TA 98 66 + 13 290 + 21 176 + 48 236 + 21 71 + 30 49 i 24 67 + 14 21 • 9 TA t o o 193 + 18 517 + 45 562 + 44 509 i 81 427 + 33 381 • 24 272 + 29 116 + 18 TA 102 283 + 6 370 + 12 315 + 45 1133 + 130 1421 + 36 759 + 60 527 + 18 215 + 21 TA 104 461 i 14 921 1 28 1035 i 124 1440 1 95 1276 i 120 955 + 21 502 t 9 240 i 21

IV TA 97a 200 + 15 240 + 14 250 + 21 262 + 9 350 + 21 279 + 12 210 + 8 79 + 29 TA 98 47 i 13 66 + 14 72 + 18 99 i 14 107 t 21 59 + 13 0 + 0 0 + 0 TA 100 190 + 14 315 + 13 416 + 24 452 + 22 420 + 54 300 1 14 235 1 20 120 + 15 TA 102 279 + 15 339 + 21 603 i 86 659 + 32 729 + 108 480 + 28 354 + 8 280 + 3 TA 104 420 + 30 445 + 29 720 + 133 895 + 58 902 + 130 545 • 166 520 • 30 345 + 5

^The number reoresents the histidine revertants in terns of iiean + S.O.. 0+0 not a single colony.

u 0) 4-> to w v> a> I

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<1> -r— >> £ 1 c <c 1 to O 1 0) Q. >,r- <c (C ««- C O) 0> <4- (ft 4-> (ft 1 O tt)'— D. o u a> 3 C C 1 0) 1 0.-0 <c i- c -r- CJ o c >» 1 c c c c 1. 1- OJ o +-> c ® O (J E (C 1 O O-r- o t- s- O (C 4-> O (C C-r- C 05 >- 1 -t- -r- "C T— O a-.- (ft 1- (D (ft 0) U4-> 1 +J 4J-r- V) JC O 1 (ft i- (ft •M «- 3 «C (ft 1 (C <C T— (/> t> CC o> -r— -1— C 0) o- r— j O 4-> cn o tf) o «•- <ft a> > (ft O > 05 0.0) 1 O 31- X O <D 1 <C JC C 0) 0.0) 1- 1 -1 E^ LU _J to od CD.t-> t- < (O l-M- D::- I

• • • 1 evj CO m f— 00 a> 1

C4-> O C <D a in

r — T —

CA (V 0) T5 I-

<0 r— o E >s CO

Fig.1: Dose-response effects of test steroid I-IV with Ames tester strains. Spontaneous revertants have been subtracted. A, TA102; B, TA104.

Steroid I (0)

Steroid II (•)

Steroid III (A)

Steroid IV (A)

5.0 10.0

STEROID Aig / P L A T E

5.0 10.0

STEROID / PLATE

Fig,2: Dose-response effects of test steroid I-IV with Ames tester strains In the presence of post-mitochondrlal supernatant (Sg fraction). Spontaneous revertants have been subtracted. A, TA102; B, TA104.

Steroid I (0)

Steroid II (») Steroid III (A.)

Steroid IV (4)

5.0 10.0

STEROID Mg I PLATE

0. cr u a I/) h-Z ^ cc Ul > UJ a:

5.0 10,0

STEROID M g ' P L A T E

CHAPTER IV

Role of Active Oxygen Species in Steroid

Induced Mutagenesis

Introduction

The active oxygen species are involved in the

pathogenicity of several degenerative diseases like cancer,

atherosclerosis, emphysema, aging, and can produce oxidative

damage to macromolecules'in somatic cells, such as, DNA

strand breakage and base alterations (Ames, 1983; Adelman et

a/., 1988; Marx, 1987; Imlay and Linn, 1988; neghini, 1988;

Pryor, 1988; ). In fact, oxygen radicals play a prominent

role during the stage of tumor promotion and also participate

in the oxidative metabolism of mutagens and in addition

possess genotoxic properties fCerutti, 1985; Ames,1986;

Pryor, 1986; Meneghini, 1988; Hochstein and Atallah, 1988).

These active oxygen species include, superoxide radical,

hydroxyl radical, singlet oxygen and peroxyl radical.

Generation of these species alongwith been

triggered by a variety of chemical and physical agents as

well as biological processes leading to mono-electronic

reductions of ground state O2 (De Flora et al. , 1989;

Halliwell and Gutteridge, 1990).

Singlet oxygen (^02), the most important in biological

system has no unpaired electrons and hence is not a radical.

Formation of occurs 1n vivo especially when several

biological pigments, such as chlorophylls, retinal, flavins

or porphyrins are illuminated in the presence of Og

(Halliwell and Gutteridge, 1984),

Superoxide radical (02"~) is formed when an electron is

accepted by the ground state oxygen molecule and is formed in

almost all living cells. ' give rise to a number of

other oxygen radical species like H2O2, ^Og and 'OH.

Several natural and synthetic chemical compounds e.g.,

flavonoids, xanthines, steroids and many other antibiotics

generate oxygen radicals by metal catalyzed and oxido-

reduction type of reactions (Brawn and Fridovich, 1981;

Rahman et al., 1989). Autoxidation of certain compounds in

vivo also give rise to oxy radicals (Simic, 1988). The

formation of carbon centered, peroxyl radicals and H2O2 have

been evidenced by the autoxidation of cholesterol (Smith,

1981; Sevilla et al., 1986).

More recently attention is shifting towards the

generation of oxy radicals in biosystems, their inhibition in

vivo, and prevention of various diseases in which oxy free

radicals play a significant role (Simic et al., 1989). These

radicals, may be generated directly or indirectly by

exogenous agents and processes in addition to their

generation by reduction of oxygen and/or peroxides in

biosystems (Simic et al., 1989).

Free radical quenchers have generally been used to *

assay the production of active oxygen species by several

agents and their involvement in various diseases fMcCord and

Fridovich, 1968), but it is also essential to assay the

generation of these radicals by some well established in

vitro free radical detection assays (Kraljic and El-Mohsini,

1978; Richmond et al. , 1981; Nakayama et al. , 1983). Oxygen

radicals have been reported to be mutagenic in S. typhimurium

test. These radicals are sensitive towards both TA102 (Levin

et al., 1982) and TA104 (DeFlora et aT., 1989). One of the

test steroids (steroid VIII) has already been reported to be

mutagenic in TA102 and this mutagenicity has been attributed

to the generation of oxygen radicals in the system (Islam and

Ahmad, 1991). Present chapter deals with the assay of active

oxygen species by the test steroids (I and VIII) and their

role in the mutagenicity in Ames tester strains.

Bacteria:

Ames tester strains used in this study and their associated genotypic markers are given in Chapter-II (Table-1 ).

Media:

The media for the Ames tester strains were essentially the same as described by Maron and Ames (1983) and have been given in Chapter-II,

Steroids:

Steroid I and VIII were used to assay their active oxygen species production.

Assay of superoxide radical Species (Og'"):

Op'" was assayed following the methoa of Nakayama et al. ( 1 983 ) with slight modifications. Different concentrations of steroid I (200, 400 and 600 nmoles) and steroid VIII (0.2 and 0.4 u mole) samples were added to 2.5 ml potassium phosphate buffer, pH 7.8 containing 50 ug NBT and 0.06^ triton X-100. Immediately after mixing the contents, absorbance was taken at 560 nm in Bausch and Lomb Spectronic-20 spectrophotometer. The reaction mixture was incubated in fluorescent light (500 lux) and absorbance was measured after different time intervals against a blank which does not contain steroid. In order to ascertain the generation of superoxide radical, SOD (50 ug/ml) was added to the buffer before addition of the steroid.

Assay of hydroxyl radical formation ('OH):

The generation of hydroxyl radicals by varying concentrations of steroids I and VIII was determined by incubating for 2 h at ambient temperature in light, a solution containing 20 mM salicylate, 0.1 mM EDTA, 0.5 mM CUCI2 and 150 mM potassium phosphate buffer pH 8.0 in a total volume of 2.0 ml. Salicylate was converted to hydroxylated products which were extracted and determined colorimetrical 1 y by Beckman DU-40 spectrophotometer (Richmond et al., 1981). The production of "OH was confirmed by adding scavengers: catalase (25 ug/ml), SOD (50 ug/ml), sodium benzoate, sodium formate, potassium iodide, mannitol and thiourea (each 4 mM final concentration separately) before incubation with salicylate mix.

Assay of hydrogen peroxide production (H2O2):

The generation of HgOo was estimated according to the method of Nakayama et a7 (f983). 2.0 ml sample containing different amounts of steroid dissolved in DMSO was mixed with equal volume of 50mM sodium phosphate buffer, pH 7.2 and incubated at 37' C for 90 min. 2.0 ml aliquot of this mixture was added to 2 ml of titanium sulphate (TiSO^) solution. The blank was prepared by employing HpSO^ in place of TiSO^. The reaction mixture was measured at 410 nm. In order to confirm that the color change was due to generation of H2O2, a separate reaction tube was run containi'ng 0.4 ml of T mg/ml catalase solution.

Detection of singlet oxygen (^Og):

Quantitation of singlet oxygen at increasing concentrations of steroids I and VIII was done in aqueous solution by the method of Kralijic and El-Mohsini (1978). This was based on a decrease in the absorbance of N,N-dimethyl-p-nitrosoani1ine at 440nm using histidine as a selective acceptor of singlet oxygen. The reaction mixture contained lOOmM potassium phosphate buffer, pH 7.8, 3 mg/ml (14.3 uM) histidine (His) and 30 uM pRNO. The mixture was irradiated in sunlight for various time periods. The extent of bleaching of pRNO was recorded spectrophotometrical ly. Riboflavin (30uM), a known singlet oxygen producer was used as a positive control fJoshi, 1985). Quenching studies with sodium azide were performed in lOOmM phosphate buffer. pH 7.0 according to Joshi (1985).

Mutagenicity testing of test steroid in the presence of oxygen radical scavengers:

To 0.3 ml phosphate buffer, pH 7.4, 0.2 ml bacterial culture (1-2 x 10^ viable counts ml~''), and different quantities of the steroid I were added. To this mixture oxygen radical scavengers namely catalase (32 ug/plate), SOD (20 ug/plate), sodium benzoate (1.04 u moles/plate), sodium formate (1.04 u moles/plate), sodium azide (0.026 u moles/plate), mannitol (1.04 u moles/plate), potassium iodide (1.04 u moles/plate), and thiourea (1.04 u moles/plate), were added separately. A positive control without scavenger was also run. These reaction mixtures were preincubated for 20 min at 37* 0 and then plated with 3.0 ml soft agar on to minimal media plates. The plates were incubated at 37* 0 for 48h. To test the effect of scavengers after the metabolic activation of steroid, 20 ul Sg mix per plate was added. Again the assay was conducted in triplicate and duolicate plates were used for each scavenger at each dose. The negative controls without steroid but containing the given amount of scavengers were also simultaneously run.

Results

Generation of Superoxide anion:

Table 1 and 2 present the data on the production of

formazan from NBT by the test steroids I and VIII. An

increasing amount of formazcin formation was observed with

increasing time of incubation vis-a-vis incre'asing

concentrations of steroids. 75.4 n moles of formazan was

produced by 600 nmoles of steroid I after 75 min of

incubation in light (Table-1). The formation of superoxide

radical was time and concentration dependent (Figs 1 and 2).

The hyperbolic nature of curves in these figures indicate

that the 0^'' producing reaction is of first order upto 60

min incubation at all the concentrations. However, the

reaction attains a zero order kinetics after 75 min in case

of both the steroids.

Formation of 02'~ was also observed during incubation

of steroid in dark, but the absorbance was reduced by 50?«$ to

that observed in light (Figs, 1 and 2). The increasing

production of formazan indicates the increase in Og'"

generation by the steroid. Addition of SOD into the reaction

mixture inhibited the formazan production.

Hydroxy! radical formation:

Tables 3 and 5 summarise the data on the concentration

of hydroxylated products formed by the steroid I and VIII in

the presence of Cu(II). This experiment was based upon the

conversion of salicylate into its hydroxyl derivative. The

data again indicates the increasing amount of hydroxyl

radical formation with the increasing concentration of the

steroids. 200 nmoles of steroid I generated 118.5 nmoles o-f

•OH (Table-3) but the steroid VIII at the same concentration

produced 72.3 nmoles of -OH (Tab1e-5).

Hydroxy! radical formation was also inhibited by the

scavengers of 02'", H2O2 and 'OH radicals (Tables 4 and 6>.

Catalase and SOD inhibited about 98% and 995 of 'OH

respectively in case of steroid I and 88% and 755

respectively in case of steroid VIII. Thiourea, benzoate and

iodide exhibited about 50% inhibition in *0H production by

both the test steroids (Tables 4 and 6),

Production of Hydrogen Peroxide:

Figure 3 shows the pattern of H2O2 produced by the

varying amount of steroid I. There was a linear relationship

between the generation of H2O2 vs dose of steroid in the

reaction mixture with the release of approximately 10 moles

of H2O2 generated per mole of steroid I.

Table 7 shows the production of H2O2 by the steroid

VIII. The H2O2 generated by steroid VIII was slightly lesser

than that of steroid I. The catalase at a concentration of

100 ug/ml completely inhibited the generation of H2O2 upto a

dose of 0.4 u moles of both steroids (Fig. 3 and Table-7).

Production of Singlet Oxygen:

The ability of steroids I and VIII to form O2 was

determined by monitoring the bleaching of p-

nitrosodimethy1 ani1ine at 440 nm (Figs. 4 and 5). The

steroids at the concentration of 100 uM showed a considerable

amount of bleaching which enhanced significantly at higher

concentration of steroids. The produced by steroid I

was double than that produced by steroid VIII at 100 uM

steroid concentration (Figs. 4 and 6). The extent of

bleaching was also a function of the energy of exposed

sunlight. Sodium azide, a well known singlet oxygen quencher

resulted in approximately inhibition of RNO bleaching

under our experimental conditions (Figs. 4 and 5). The

figures also show the RNO bleaching by riboflavin, run as

positive control,

Mutagenicity of test steroids in the presence of free radical

scavengers:

The mutagenic activity of steroid I in the absence of

scavengers was dose dependent and the his"^ revertants

increased after metabolic activation with both the tester

strains (Tables 8 and 9). The free radical scavengers were

neither detected to be mutagenic nor toxic with TA102 and

TA104 under our experimental conditions (results not shown).

All the scavengers of active oxygen species had marked

effect on the mutagenic potential of steroid I with the

SalmoneT Ta typhimurium TA102 and TA104 strains both in the

presence and absence of Sg fraction (Tables 8 and 9). The

scavenging effect was apparently more pronounced in the

absence of Sg fraction. Sodium azide (0.026 umoles/plate) »

had almost 45-60% inhibitory effect on the mutagenicity of

steroid I against TA102, without metabolic activation but the

inhibitory effect of azide was around 25-45% after metabolic

activation fTable-8). This indicates that the is also

playing a role in the steroid I induced mutagenesis.

Moreover, the percent relative activity of both the tester

strains was inhibited to less extent by the oxygen radical

scavengers in the absence of Sg fraction as compared to that

in presence of Sg fraction (Tables 8 and 9).

Discussion

Almost all compounds that produce active oxygen species

have been reported to be mutagenic in SaTmonella typhimurium

strains TA102 (Levin et al., 1982a) and in TA104 (De Flora et

al., 1989). The mutagenic response could be ascribed both to

the superoxide radical and to hydrogen peroxide (De Flora et

al., 1989). However, several authors have attributed the

true damaging species to be a highly reactive hydroxy!

radical derived from 02*" (Fridovich, 1978; Halliwell et al.,

1980; Halliwell, 1981; Nassicalo et al. , 1989). From our

data it is now clear that the test steroid I (3^ -Acetoxy-5

«C-cholestano [6 ©c ,6-d], 1 '-3'-oxathi ol ane -2'-thione and

steroid VIII (3P -Acetoxy-5 oQ -cholestano r5,6-b]-N-

phthalimidoaziridine) can also produce superoxide radical in

the absence of other reducing agents. Such observations have

been reported with several mutagenic compounds (Wong et al. ,

1984a, 1984b, Chrfsey et al., 1988). Moreover, the

superoxide and H2O2 can also generate the hydroxy! radical by

Haber-Weiss (1) and Fenton (2) reactions respectively.

Og'" + H2O2 — > -OH + OH" + O2 (1)

H2O2 + FeflD/CufI) — > -OH + OH" + FefIII)/Cu(II) (2)

The superoxide anion may also lead to the formation of

a peroxide ion (02^') which on protonation at neutral pH

would give rise to H2O2. Alternatively, in aqueous solution,

the superoxide anion undergoes dismutation to form H2O2 and

©2 fHalliwell and Gutteridge, 1984; Marx, 1987).

202"" ^ "2^2 ^2 ^^^

Our results summarized in tables 1,2,3,4 and 7 as well

as in Figure 3 clearly suggest that both the oxathiolane and

aziridinyl steroids give rise to superoxide anion (Og'")

which then form 'OH and H2O2. These results are in agreement

with the mechanism presented in reactions 1,2, 3 and the

findings of Islam and Ahmad (1991). A possible source of -OH

and 02*" as well as H2O2 by steroid I is shown in (Fig. 6).

The steroid VIII has been shown to generate the active oxygen

species by a similar mechanism (Islam and Ahmad 1991). Other

steroidal derivatives may also generate oxygen radicals in

almost similar manner (Islam et al., 1991). The homolytic

fission of the acetoxy group from the steroidal moiety might

further give rise to H2O2, 'OH as well as carbon centered

radicals (Islam and Ahmad, 1991). It is suggested that the

generation of from acetoxy moiety attached to

3rd position of steroidal ring (Fig. 6 and Islam and Ahmad,

1991) increases the mutagenic potential of the oxathiolane

and aziridine steroids (Islam and Ahmad, 1991). Islam and

Ahmad (1991) have also suggested that the attachment of

individual moieties or groups with steroidal nucleus make

them remarkably mutagenic with the probable involvement of

various oxygen radical species in the biological system. The

formation of carbon centered and peroxyl radicals have been

evidenced by the autoxidation of cholesterol (Sevilla et a/.,

1986). H2O2 has also been reported to be one of the products

of cholesterol autoxidation (Smith, 1981). The possibility

of autoxidation of steroidal moiety and thus giving rise to

active oxygen species and H2O2 also can not be ruled out.

Moreover, the active oxygen species in turn, have also been

shown to oxidize the cholesterol (Gumulka et a/., 1982).

Detailed studies have shown that singlet oxygen (^©2) was the

most active agent taking part in cholesterol oxidation (Araki

et al. , 1984). Such type of cholesterol oxidation process

yielded biologically active oxysterols (Smith and Johnson,

1989). These oxysterols have been reported to be uniformly

toxic in a wide variety of in vitro bioassays (Smith, 1981)

and were also toxic to Salmonella typhimurium (Ansari et a/.,

1982; Smith and Johnson, 1989). The test steroids under our

experimental conditions were also found to generate singlet

oxygen (^02) (Figs. 4 and 5). Singlet oxygen may arise due

to variation of Haber-Weiss reaction as indicated in equation

(4) (Kellog and Fridovich, 1975; Badway and Karnovsky, 1980).

transition metal O G ' + H 2 O 2 > O 2 + ' O H + 0H~ ( 4 )

The interconversion of reactive oxygen species also

occurs in the active oxygen generating systems (Joshi, 1985).

Singlet oxygen (^02) has been reported to be more potent in

DNA, protein and cell membrane damage fPathak and Joshi,

1983; 1984). Benzo[a]pyrene, a well known mutagen and

carcinogen was shown to produce singlet oxygen and its

mutagenicity may be due to this fact (Wei et al., 1989).

Our results show a significant reduction in the

generation of oxygen radical species by the test steroids on

treatment with oxygen radical scavengers. This reduction in

radical generation strongly suggests that the test

oxathiolane and aziridinyl steroids generate active oxygen

radicals (Richmond, 1981; Nakayama et al., 1983; Marx, 1987,

Islam and Ahmad, 1991).

A significant decline in the number of his" revertants

of Ames tester strains, TA102 and TA104 in the presence of

oxygen radical scavengers (Tables 8 and 9) further confirms

the involvement of active oxygen species in the mutagenicity

of oxathiolane steroids. These results are consistent with

those reported earlier for other steroids especially the

aziridinyl derivative (steroid VIII) with TA102 (Islam et

al., 1991; Islam and Ahmad, 1991). The results presented in

table 8, when compared with the earlier reports from our

group (Islam and Ahmad, 1991) show that the relative activity

was higher in case of steroid I (oxathiolane derivative) than

that in steroid VIII (aziridinyl directive). Moreover, the

mutagenic potential of oxathiolane steroid with TA102 (Table-

8) was significantly reduced in the presence of sodium azide,

a singlet oxygen quencher (Foote et al., 1972). These results

are in conformity with the findings of Wei et a7. (1989).

In conclusion our findings strongly suggest the active

involvement of singlet oxygen in the mutagenic behaviour of

certain steroids in addition to the role played by other

oxygen radical species as is obvious by this work and our

earlier reports (Islam et al., 1991; Islam and Ahmad, 1991).

TABLE-1: Generation of superoxide anion (02* ) by steroid I in terms of the conversion of nitroblue tetrazolium to a formazan at different incubation times and varying concentrations of steroid I

Incubation Time Steroid Condition Formazan formation fmin) fn moles) fn moles)

15 200 light 18.4 200 light + SOD 0.0 400 light 41 .6 400 light + SOD 0.0 600 light 47.4

30 200 light 31 .0 200 light + SOD 0.0 400 light 52.0 400 light + SOD 0.0 600 light 56.8

45 200 light 38.8 200 light + SOD 0.0 400 light 55.2 400 light + SOD 0.0 600 light 60.2

60 200 light 44.4 200 light + SOD 0.0 400 light 60.2 400 light + SOD 0.0 600 1 ight 75.4

75 200 light 50.4 200 light + SOD 0.0 400 light 65.6 400 light + SOD 0.0 600 light 75.4

TABLE-2: Generation of superoxide anion (02'"^ terms of conversion of nitroblue tetrazolium to a formazan at different incubation timings and varying concentrations of steroid VIII.

Incubation Time fmin)

Steroid fn moles)

Condition Formazan Formation fn moles)

15 200 light 8.2

400 light 17.2

400 light + SOD 0.0

30 200 light 8.6

400 light 19.4

400 light + SOD 0.0

45 200 light 27.4

400 light 45.8

400 light + SOD' 0.0

60 200 light 41 .6

400 light 56.8

400 light + SOD 0.0

75 200 light 52.0

400 light 62.0

400 light 0.0

TABLE-3: Production of the hydroxy! radicals with varying concentrtions of the steroid I in light.

Steroid fn moles)

Hydroxylated product (n moles)

50 63. ,7 100 82. ,8 150 100. ,9 200 108. .6 300 118, .5'

TABLE-4: Effect of quenchers on the formation of hydroxy! radical by the steroid I.

Quenchers Hydroxylated Product formed (n moles)

Inhibition

Control 103. 70 0. 0 Control + SOD (16 ug/ml) 2. 15 97. 7 Control + Catalase (25 ug/ml) 0. 92 99. 1 Control + Mannitol (4 mM) 52. 60 49. 2 Control + Sodium formate (4 mM) 72. 90 29. 7 Control + Sodium benzoate (4 mM) 60. 60 41 . 5 Control + Thiourea (4 mM) 57. 50 44. 5 Control + Potassium Iodide (4 mM) 58. 80 43. 3

Control = Salicylate + Oxathiolane Steroid (200 n moles) + CuClp ^200 uM)^ Concentration of scavengers shown are the final reaction concentration.

TABLE-5: Production of the hydroxy! radicals with varying concentrations of the steroid VIII in light.

Steroid (n moles)

Hydroxy!ated fn moles)

Product

50 100 150 200

51 .4 60.3 64.3 72.3

TABLE-6: Effect of quenchers on the production of hydroxy! radicals by steroid VIII.

Quenchers Hydroxy!ated Product formed

(n moles)

Inhibition

Control 72. 30 0 .0 Control + SOD (16 ug/ml) 17. 80 75 .30 Control + Catalase (25 ug/ml) 8. 90 87 .69 Control + Mannitol (4 mM) 38. 15 47 .20 Control + Sodium benzoate (4 mM) 41 . 50 42 .60 Control + Thiourea (4 mM) 35. 40 51 .00

Contro! = Salicylate + Steroid VIII (200 n moles) + CuCl (200 uM) concentration of scavengers shown the final reaction concentrations.

TABLE-7: HgOg Production by steroid VIII.

Steroid fu moles)

H G O G

fu moles) in presence of catalase

0 . 0 5 0 . 1 9 5 0 . 0

0 . 1 0 0 . 7 4 2 0 . 0

0 . 1 5 1 . 2 0 0 0 . 0

0 . 2 0 1 . 5 3 0 0 . 0

0 . 2 5 1 . 8 9 0 0 . 0

0 . 3 0 2 . 3 5 0 0 . 0

0 . 3 5 3 . 0 0 0 0 . 0

0 . 4 0 4 . 2 0 0 0 . 0

TABLE-8: Effect of scavengers of reactive oxygen species on the mutagenic activity of the steroid I in S. typhlmurlum TA102 strain.

Scavengers Amount of Histldine"'' revertants/ Relative activity^ Steroid plate + SD (%) (ug/plate)

No Scavenger o.o'' 303 + 16 293 + 15 100 100 0.5 610 + 89 692 ± 101 100 100 1.0 855 ± 31 980 + 29 100 100

Superoxide 0.0^ 271 + 51 315 ± 9 89.44 > 100 dismutase 0.5 392 + 45 468 + 43 39.41 38.34 (20 ug/plate) 1.0 422 + 42 480 + 15 27.35 24.01

Catalase 0.0^ 294 ± 37 293 ± 64 97.02 100 (32 ug/plate) 0.5 395 + 75 402 + 83 32.89 27.32

1.0 404 + 15 412 ± 49 19.92 17.32

Mannitol 0.0^ 287 ± 35 314 ± 28 94.72 > 100 (1.04 u moles/ 0.5 382 + 23 420 + 53 30.94 26.56 plate) 1.0 419 + 20 437 ± 78 24.09 17.90

Formate 0.0^ 268 + 54 320 + 43 88.45 > 100 (1.04 u moles/ 0.5 355 +16 4 4 0 + 5 8 28.33 30,07 plate) 1.0 375 + 38 466 + 78 19.38 21.25

Benzoate 0.0^ 235 + 31 293 + 20 77.56 100 (1.04 U moles/ 0.5 211 + 31 373 + 43 28.33 20.05 plate) 1.0 362 + 17 400 ± 11 23.00 15.55

Thiourea 0.0^ 283 + 14 325 + 15 93.39 > 100 (1.04 u moles/ 0.5 382 + 66 445 + 49 32.24 30.07 plate) 1.0 402 i 30 493 ± 37 21.55 24.45

Iodide 0.0^ 288 + 41 283 ± 35 95.04 96.58 (1.04 u moles/ 0.5 456 +119 480 + 133 54.72 40.37 plate) 1.0 510 + 48 460 + 52 40.21 25.76

Azide O.O'' 270 + 34 255 + 16 89.10 87.03 (0.026 u mole/ 0.5 340 + 50 510 ± 75 22.80 63.90 plate) 1.0 360 + 39 546 + 41 16.30 42.35

T - C ( + ) ® % relative activity x 100

T - C ( - )

where T is steroid treated and C is solvent treated counts of TA102 cells. (+) indicates, 'in presence of scavenger' and (-) indicates, '1n absence of scavenger'.

The Zero dose indicates the value of solvent used as control.

TABLE-9: Effect of scavengers of reactive oxygen species on the mutagenic activity of the steroid I in S. typhimurium TA104 strain-

Scavengers Amount of Histidine"'' revertants/ Relative activity^ Steroid plate ± SD f%) (ug/plate)

No Scavenger 0.0^ 495 + 70 513 + 54 100 100 0.5 875 ± 85 1065 + 212 100 100 1.0 1190 ± 44 1450 + 65 100 100

Superoxide 0.0^ 416 + 39 493 ± 64 84.04 96.10 dismutase 0.5 632 +103 770 + 52 56,84 36.83 (20 ug/plate) 1.0 820 + 35 808 ± 24 58.12 33.61

Catalase 0.0^ 477 ± 83 495 ± 11 96.36 96.49 (32 ug/platej 0.5 660 +-13 603 ± 58 48.15 34.05

1.0 757 ±124 782 + 14 40.08 30.62

Mannltol 0.0^ 490 + 29 610 ± 14 98.98 > 100 (1.04 u moles/ 0.5 692 + 93 716 + 14 53.15 19.20 plate) 1.0 847 ± 37 844 + 37 51.36 24.97

Formate o.o' 434 ± 23 503 + 15 87.67 98.05 (1.04 u moles/ 0.5 585 ± 60 686 + 95 39.73 33.15 plate) 1.0 718 + 88 770 + 22 41.15 29.16

Benzoate 0.0^ 495 + 21 582 ± 40 100.00 > 100 (1.04 u moles/ 0.5 576 ±110 716 ± 108 21.31 24.27 plate) 1.0 706 ± 11 836 ± 64 30.35 27.10

Thiourea o.o'' 443 ± 17 530 ± 103 89.49 > 100 (1.04 u moles/ 0.5 532 ± 55 604 ± 42 23.42 13.40 plate) 1.0 620 ± 49 674 ± 84 25.46 12.48

Iodide 0.0^ 462 ±141 499 ± 21 93.33 97.27 (1.04 u moles/ 0.5 590 ±110 680 ± 112 33.68 32.78 plate) 1.0 730 ±128 808 ± 18 38.56 32.97

T - C ( + ) ^ % relative activity x 100

T - C ( - )

where T is steroid treated and C is solvent treated counts of TA102 cells. (+) indicates, 'in presence of scavenger' and (-) indicates, 'in absence of scavenger'.

The Zero dose Indicates the value of solvent used as control.

Fig.l: Generation of superoxide anion (Og"") by steroid I In light. Effect of incubation in dark and in presence of superoxide disrautase (SOD).

Concentrations of steroid I used were:

200 n moles CX); 400 n moles (•); 600 n moles (0);

400 n moles in dark ("A); 400 n moles in presence of

SOD (a") and solvent as control (*).

E c o VD to

UJ U z < (Q cr S CD <

30.0 45.0 60.0 75.0

TIME ( min)

Fig.2: Generation of superoxide anion (©2" ) by steroid VIII in light. Effect of incubation in dark and in presence of SOD.

Concentrations of steroid VIII used were: 200

n moles (0); 400 n moles (•); 400 n moles in dark

(Ai; 400 n moles in presence of SOD (1 ) and solvent

as control (*).

V A AZAO

E c o vo IT)

UJ U z < 03 CC o U) a> <

TIME ( mm )

Fig.3: Production of H2O2 as a function of steroid I concentration.

Steroid I : (t)

Steroid I + Catalase (100 ug/ml) : (i)

o £ A <S( 0 fNI 1

Steroid ( XL mole )

Fig.4: Formation of singlet oxygen at various concentrations of steroid I.

RNO + Histidine (His)

RNO + His + Solvent

RNO + His + 100 uM Steroid I

RNO + His + 100 uM Steroid I + 50 mM

Sodium azide

RNO + His + 200 uM Steroid I

RNO + His + 200 uM Steroid I + 50 mM

Sodium azide

RNO + His + 30 uM Riboflavin (positive control)

e c o >t

UJ u z < OQ a. o \r> m <

J / Cm 2

Fig.5: Formation of singlet oxygen at various concentrations of steroid VIII.

RNO + Histidine (His) : (0) RNO + His + Solvent : ( 1 ) RNO + His + 100 uM Steroid VIII : (A) RNO + His + 100 uM Steroid VIII + 50 uM : Sodium azide : (f) RNO + His + 30 uM Riboflavin : («) (positive control)

UJ 2 < CO cc o 1/1 CO <

J / Cm^

B 2 o V-a VI o 5

rt n o

+ X 2 0 5 1

0 pn 1

I I 0 1

J3 n r c I o —*

O =">-1 I o m

o—v/1 c I 3 u

•f •o I

I i_> (n X

X u 0=U

i 2 m I u

O-u o-u

I O - u

llWHlO ^ y.

U ~ I/)

•P •i-t o •H C CU 00 rt 4-> 13 E

X) <U o ^ c •rt v-* XI

o cu 4J C/}

LW o s cn •H c cd x: o cu e tu r-t • r - l cn CO O a

ao •rt ClH

I/) £

CHAPTER V

Steroids as the Inducers

of SOS Response in E.coli

and A phage systems

Introduction

DNA damage or modifications by physical and chemical

agents pose a serious challenge to a cell, because of the

possibilities that it may give rise to mutations, aging or

lead to cell death (Waring, 1981). Moreover, the loss of

viability in E. coli and bacterio-phages due to DNA damage

have been occasionally attributed to the oxygen radical

species generated in the bio-system (Simic et al. , 1989) or

by the reduction of some exogenous chemicals in the bio-

system (Loeb et al. , 1988; Imlay and Linn, 1988). The

capacity of a system to repair such damage is basic to the

ability of an organism to withstand the biological stress.

Correctly repaired genetic damage has little effect on the

biological functions of a system (Walker, 1985).

At least in E. coli, the DNA lesions are of two types

on the basis of the processing, necessary to cause mutations.

The first and simple class, exemplified by base

modifications, appears to give rise to mutations by simply

mispairing during normal DNA replication (Drake and Baltz,

1976; Dodson et al., 1982; Loeb & Kunkel , 1982; Walker,

1984). In contrast the second and much larger class

exemplified by lesions introduced by physical and chemical

agents requires the participation of a special inducible

system termed as SOS response (Radman, 1975; Witkin, 1976;

Walker, 1984; Walker, 1985; Dale et a7., 1992). This system

includes the induction of a transitory mutagenic DNA repair-

system, the activation of inducible prophage and of several

other functions involved in cell division and DNA metabolism

(Radman, 1974; Witkin, 1976; Strauss, 1989). The SOS network

controls the expression of genes whose products are known to

play roles in excision repair, daughter strand gap repair and

SOS processing fWalker, 1985).

It has been clearly shown that a number of genes coding

for DNA repair proteins can be induced by DNA damage. Thus

several methods have been developed to estimate the extent of

DNA damage for determining the mutagenic potential of a

chemical, based on the expression of SOS genes in treated E.

coli cells (Moreau et a7., 1976; Weymouth and Loeb, 1978;

Bagg et al. , 1981; Kenyon and Walker, 1981; Quillardet et

al., 1 982 ; Kunkel et a 7, 1 983 ). In addition, phage

inactivation offers a better parameter to study the

involvement of reactive oxygen species in genotoxicity

(Lusthof et al., 1988).

The work presented in this chapter, was therefore,

planned to estimate the extent of genetic damage brought

about by steroid treatment and to understand their mutagenic

behaviour in terms of SOS function in E. coli and phage . We

have used lambda phage and the SOS defective strains of E.

CO77 which might serve as convenient models for this purpose,

because, a slight change in the environment could be

reflected in their plaque and colony forming capacities.

Several workers have used bacterial transformation with

plasmid and phage DNA as a tool for studying the DNA

modification and DNA-drug interaction (Bucala et a1., 1984,

1986; Palu et al., 1987). We have, therefore, exploited the

transformation assay to understand the mechanism of steroid

induced alterations and mutations in DNA under in vivo

conditions.

Bacteria, phages, lysogen, media, buffers and test steroids used in this study are given in Chapter-II. The relevant genetic markers associated with each E. coJi and

Astrains are also given in Chapter II (Tables 1 and 2).

Survival of E. coli strains was evaluated in the presence of steroid I, II, III and IV while the phage inactivation and weigle mutagenesis was studied with steroid I and VIII. Induction of prophage was studied employing steroid I.

Treatment of E. coli K-12 with test steroids:

The E. coli K-12 mutants as well as the isogenic wild type strains were harvested by centrifugation from exponentially growing cultures f1-2 x 10® viable counts/ml). The pellets so obtained were suspended directly in 0.01 M MgSO^ and treated for different time period with 0.5 ug steroid/ml culture. The treated cells were then plated to assay colony forming ability. Plates were incubated over night at 37"C. Solvent as control was run simultaneously,

A -prophage induction in the presence of steroid I:

AgJ857/AB1 157' (1-3 xlO^ viable counts ml"') was centrifuged washed and resuspended in

Exponentially grown lysogen » counts ml" ) was centrifuged

0.01M MgSO^ solution. The resuspended culture was treated with the test steroid at 32^C for 3 h. The cells were washed and resuspended in nutrient broth with and without chloramphenicol (100 ug ml" ) and incubated for next 3 h at 32'^C. Aliquots were taken out at regular intervals, suitably diluted and plated with excess of C600. The plaques were scored after 0/N incubation at 42®C. Simultaneously, the control was also run taking the solvent only.

Effect of steroid treatment on the transformation, efficiency of E. coli cells:

Preparation of competent cells: Exponentially grown (1-3 x 10^ viable counts/ml) of E. coli AB1157 (w.t.) and AB2463 (recAl cells were harvested and washed with MgSO^ solution. The chilled cells were made competent according to Mendal and Higa (1970) by treating with 0.05 M Tris-CaCl2 buffer on ice for 20 min. The competent cells were stored on ice for 4 h and used for transformation with treated pBR322 DNA.

E. coli cells which have been treated with steroid and made competent, were stored on ice upto 1 h only before transformation step to minimize the loss of SOS response if any, brought about by the steroid treatment.

Treatment of plasmid pBR322 with steroid I and VIII: About 2 ug of plasmid DNA was separately treated with steroid I and VIII (100 uM) as well as solvent (as control) for 4 h in light in presence of 100 uM Cu(II) in a total volume of 40 ml rxn mix. The treated pBR322 DNA- steroid mix was dialysed against 100 volumes of 0.01 M TN buffer (pH 7.5) containing 10-15^ formamide for 4 h at 20®C.

Treatment of E. coli cells with steroid I and VIII: The transformation experiment involving the cell treatment was done as follows: Exponentially grown cells of E. coli were harvested and resuspended into 0,01 M MgSO^. These cells were treated with 1.0 ug/ml of steroid I and VIII as well as solvent (as control) in presence of 100 uM Cu(II) and were incubated for 4 h in fluorescent light at room temperature.

For comparison the experiment involving the Benzo[a]pyrene (25 uM) was simultaneously carried out under similar conditions.

Transformation experiment: To 200 ml of competent E. coli cells about 2-3 ug of treated and untreated plasmid DNA was added separately in chilled microfuge tubes. These tubes were incubated for 60 min on ice and transferred to a water bath preheated to for 2 min. The tuiDes were again incubated on ice for 20 min. Then 1 ml of nutrient broth was

added to each tube and incubated at 37* 0 for 2 h to permit phenotypic expression of olasmid encoded antibiotic resistance genes. 0.1 ml aliquotes fsuitably diluted) were plated on nutrient agar ^suoolemented with 50 ug ml" amp, 10 ug ml" tet and 50 ug ml" amp plus 10 ug ml"' tet. To obtain the viable counts the cells were also plated on the plain nutrient agar. The plates were incubated 0/N at 37* 0. the colonies on each plate were scored. To some tubes universal random primer (10 ul ) was added alongwith untreated plasmid DNA. The transformation and mutation frequency at ampicillin and tetracycline loci of the transforming plasmid were calculated.

Extracellular steroid treatment of phage X :

Phage A virions (10^ PFU/ml) were separately treated with the test steroids I and VIII (50 uM) in the presence of 100 uM Cu(II) at room temperature in fluorescent light (500 lux). Aliquots of 0.1 ml were withdrawn at regular intervals, suitably diluted in the dilution buffer and allowed to adsorb on the radiation sensitive and wild type host strains at 37^C for 20 min. The infective centers were plated on nutrient agar by the double layer method. Phages were counted after overnight incubation of plates at 31^. Solvent treated controls were also run simultaneously.

Intracellular steroid treatment:

The radiation sensitive and wild-type E. col i cells were harvested from exponential cultures and resuspended in dilution buffer. Infective centers were prepared by adsorbing phage A at high multiplicity of infection (5:1). Unadsorbed phages were removed by centrifugation and washing twice with the dilutjon buffer. The complexes were resuspended in 0.01M MgT^O^ DH 8.0 and treated with the 50 uM of steroid I and steroid vIII separately in presence of 100 uM Cu(II) at room temperature in fluorescent light (500 lux). Aliquots were withdrawn at regular intervals suitably diluted and plated with 0.3 ml of 0/N grown wild type E. co77 cells and top agar by the overlay technique. Plaque forming units (P.P.U.S.) were counted after overnight incubation of plates at 37°C.

In some experiments the desired concentrations of active oxygen radical scavengers were also supplemented during the steroid treatment. Solvent treated controls were also run simultaneously.

steroid I and VIII induced c mutation of "X d*":

Bacteriophage Ac"*" was treated with steroid I and VIII (1.0 mg/ml) as free particles as well as intracel lularly in the wild type and recA strains of E. coli K12. The clear plaques (c mutants) were scored according to the method of Defais et al (1971). In order to study weigle mutagenesis, exponentially grown wild type E. coli (AB1157) cells and phage Xc"*" particles were separately treated with steroid I and VIII. The infective centers were then prepared at low multiplicity of infection.

Results

Survival of E. coli K-12 treated with test steroids:

The survival patterns of wild type and mutant E. coli

strains in presence of steroids I-IV are shows in Figs.1-4

respectively. The mutant strains of E. coli like recA, recB

recF, lexA and rer were more sensitive towards steroids I-IV

as compared to w.t. strain. The uvrA mutant also showed a

slight decline in its survival than that of the wild type

strain. LexA mutant was found to be highly sensitive and its

survival was reduced by 85* after 2 h of steroid I-IV

treatment. After 6 h treatment survival of lexA strain was

found to be 2% only. In case of recA mutants the loss in

survival was around 40-60* by the steroid treatment. The

survival of recF and recA was almost same (35%) after 6 h of

steroid I-IV treatment (Figs.1-4).

X -prophage induction in the presence of steroid I:

Table-1 shows the induction of A -prophage as a result

of steroid treatment of lysogen during post treatment in

liquid holding recovery in the nutrient broth. A fraction of

the lysogenic population exhibited induction of the lytic

cycle during liquid holding at 32^C. The prophage induction

was dose dependent. At 2 ug/ml steroid treatment the fold

induction was 5.4 after 3 h incubation, where as it was 3.3

fold at 1 ug/ml steroid I treatment. There was insignificant

increase in the prophage induction over and above the

background level in the solvent treated control as well as in

the chloramphenicol-supplemented treated samples.

Effect of steroid I treatment on the transformability of E.

The Table-2 describe the effect of steroid I and VIII

as well as Benzo[alpyrene treatment on the transformation

capacity of E. coli AB1157 fw.t) and SOS repair defective

AB2463 (recA) strains. A pronounced decline was observed in

the colony forming units of recA mutant on ampicillin plus

tetracycline (A + T) supplemented nutrient agar plates as

compared to w.t. E. coTi strain. The reduction in the

transformation frequency was observed to be more pronounced

when both pBR322 plasmid as well as E. coli cells were

treated with the test compounds.

Benzo [a] pyrene a well known carcinogen was used as a

positive control. B[a]p reduced the transformation

efficiency of recA cells to a remarkable extent as compared

to wild type E. coli cells.

Tab1e-3 shows the steroid and B[alp induced

mutagenicity in E. coli after treatment. The mutation

frequency in case of w.t. cells was found to be remarkably

significant when both pBR322 and bacterial cells (Weigle

Mutagenesis) were treated with the steroids as compared with

the background mutation obtained with solvent control. But

the mutation frequency in recA cells was not increased above

the background level. Moreover, the mutation frequency was

enhanced to a considerable level when steroid treated cells

were transformed with pBR322 plus universal random primer

(Table-3).

Survival of ^ -phage on steroid treatment:

Extracellular treatment of Xc"*" with the test steroid

upto 4h at the dose of 50 uM had no significant effect on the

plaque forming ability. Survival was, however decreased in

po7 A and rer mutants of E. coli upto 30% after 6h. The recA

exhibited a decline of 50% after 6 h. There was

insignificant decline in PFU within the w.t. and uvrA

backgrounds (Fig.5). Almost similar results were obtained

with "Kvir when treated extracel lularl y with the steroid I

(Fig.6). The survival of }iVir in poTA host decreased upto

3551$ where as recA host enabled 55% recovery of 6h treated

"Kvir particles.

When A c"*" was treated intracellularly with the steroid

I and VIII at the dose of 50 uM the decline in PFU was much

pronounced than that on extracel1u1ar treatment. The

reduction in PFU of A -complexes with radiation sensitive

mutants poTA, recA, lexA and rer was much more pronounced

compared with the wild type strain on steroid I treatment.

Ac"*" - uvrA complex on the other hand, was resistant to steroid

treatment in comparison with other mutants (Fig.?). The

effect of steroid VIII treatment to "K-E. coli complexes

(Fig.8) was slightly less than with the steroid I treatment

(Fig.7). The X-complexes with polA, recA and TexA

mutants on steroid VIII treatment displayed a loss of PFU

upto A0% (Fig.8).

Survival of Tired and }\bio1 as a result of steroid treatment:

Treatment of "k-red-E. coli complexes with steroid I

showed a pronounced decline in PFU (Fig.9). "hred-recA and

T^red-lexA complexes were found to be more sensitive than the

Ared-wild type complex and more than 100 fold loss in PFU was

observed in the ^ recf-radi at i on sensitive mutants as

compared to rec^-wild type complex on 6h treatment (Fig.9).

The loss in PFU was much less pronounced in case of T^red-

uvrA and red-rer complexes as compared to "hred-recA and

}\red-lexA complexes.

Almost similar results were observed with the treatment

of steroid VIII to the X -E. coli complexes at the same dose

(Fig.10).

The survival of "K bio1 on intracellular treatment was

also reduced to a significant extent. The loss in PFU was

remarkable in case of "Xbiol-rer and "hbiol-uvrA complexes

compared with "Kbiol- w.t. complexes (Fig.11).

Effect of active oxygen radical scavengers on the phage

inactivation by steroid I and VIII:

Tables 4 and 5 show the fold inactivation of red-recA

complexes in presence of active oxygen scavengers. The

effect of catalase was significant. The 'OH and

scavengers also had a highly favourable effect on the plaque

forming ability of steroid treated complexes (Tables 4 and

Steroid induced c mutation:

The results given in tables 6, 7 and 8 indicated

significant increase in the clear plaques from the turbid

ones above their spontaneous level on extracellular treatment

of lambda. However, a more significant but moderate increase

in the c mutants was observed on the intracellular treatment

of lambda w.t. E. coli complexes. Intracellular treatment of

}i-recA complex did not exhibit any induction of clear plaques

CTable-e) above spontaneous level. On the other hand, a

remarkable level of mutation was observed when steroid I and

VIII treated phage particles were allowed to adsorb on the

respective steroid treated wild-type bacteria (weigle

mutagenesis) (Tables 7 and 8).

Discussion

Cellular DNA damage occurs following the exposure to UV

light. Ionizing radiations, various environmental chemicals

as well as reactive oxygen species (Lowen et a1. , 1982; Wong

et a1., 1984a,b; Friedberg, 1985a,b; Dale et al., 1992K If

left unrepaired, the DNA damage can result in cell death,

mutation and neoplastic transformation . Prokaryotes and

eukaryotes have evolved systems for reversing DNA damage and

several classes of DNA repair enzymes have been identified

(Friedberg and Bridges, 1983).

Since it is always desirable to carry out various types

of tests to predict the actual behaviour of the test compound

(Maron and Ames, 1983), we have therefore, employed the E.

coli and lambda systems to test the genotoxicity of

relatively potent steroids detected on the basis of Ames test

(Chapter-Ill).

A wide variety of chemical agents have been reported

which induce the SOS response (Bagg et a/., 1981; Quillardet

et a7., 1982; Dale et al., 1992). It is postulated that this

inducible error-prone repair pathway presumably involving

recA and lexA genes could potentially operate on several

types of lesion in DNA (Walker, 1985; Strauss, 1989), The

recA protein is activated when it binds to gaps caused by the

replication fork encountering lesions (Roberts and Devoret,

1983). The recA, recF, lexA and rer mutants of E. coli were

found to be highly sensitive to the test steroids (Figs.1-4)

suggesting damage to the exposed cells as well as the role of"

recA, lexA, recF and rer genes to cope with the hazardous

effect. Many chemicals like aziridinyl benzoquinones

(Lusthof et a7,, 1988) and some steroidal derivatives (Islam

et aU, 1991; Islam and Ahmad, 1991) also decreased the

survival of SOS defective bacterial strains, thereby

suggesting the induction of SOS repair system. In addition,

the recF has been suggested to play role in the formation,

stabilization or utilization of SOS - inducing signal.

However, recF gene product also participates more directly in

the mechanism of daughter strand gap repair (Ganesan and

Seawell, 1975; Rothman and Clark, 1977; Walker, 1985). The

rer gene is an inducible gene and regulates the coordination

between repair and replication of damaged DNA (Srivastava,

1976, 1978). The idea that the SOS response is initiated in

E. coTi as a result of steroid treatment gains further

support from the induction of prophage in the lysogen

(Table-n and by Ames testing studies (Chapter-Ill) with

Salmonella strains (Little et al., 1989). Moreover, the SOS

repair, similar to our case has been shown to essentially

require the de nove protein biosynthesis for its initiation

fTable-1, Witkin, 1976). The induction of prophage by other

steroidal derivatives (Islam et al., 1991; Islam and Ahmad,

1991) and by non-physiological pH (Musarrat and Ahmad, 1991)

has been reported earlier by our group. Moreover, the

prophage induction by physical agents like UV and X-rays is

well established and considered to be one among the many SOS-

response inducing systems (Witkin, 1976).

Several workers have used the transformation capacity

of bacteria with the plasmid DNA as a tool to study the drug-

DNA interaction and/or DNA damage (Chakraborty et al., 1975;

Haque, 1979; Yuqin et al., 1983; Palu et al., 1987). Our

results show a marked degree of reduction in bacterial

transformation on steroid I and VIII as well as

benzo[alpyrene treatment (Table-2). These results lead us to

suggest that DNA is modified on steroid treatment which lead

to marked biological and structural alterations. Bucala et

al. (1985) have observed the loss in transformation capacity

in E. col i cells on the gl ucose-6-phosphate treatment of

PBR322 plasmid DNA. Moreover, these workers have suggested

that DNA damaging agents induce unstable mutants whose

progeny are lost and escape detection. They have reported

that the plasmid pRB322 was mutated on glucose-6-phosphate

treatment and suggested that the mutations were of single or

multiple deletion, insertion or duplication type. The

significant degree of mutation frequency at the ampicillin

and tetracycline loci of pBR322 in wild-type E. col i on

steroid or BCa]p treatment (Table-3) indicates that these

mutations are the result of SOS processing (Walker, 1984K

The loss of viability in certain radiation sensitive E.

coli K-12 mutants and reactivation of inducible prophage with

the concomitant induction of SOS response on treatment with

some steroids have recently been demonstrated by our group

dslam et al. , 1991; Islam and Ahmad, 1991), Steroid

treatment resulted in the decline of the plaque forming units

fPFU) of phage X (Figs,5-11). This decline was pronounced on

the intracellular steroid treatment, especially when

phage A was complexed with radiation sensitive E. coli

mutants like recA, lexA rer and pal A. The recA and polA

mutants of E. coli have also been reported to be sensitive to

ionizing radiations as compared to w.t, bacteria and were

unable to repair single strand breaks (Kapp and Smith, 1970;

Town et al., 1971; Ahmad and Srivastava, 1980). As is

evident from figs. 5 and 6, the phages ^ c"*" (w.t.) and

},vir displayed a significant sensitivity to steroids on the

recA, lexA, rer and polA backgrounds as compared to that in

wild type strain. Moreover, Xbio^ and "K red were highly

sensitive to the test steroids suggesting the role of red^

gene of bacteriophage A in the repair of steroid induced DNA

lesions (Figs 5, 7, 8, 9, 10 and 11). In addition, X reef and

Xbiol exhibited roughly similar sensitivity to steroid I

suggesting that the gam gene might not have a significant

role to play in this type of repair process. The red gene

plays a role in normal recombination process. It is also

considered to exert a complementary effect to the recA gene

of E. coli in the repair of radiation, mild heat injuries and

alkali induced damages (Srivastava, 1973; Ahmad and

Srivastava, 1980; Musarrat and Ahmad, 1991"). Steroid I and

VIII treatment to "h-E. coli complexes had a pronounced

effect on the plaque forming ability of "K red especially

when it was complexed with radiation sensitive mutants of E.

coli as compared to wild type strain (Figs. 9 and 10). These

results thus indicate the active involvement of red gene in

the repair of steroid induced lesions. The red gene function

is supposed to control the repair of ssbs in phage DNA, so

does the polA^ gene of E. coli. Therefore, single strand

bre^aks are considered to be lethal lesions in the absence of

red and polA genes (Srivastava, 1973). ssbs are also induced

in DNA by the ionizing radiations a well known source of

hydroxyl radicals (Dizdaroglu et al. , 1991). Moreover, 'OH

radicals per se induce nicks in the DNA molecules fTullius,

1987).

A considerable reduction in the ^ phage inactivation

in the presence of oxygen radical scavengers fTables-4 and 5)

suggest the role of active oxygen species in the steroid

mediated phage inactivation. The active involvement of

oxygen radicals in the steroid I and VIII induced

mutagenicity has been reported by our group (Islam and Ahmad,

1991; Chapter III). Furthermore, the mutagenesis by agents

that produce active oxygen species has been demonstrated both

in prokaryatic and eukaryotic cells (Farr et al. , 1986; Loeb

et al., 1988; Wei et al., 1989; De Flora et a?., 1989),

A-E. coli uvr complex was found to be less sensitive

to steroid treatment as compared to other A-radiation

sensitive E. coli mutant complexes (Figs.5-8). The rect gene

function of \ and uvr^ of E. coTi have been reported to act

independently fSrivastava, 1973). In view of these findings

it can be suggested that excision repair might not be an

active mechanism for the repair of steroid induced lesions

in DNA molecule. Similar observations were also reported

earlier in case of alkali induced lesions of DNA molecule

(Musarrat and Ahmad, 1991). The contribution of red, recA,

lexA and rer genes appeared to be almost similar (Figs.5-11).

The recA and lexA genes are believed to initiate the error-

prone repair process in cells exposed to radiation and

hazardous chemicals, and thus enhance the mutation frequency

of the system (Radman, 1975; Witkin, 1976; Walker, 1985,

Dale et al., 1992).

An increased frequency of c mutation was observed on

the intercellular treatment of Ac"*" (Table 7), this c

mutation was more pronounced when both the host and lambda

were treated separately, also termed as weigle mutagenesis

(Table 8).. A similar type of mutation frequency was also

obtained when the steroid I, VIIT and Benzo [a] pyrene

treated wild type E. coT i was transformed with steroid I.

VIII and Benzo [a] pyrene treated pBR322 plasmid DNA (Table-

3). Such enhancement in mutation frequency is well

established in the case of radiation (Witkin, 1976) as well

as alkali (Musarrat and Ahmad, 1991) induced mutagenesis.

Interestingly enough, the untreated phage or plasmid

DNA introduced into the steroid treated host cells resulted

in the enhancement of mutation on the phage and plasmid DNA

markers. Similarly, simultaneous introduction of universal

random primer alongwith the untreated plasmid DNA brought

about the enhanced mutation at amp and tet loci of plasmid.

These findings lead us to suggest that the diminished proof

reading activity of DNA polymerase(s) brought about by the

induction of the SOS response in the steroid treated E. coli

(Islam and Ahmad, 1990; and this work) may be the major cause

of mutagenesis. Villani et al (1978) has suggested that

damaged DNA induces an inhibitor which inhibits or reduces

the proof reading 3'-5' exonuc1eo1ytic activity of

constitutive DNA ploymerases. Moreover, Lu et a7 f1986) have

provided evidence that RecA protein binds to the £-subunit

of DNA polymerase III and thus reduces the proof reading

activity of the enzyme. Our result with rec~ cells further

substantiates the findings of Lu et al. f1986).

Another significant finding is the higher mutation

frequency with treated plasmid in the untreated host compared

with that in reverse treatment system (table-3) which clearly

implies that misreplication brought about by the induction of

SOS response is not the only factor to give rise the mutation

rather mis-repair of the steroid-induced DNA damage is also

essentially involved. This is important in ruling out the

possibility of steroid induced cellular alterations without

the involvement of DNA damage. In v.itro studies further

support this idea (Chapter-VI).

In view of the present work, it seems beyond doubt that

the steroid treatment to E. col i and phage A both

extracellularly and intracellularly induces lesions in DNA

that are repairable in the host strains having active repair

machinery derived by the host and / or phage genes initiating

the SOS response and thus bring about the mutations in

bacterial as well as phage DNA. The induction of DNA lesions

may be due to the active oxy radicals generated by the test

steroids (Table 4 and 5;Chapter IV; Islam and Ahmad, 1991).

TABLE-1: Induction of prophage in lysogen by steroid I.

ACI857 E. coli (AB1157)

Time of Solvent Fold Induction In Presence Incubation Treated of Prophage after of in Nutrient Control Steroid I Treatment Chloramphenicol Broth (h) (P.F.U.) (100 ug/ml)

ug/ml 2ug/ml

0 1 .00 1.00 1.00 1 .000

1 1 .00 1.35 2.30 0.980

2 1 .70 2.50 5.02 1.000

3 1 .95 3.34 5.38 1 .020

LU <4-o >. o c 0) 3 u 0) L. «»-

c o •r" CB e L. o (0 c CB i.

0) jC •P c o c 0) E . tJ < <8 2 0) O T3 f-•O E

t: W O CB I- 1-0) a +3 OS CM CM CO o a: CD •p a o Q) £ -1-

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I- ® ~ er. t-

®0 W m «

— e ®-r- (0 OX " ^ a I! ® O 3«M CO

I—OC 3 CD Hj o Q.

TABLE-4: Effect of oxygen radical scavengers on the suY-vival of A phage with in the steroid I treated

Ared-recA E. coli complexes.

Incubation conditions PFU/ml

Untreated 4.6 X 10' Steroid I + Cu(II) (Control) 1 .8 X 10' Control + Mannitol (2.0 mM) 7.0 X 10 Control + Thiourea (2.0 mM) 2.9 X 10 Control + Benzoate (2.0 mM) 5.8 X 10 Control + Formate (2.0 mM) 4.3 X 10 Control + Azide (0.05 mM) 0.85 X 10 Control + Catalase (16 ug/ml) 0.40 X 10

The final concentrations reaction mixture was 50 uM

of steroid-I and Cu(II) and 100 uM respectively.

i n the

TABLE-5: Effect of oxygen radical scavengers on the survival of phage with in the steroid VIII treatedAred-rec>1 E. coli complexes.

Incubation conditions PFU/ml

Untreated 5.0 X 10 Steroid VIII + Cu(II) (Control) 1.2 X 10 Control + Mannitol (2.0 mM) 4.3 X 10 Control + Thiourea (2.0 mM) 4.8 X 10 Control + Benzoate (2.0 mM) 2.7 X 10 Control + Formate (2.0 mM) 0.8 X 10 Control + Azide (0.05 mM) 1 .5 X 10 Control + Catalase (16 ug/ml) 8.9 X 10

The final concentrations of steroid VIII and Cu(II) in reaction mixture was 50 uM and 100 uM respectively.

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Flg.1: Survival of f. coll K-12 cells exposed to steroid I

Wild-type

• • 0 0

TIME ( H )

Fig.2: Survival of E. coll K-12 cells exposed to steroid II

Wild-type

3 LL U

cc D CO I— z Ui o Q: LU a.

Fig.3: Survival of E. coli K-12 cells exposed to steroid III

Wild-type

A — ^

TIME ( h )

Fig.4: Survival of E. coll K-12 cxlls exposed to steroid IV

Wild-type

§ i 0 0

• -A

TIME ( h )

Fig.5: Propagation of extracellularly treated with steroid I in various E. coll host strains.

Wild-type 0 0

recA t •

lexA 0 9

rer * f

uvrA X — X

polA* A — A

po1A~ A —

Z) u. a.

< > > q: 3 to

liJ u a: LJ a.

100 80

10 - J L_ 2.0 A.O

TIME ( h ) 6.0

Fig.6: Plaque forming ability of steroid I treated A vir particles in various E. coll host strains.

Wild-type : 0 — 0

recA •—f

poTA : Br—A

UJ 20 o cr Ui 0.

Time (h )

Fig.7: Survival patterns of Intracellularly treated \ c*"

with stero1d-I in various E. coll host strains.

Wild-type

polA*'

po 1A~

e — 9 » — *

A—Ik

D U. a.

< > > cc

z lU L> cr UJ cl

Fig.8: Survival patterns of intracellularly treated A c* with steroid VIII in various E. coll host strains.

Wild-type 0 0

recA • • lexA 0 9

uvrA X — x

polA^ A — A

po 7/4" A—A

1 > cr 3 if)

UJ u cc UJ CL

100 80

10 _L 2.A A.O

TIME (h )

Fig.9: Survival patterns of Intracellularly treated \ red with steroid I In various E. coll host strains.

wnd-type

0 — 0

f — •

* — 4

X—^x

3 U. a. it 10 -

en 3 in

z Ul u tr UJ Q.

2.0 A.O TIME ( h )

Fig.10: Survival patterns of intracellularly treated \ red with steroid VIII in various E. coTi host strains.

Wild-type

0 — 0 • — •

X ™ X

D U. Q.

I-z UJ u cr UJ Q.

2.0 A.O

TIME (h )

Fig.11: Survival patterns of intracellularly treated blol with steroid-I in various E. coll host strains.

Wild-type

0 — 0

X — X

Z) u. a.

< > > a: Z) (/I

2 LU U cr UJ Q.

2.0 A.O

TIME ( h )

CHAPTER VI

Steroid Induced DNA

Lesions (In vitro and In vivo

Studies)

Introduction

DNA damage has been proposed as a potentially useful

parameter in screening chemicals for their genotoxic

properties, since many chemical carcinogens and mutagens have

been shown to produce DNA damage in mammalian cells fPetzold

and Swenberg, 1978; Schiwarz et al., 1979; Stitch et al. ,

1979; Ames, 1983). Some forms of DNA damage have been

suggested as the initiators of chemical carcinogenesis

(Brooks, 1977; Lutz, 1979). One particularly common type of

DNA damage is the DNA strand breakage, produced by the

exposure of DNA to a variety of physical and chemical agents

(Daniel et al., 1985). Oxygen radicals have been reported to

attack DNA either at sugar or base giving rise to a large

number of products (Hutchinson, 1985). Hydroxyl radical

generates single strand breaks in duplex DNA (Hertzberg and

Dervan, 1984). In fact, such single stranded DNA breaks

accumulate during exposure of bacterial and mammalian cells

to active oxygen radicals, ionizing radiations or ozone

(Ananthaswamy and Eisentark, 1 977; deMe11o-Fi1 ho and

meneghini, 1985; Birnboim and Kanabus-Daminska, 1985).

These strand breaks may be useful as an analytical

parameter for assessing the exposure of genetic material to

genotoxic chemicals (Stitch et al., 1 979; Freeman et al. ,

1986). Moreover, since certain steroidal derivatives have

been shown to generate single strand breaks in the DNA vis-a-

vis their role in the production of oxygen radical species

has been demonstrated (Islam S., Ph.D. thesis, 1991), it

seemed worth-while to study the DNA damage in terms of ssb

formation induced by test steroid I. Our purpose was to

demonstrate the presence of ssbs, if any, employing these in

vivo and in vitro techniques which could be relatively rapid

and simple nonetheless quite fool-proof. Thus for testing tv&,

in vitro ssb formation, the pBR322 plasmid and ss phage Ml 3

DNA were subjected to steroid treatment and the mobilities of

the treated DNA under electric field was determined. This

study was further substantiated by determining the S^

nuclease susceptible sites in the steroid treated calf thymus

DNA. The in vivo steroid break formation was tested using

the ligase and DNA polymerase I defective mutants of E. coli.

Certain experiments were so designed that could also give the

information r e g a r d i n g the possible involvement of oxygen

radical species

The chemicals, buffers and solutions used in this study have been described in Chaptor-Il.

Bacterial strains:

The lig7 and ooTAI mutants of E. coli K12 were employed alongwith the wild-type strain whose characteristic genetic markers have been given in table 1 of Chapter-II. Each strain was tested for its associated genetic marker before use.

S^ nuclease assay of steroid I treated DNA:

in TNE buffer was treated with Calf thymus DNA I in the presence of CUCI2 at desired concentrations, mixture was incubated at room temperature for different periods in fluorescent light. In some experiments radical scavengers and other transition metal ions included (The role of free radical scavengers has discussed in detail in Chapter-IV). The steroid treated was dialyzed against 100 volumes of TN buffer at 4®C.

steroid The time free were been DNA The

to S^ aci d

treated DNA sample so obtained was then subjected digestion. The enzyme assay was done in terms of soluble nucleotides released from DNA as a result of enzymic hydrolysis. The S^ nuclease reaction mixture in a total volume of 1.0 ml contained 500 ug substrate (native, denatured or steroid/solvent - treated DNA); 0.5 M acetate

30 units of S^ nuclease. The mix buffer, pH 4.5; ImM ZnSO^; was incubated for 2 .h at AS' C, The reaction was terminated by adding 0.2 ml bovine serum albumin and 1.0 ml perchloric acid (ice-cold). The tubes were immediately transferred to 0* 0 for at least 1 h before centrifugation to remove the undigested DNA and precipitated proteins. Acid soluble DNA nucleotides were determined in the supernatant using diphenyl amine method of Schneider (1957).

Determination of melting temperature (Tm):

Tm was determined as described by Hasan and Ali (1991). 500 ug calf thymus DNA was treated with different DNA/bp steroid molar ratio of 1:0.25, 1:1 and incubated 0/N at 37* 0, The Tm of native, solvent/steroid-treated DNA was determined by Shimadzu UV 240 spectrophotometer coupled with a temperature programmer and controller assembly. The polymer in TN buffer was heated at the rate of 1' C per min. initiating from 30* 0 upto 95®C. The absorbance was read at 260 nm and the percent denaturation evaluated with increase in temperature.

Percent {%) denaturation was calculated as:

Absorbance - Absorbance (observed) (initial)

Absorbance - Absorbance (at 95*^0 (observed)

Steroid I and VIII treatment to calf thymus DNA:

500 ug of calf thymus DNA was treated with different DNA/base pair steroid molar ratio (bp/molar) of 1:05 and 1:1

and incubated at 37* 0 0/N. The treated DNA was dialysed against TNE buffer in cold before the alkaline treatment and centrifugation. The solvent (DMSO: Acetone: 90%: 10%) treated DNA was also processed in a similar way.

Velocity sedimentation of steroid treated calf thymus DNA:

Steroid I and VIII as well as solvent treated DNA solutions f0.15 ml) were denatured by the addition of an equal volume of 0.2 N NaOH. After 10 minutes delay to permit denaturation of the DNA, 0.2 ml of each sample was layered on top of a 3.8 ml alkaline sucrose gradient (5-20% sucrose in 0.1 N NaOH and 0.01 M EDTA). The gradients were centrifuged at 37,000 rpm for 10.5 hours at room temperature in a SW-60Ti swinging bucket rotor of a Beckman L8-60M ultracentrifuge. After centrifugation 5 drop fractions were collected and diluted to about 0.3 ml by double distilled water. The diluted fractions were transferred to microcuvettes and the absorbance at 260 nm was read in a DU-40 Backman double beam spectrophotometer. Data are plotted as absorbance at 260 nm (ApRrt) against fraction number according to Kapp and Smith (1970).

Neutral sucrose gradient sedimentation of steroid treated DNA:

A homogeneous peak of calf thymus DNA obtained by prerunning was pooled out and 150 ug of this DNA was treated overnight with steroid I, VIII (at DNA base pair molar ratio of 1:0.5 and 1:1) and solvent separately at room temperature. After treatment the DNA samples were dialysed against TN buffer in order to remove the unreacted steroid. Then 0.2 ml DNA sample was layered on the top of 3.8 ml of 5-20% neutral sucrose gradient (in 0.01M TNE buffer, pH 8.0). The gradients were centrifuged at 1 1000 rpnrt for 14b at room temperature in SW-60Ti swinging bucket rotor of a Beckman L8-60M ultracentrifuge. After centrifugation, 5 drop fractions were collected and read in a DU-40 double beam spectrophotometer according to Kapp and Smith (1970). The data are plotted as absorbance at 260 nm (AggQ) against fraction number. Molecular weight of modified and un-mofidied calf thymus DNA was calculated according to Bergi and Harshay (1963).

Steroid I treatment to pBR322 plasmid and M13 phage DNA:

The reaction mix (25 ul) contained lOmMTris-HCl buffer (pH 7.5), 3-5 ug DNA, 0.2 mM Cu(ll) and varying concentra-tions of steroid I. After incubation in light at room temperature for 4 h, 8 ul of a dye solution containing 10 mM EDTA, 0.05% bromophenol blue, and 50% (V/V) glycerol was

added. This mix was then subjected to electrophoresis in submarine 1 agarose gels. The gels were stained with ethedium bromide (0.5 mg/1) and viewed and photographed under a UV transi1luminator.

Treatment of E. coli with test steroids:

The polAI and Tig? mutants of E. coli K12 as well as the wild type strain was harvested bY centri f ugat i on from exponentially grown cultures (1-3x10® viable counts ml '). The pellet so obtained was suspended directly in 0.01 M MgSO^ solution and treated with steroid I and VIII separately at a dose of 50 uM in the presence of 100 uM Cu(II) at room temp in light. Samples were withdrawn at regular intervals, suitably diluted and plated to assay colony forming ability. The plates were incubated overnight (0/N) at 37*^0. Solvent controls were run simultaneously.

Results

Effect of transition metal ions in light and dark on steroid I

mediated S hydrolysis of calf thymus DNA:

Several transition metal ions were used in steroid

mediated S hydrolysis of DNA. Among these metal ions, only

Cu(II) and Fe(III) had a profound effect on the S^ hydrolysis

of calf thymus DNA in presence of fluorescent light (Table-

21. Although Cu(II) in dark could also influence the S^

hydrolysis to a significant extent, but incubation in light

had a remarkable stimulatory effect on steroid induced damage

to DNA molecule (Table-2). On the other hand Co(II), Mn(II)

and Ca(II) exhibited a weak stimulatory effect on the steroid

mediated S^ hydrolysis of calf thymus DNA even in the

presence of light (Table-2). Since the Cu(II) was found to

be the most effective ion, thus it was used in subsequent

experiments.

S^ nuclease hydrolysis of steroid treated calf thymus DNA:

Fig.1a shows the pattern of steroid induced S^

hydrolysis in the presence of Cufll). Production of acid

soluble nucleotides was increased with the increasing dose of

test steroid and attained plateau at the dose of 200 uM.

Heat denatured DNA as positive control was normalized to 100%

hydrolysis. The values shown in the figure have been

obtained after subtracting the corresponding solvent values.

Fig. lb shows the effect of the Cudll ion concentration

on the steroid mediated S hydrolysis. The DNA was treated

with 0.2 mM steroid in the presence of varying concentrations

of Cu(II). We observed a linear relationship between Cu(II)

ion concentration Vs S^ hydrolysis upto 0.4 mM Cufll)

concentration followed by a plateau.

The S^ hydrolysis reaction was time dependent also

(Fig.1c) and increased upto 6 h attaining a plateau beyond

this incubation time.

Involvement of active oxygen species in steroid induced DNA

damage:

The steroid mediated S hydrolysis of calf thymus DNA

was inhibited by various oxygen radical scavengers (Table 3).

Hydroxyl radical scavengers like mannitol, sodium formate and

sodium benzoate resulted in significant decrease of S^

susceptible sites. Other active oxygen radical scavengers

such as SOD, catalase and sodium azide also brought about

significant inhibition in the S hydrolysis of DNA. SOD for

instance, resulted in 60% reduction in the DNA hydrolysis by

S^ nuclease. Incubation of reaction mix in dark without any

scavengers also had an inhibitory effect (40% approx.) on S

hydrolysis to a large extent (data not shown).

Degradation of calf thymus DNA treated with test steroid in

the absence of S^ nuclease:

Table-4 and 5 summarise the data on the steroid induced

DNA degradation in the absence of S^ nuclease under various

experimental conditions. Table-4 shows that the steroid in

the absfence of Cufll) brought about a very weak DNA

hydrolysis even at high doses but the incorporation of Cu(II)

enhanced this DNA degradation significantly at low steroid

doses also. 26% DNA degradation was observed at 300 uM

steroid and 400 uM Cufll) in the absence of S^ nuclease. The

solvent plus Cu(II) (as control) did not result in the

significant DNA hydrolysis (Table-5).

Sedimentation velocity gradient of steroid treated DNA for

detection of strand breaks:

Fig.3 shows the sedimentation profiles of steroid I

treated calf thymus DNA at the DNA bp/steroid molar ratio of

1:0.5 and 1:1 in the alkaline sucrose gradient (panels B and

C). There was a considerable shift towards top in the peak at

260 nm with the increasing steroid DMA ratio as compared to

solvent (as control) treated calf thymus DNA (panel A).

Fig.4 shows the sedimentation profiles of steroid VIII

treated calf thymus DNA. Panel A shows the profile of

solvent treated DNA. Panels B and C are 1:0.5 and 1:1 DNA

bp/steroid molar ratio treatments respectively. A remarkable

shift'in the peak and increase in the absorbance is observed,

indicating a decrease in the mol. wt. of calf thymus DNA with

the induction of ss breaks with the increasing steroid

treatment (Fig.3),

Figs.5 and 6 show the shift in DNA peak at 260 nm on

steroid I and VIII treatment (Panels B and C). Panel A of

Figs. 5 and 6 shows the peaks of solvent treated DNA with the

average mol. wt. to be 9.6 x 10^ dalton. The arrow in the

Figs. 5 and 6 show the peak position of DNA run as standard

mol. wt. marker. The mol. wt. of steroid treated DNA was

calculated and found to be 5.2 x 10^ and 1.8 x 10^ dalton in

case of steroid I treatment at DNA: steroid/bp molar ratio of

1:0.5 and 1:1 respectively and 6.7 x 10^ dalton and 3.2 x 10^

dalton in the case of steroid VIII treatment on the same DNA

steroidy/bp molar ratios.

Steroid induced cleavage of plasmid and phage DNA:

The test steroid converted increasing amount of

supercoiled DNA to relaxed and linear forms with increasing

doses of steroid (Fig.7). The lane A shows the solvent

treated pBR322 DNA having almost equal populations of

supercoiled and linear forms run as control. The lane B

indicates the electrophoretic pattern of steroid treated

pBR322 at 100 uM concentration in which the uppermost band

consisting of related circular population is more visible and

linear species is in abundance compared to the supercoiled

population. The DNA treated at 200 uM steroid concentration

is depicted in lane C which clearly shows maximum proportion

of linear species.

Fig.8 shows the agarose gel electrophoretic pattern of

single stranded M13 mp8 phage DNA treated with increasing

concentrations (50 uM to 300 uM) of the test steroid (lanes B

to E). Almost total population had undergone fragmentation at

300 uM steroid concentration (lane E) in the presence of

Cu(II), Lane A shows solvent treated Ml3 phage DNA run as

control.

Survival of E. coli strains treated with test steroid:

The survival pattern of polA and lig mutants at 50 uM

steroid I (Fig.9) and steroid VIII (Fig.10) treatment is

shown. Both the mutants exhibited a remarkable reduction in

their colony forming units (C.F.U.) as compared to the wild

type strains on treatment with both the test steroids.

Discussion

It is a w e n known fact that S^ nuclease hydrolyses

single stranded regions in duplex DNA and also detects

locally altered structures (minor distortions) introduced in

DNA by physical and chemical agents (Shishido nd Ando, 1982).

Our results shown in Fig.1 indicate that the steroid treated

calf thymus DNA at low concentrations is a relatively better

substrate for S^ nuclease (Fig.1a) compared to that at higher

concentrations though the S susceptible sites are more at

higher dose. Moreover S^ hydrolysis of steroid treated DNA

was increased by Cufll) which was found to be the most

effective ion among all the transition metals used (Fig.lb,

Table-2), Earlier workers have also reported the maximum

scission/degradation of DNA in the presence of Cu(II) by

flavonoids and many other DNA damaging drugs (Wong et al.,

1984b; Ehrenfeld et al., 1987; Quinlan and Gutteridge, 1987;

Rahman et al. , 1989).

The generation of oxygen radicals in the proximity of

DNA is well established as a cause of strand scission

(Gutteridge and Halliwell, 1982; Wong et al., 1984b;

Ehrenfeld et al., 1987). The copper ions were also found to

be involved in the H2O2 induced DNA breakage (Sagripanti and

Kraemer, 1989; Yamamoto and Kawanishi, 1989). The

significant inhibition of steroid induced S^ hydrolysis in

the presence of oxygen radical scavengers (Table-2), thus

indicate the active involvement of oxygen radicals in DNA

damage.

Further studies showed that the steroid at moderate t>»M

doses itself brought about significant^degradation (Table-3)

and this degradation was enhanced by the addition of Cu(II)

into the reaction mixture (Table-4). The oxygen free

radicals especially singlet oxygen generated by the test

steroids fChapter-IV) might play an essential role in the DNA

degradation. is considered to be extremely reactive

species in DNA damage at least under in vitro conditions

(Joshi, 1985).

Our results also indicated a reduced Tm upon treatment

of DNA with steroid I (Fig.2). These results can account for

the destabi 1 i zation of the duplex DNA, with the possible

formation of single strand breaks. The increasing

concentration of steroid further reduced the melting

temperature of steroid treated DNA, thus indicating a direct

relationship with the steroid concentration and DNA strand

breaks. Rahman et al (1990) have reported that methyl

glyoxyl treatment also reduced the Tm of calf thymus DNA at

lower concentrations thus suggesting for the induction of ss

breaks in DNA. In addition, low doses of steroid VIII have

also been shown to decrease the melting temperature of calf

thymus DNA and also induce single strand breaks (Islam S, f

Ph.D. thesis, 1991").

The velocity sedimentation analysis of steroid treated

DNA clearly indicated the decrease in the molecular weight of

the steroid treated DNA (Figs.3, 4 and 5,6) as compared to

that of the solvent treated control DNA (panel A of Figs.

3,4,5 and 6), thus suggesting for the presence of single and

double strand breaks in the steroid treated DNA molecule. The

induction of single and double strand breaks in the calf

thymus DNA by ionizing radiations and their detection by

sedimentation velocity centrifugation technique is well

documented (Kapp and Smith, 1970: Srivastava, 1974; Lehman

and Stevens, 1977; Lliaks, 1991).

Supercoiled plasmid DNA is a convenient substrate for

rapid and sensitive assay of the reactions inducing nicks

(Rehman et a7., 1990). The conversion of supercoiled plasmid

DNA molecules to relaxed and linear forms is evident in our

system (Fig.7). Complete fragmentation of single stranded

phage M13 DNA was found to occur at moderately high dose of

steroid I (Fig.8). These results lead us to suggest that the

extensive nicking in DNA due to the production of highly

reactive -OH by the test steroids (Chapter-IV). It is a well

documented fact that 'OH radicals induce nisks in the DNA

molecule by extracting a hydrogen atom from a deoxyribose in

the backbone, which then by secondary reactions breaks down,

cleaving the chain (Hertzberg and Dervin, 1984) at several

sites randomly (Tullius, 1987).

E. coli DNA repair defective polAI and Tig? mutants

were found to be sensitive to steroid treatment compared to

wild type strain, thereby suggesting the role of these genes

in the repair of steroid induced DNA damage (Figs.9 and 10).

Earlier studies have shown that recA and polA mutants of E.

coli K12 were sensitive to ionizing radiations, inducing

single strand breaks into the bacterial genome (Srivastava,

1973; 1974). We have discussed at length regarding the

probable generation of reactive oxygen species in Chapter-IV

and also shown the induction of SOS response in the steroid

treated SaTmonella and E. coli strains (Chapters III and V).

Moreover, the sensitivity of lig7 and polAI mutants of

E. coli to the steroid treatment clearly indicates the direct

and/or indirect strand breakage of DNA brought about by test

steroids. Furthermore, the DNA nicking and degradation might

be due to the generation of oxygen free radical species.

Although the presence of ssbs under in vivo conditions

would not pose a great threat to the survival of treated

bacterial cells, since the nicked DNA will be subject to fast

rejoining by DNA polymerase I and DNA ligase (Town et al,

1971; 1972; Srivastava, 1974; Pauling and Beck, 1975; Ward,

1988"), but the accumulation of breaks would give rise to the

generation of double strand breaks within the genome and even

a single double strand break is expected to be potentially

lethal lesion and would trigger for the SOS repair (Lliaks,

1991). Our DNA degradation studies further substantiate our

findings (Tables 3 and 4).

TABLE 1: Effect of transition metal ions (0.4 mM) on the S^ hydrolysis of calf thymus DNA by steroid 1 treatment (0.2 mM) in the presence of light and dark.

Reaction mixture (Sample)

DNA Hydrolysed

Native DNA 4. 90 DNA + Steroid I (light) 20. 47 DNA + Steroid I (dark) 12. 40 DNA + Steroid I + Fedl ) (light) 25. 35 DNA + Steroid I + Fedl ) (dark) 2. 71 DNA + Steroid I + Fedl (light) 20. 66 DNA + Steroid I + Fedl (dark) 6. 10 DNA + Steroid I + CudI (light) 47. 00 DNA + Steroid I + CudI (dark) 26. 30 DNA + Steroid I + CodI (light) 13. 56 DNA + Steroid I + Mndl (light) 12. 73 DNA + Steroid I + Nidi (light) 8. 56 DNA + Steroid I + Cadi (light) 2. 84

TABLE-2: Inhibition of S^ nuclease hydrolysis of DNA after treatment with steroid I (0.2 mM) and Cu(II) mM) in the presence of scavengers.

Scavengers Ihibition of S 1 nuclease Hydrolysis {%)

Superoxide dismutase (100 ug/ml) Catalase (100 ug/ml) Mannitol (50 mM) Sodium formate (50 mM) Sodium benzoate (50 mM) Sodium azide (50 mM)

60.0 73.3 71 .1 73.3 63.0 28.9

Concentrations of the concentrations.

scavengers shown are final

TABLE-3: Degradation of calf thymus DNA treated steroid I i n the absence of S^ nuclease.

ug acid soluble DNA nucleotides

% DNA % DNA degraded degraded after subtraction

from control

Denatured DNA

DNA (as

+ Solvent Control)

DNA + Steroid (dose of steroid uM)

50 100 200 300 400

37.5 42.5 45.0 53.7 6 0 . 0

7.8 8.92 9.45

11 .28 12.60

0.0 0.53 1 .05 2.88 4.20

TABLE-4: S-j nuclease independent degradation of calf thymus DNA treated with varyjng concentration of steroid I in presence of Cu(II)

ug acid soluble DNA nucleotides

% DNA degraded

% DNA degraded after subtraction from control

Denatured DNA 481.0

DNA + Solvent + 46.0 Cu(II) (as Control)

DNA + Steroid + Cu(II) (dose of steroid uM)

50 100 200 300 400

112.5 127.5 157.5 172.5 165.0

23.38 26.50 32.74 35.86 34.30

13.82 16.94 23.18 26.30 24.74

* The concentration of Cu(II) used was 0.4 mM,

Flg.1: S^ dependent hydrolysis of calf thymtis DNA after treatment with steroid I

(a) effect of varying concentrations of steroid I in presence of Cu(II) (400 uM).

(b) effect of varying concentrations of Cu(II) in presence of 200 uM steroid I.

(c) Kinetics of S hydrolysis of DNA in presence of steroid I (200 uM) and 400 uM Cu(II).

50 100 200 300

STEROID (>iM ) AOO

50 100 200 3 0 0

C u d I ) ( > J M )

AOO 500

o LJ > _J 0 a > 1 < z o

A 5 6 7

T I M E ( h )

Fig.2: Thermal melt^1ng.>prof 1 le of native and steroid I treated DNA ' ^ \ r

( \ V V

Native DNA \ \ \ : 0 0 ; \ V.

Steroid-I treated WJA: '"

1:0.25* \ © 9

1:1 > •

*(1:0.25 = 500 ug DNA : 30 ug steroid I )

< a: 3

Z UJ u cr LjJ a.

-SelcHmentatlcm profiles of solvents and steroid I treated calf thymus DMA on alkaline sucrose gradients (5-20%).

Solvent treated Panel A

Steroid trecited:

Panel B I

Panel C

e c o u>

UJ u z < CQ cc O CO CD <

6 8 10 12 U 16 18 20. 22 F R A C T I O N N U M B E R

Fig.4: Sedimentation profiles of solvent and steroid VIII treated calf thymus DNA on alkaline sucro^se gradients (5-20%X,

Solvent treated : Panel A

Steroid treated:

1:0.5 : Panel B

1:1 : .Panel C

e c o <£> (N

UJ u z < CO or o I/) QO <

1 1 1 1 1 1 .

1 i 1 8 10 12 lA 16

FRACTION NUMBER 18 20 22

Fig.5: Sedimentation profiles of solvent and steroid I treated calf thymus DNA on rteutral (pH 8.0) sucrose gradients (5-20%). '

Solvent treated Panel A

Steroid treated:

Panel B

Panel C

(arrow indicates the mol-wt- marker).

DNA peak position run a.s

e c o {£> CM

S 0.5 z < GO Q: o (/) CQ <

8 12 16 20 2A FRACTION NUMBER

Fig.6: Sedimentation profiles of solven^\and steroid VI treated calf thymus DNA on neutral \sucrose gradier (5-20%). ' ^ '

So 1 vWt't reated

Steroid treated:

1:0.5 ..

Panel A

.P.ane.1

Panel C

(arrow Indicates th€ '

mol.wt. marker)^^

DNA peak position run $s

6 c o (X> <N UJ a z < QQ CC O if) QQ <

X 0 8 12 16

FRACTION NUMBER 20 2A

rig.7: Agarose gel elcctropljorecis of pDR3J>2 plassr.lcf P..,. incubstect v:1tfi £00 rM OuClI) €irX vtryih'-concentrations cT ciesvld I.

lane A

lane B

lane C

Solvent

100 uM store id

2C0 uf: stcroU

•Jh. position of supercoiled (SC), linear (LIK) and

(.IrcuUs relaxed (OC) DNA are 1ndiG<xOfcw.

Fig.8: Agarose gel electrophoresis of steroid I treated single stranded M13 mp8 phage DNA

lane A : Solvent + 200 uM Cu(II)

lane B to E 50, 100, 200 and 300 uM steroid I + 200 uM Cu(II) respectively.

A e C D E

F1g.9:- Effect of the steroitj I treatment on the survival of E. coll K12 strains.

Wi-fd type (w.t.) : 0 0

pol^ : A — - A . A

po 1A~ • •

Jl9~ : A -A

Time (h )

Fig.10: Effect of the steroid VIII tpeatment on the survival of E. coll K12 strains.

Wild type (w.t.)

po 1A~

A — ^

60 o -

40 > > cc D (n

20 z u) o oc UJ a

TIME ( h )

CHAPTER VII

General Discussion

The Salmonella test was first of all recognised as an

appropriate carcinogenicity testing system in the study of

300 chemicals most of which were known carcinogens (McCann et

al. , 1975a; McCann and Ames, 1^76, 1977). Since then, the

test has been established for the evaluation of the risk of

environmental chemicals (Ames, 1984). Although the validity

of the test has been questioned in terms of the actual

carcinogenic behaviour of the test compounds, yet a

remarkable correlation was obtained with several mutagenic

compounds tested by the Salmonella system (McCann et al.,

1975a; Flessel et al., 1987; Ashby and Tennant, 1988). Our

results indicated a high degree of mutagenicity of the test

steroids with GC—>At transition mutants, TA102 and TA104

(Levin et al., 1982a) compared with those having GC base

pairs at the critical site of mutation suggesting that the

test steroids preferentially act upon AT base pairs to bring

about transition mutation (Table-1, Figs.1 and 2 of Chapter-

Ill). Interestingly enough, the testing strains responded

significantly even in the absence of post mitochondrial

supernatant (Sg fraction). The addition of Sg fraction

further enhanced the mutagenic potential of steroids (Table-2

Chapter-Ill) suggesting that the metabolic products of the

test steroids might be even more mutagenic. Similar

findings, were also obtained with other synthetic steroids

(Islam et al., 1991; Islam and Ahmad, 1991).

The steroidal derivatives having the groups attached to

3rd position were highly mutagenic than those having no

groups linked at this position (Table-1, Chapter-IIIK

Oxathiolane steroidal derivative having acetoxy group at

position 3 was most potent mutagenic compound after metablic

activation among the test steroids fFig.2, Table-3 of

Chapter-Ill). Moreover, the mutagenicity was enhanced with

the increasing number of oxygen atoms in steroidal derivative

(Table-1, Chapter-Ill), thereby indicating the probable role

of oxygen atoms for the enhanced mutagenicity of the

steroids.

Our results are consistent with the idea that

electronegative group at the 3rd position of the steroidal

nucleus often enhances the e1ectrophi 1 ic nature of

susceptible moiety present at the 6th position which in turn

upon interaction with some nucleophile in the system

generates active oxygen radical species (Simic et al., 1989).

Although the presence of electrophi1ic group in the test

steroids seems to be desirable in certain cases, it was not

probably an essential requirement for these compounds to

become mutagenic. In such cases, the bulky groups present at

the 3rd and 6th positions might create steric hindrance due

to which the overall structure becomes unstable and the

groups get cleaved from the parental nucleus. The cleaved

group then probably interact with some reactive species in

the system and generate oxygen radical species. Moreover,

the association of groups with the steroidal nucleus seems to

be necessary for the mutagenic potential of test steroids as

the parent steroid did not exhibit a significant mutagenicity

(Table-1, Chapter-Ill). These findgins are in agreement with

those reported by Islam and Ahmad (1991).

The tester strains TA102 and TA104 have been reported

to detect a variety of oxidants and other agents as mutagens

(Levin et al. , 1982a; DeFlora et al. , 1989). All the test

steroids displayed high mutagenicity with these strains.

Moreover, a significant amount of inhibition in the mutation

frequency of these strains with the potent test steroid was

found in the presence of the scavengers of active oxygen

species as well as of H2O2 (Tables 8 and 9, Chapter-IV).

This finding further provides support for the production of

active oxygen species viz., O^'', 'OH and as well as of

H2O2 in the test system. It is noteworthy that the

generation of these active oxygen species have also been

found under some in vitro conditions (Chapter-IV). In vitro

generation of active oxygen species was also demonstrated by

earlier workers with a variety of mutagenic and carcinogenic

compounds (Nakayama et al., 1983; Wei et al., 1989; Rahman et

al., 1990).

Several authors have attributed the true damaging

species to be a highly reactive -OH derived from 02*" and

Pi'^sumably formed by the Haber-Weiss reaction

(Fridovich, 1978; Helliwell, 1982, Nassi-calo et al., 1989).

02*" + HgOg > -OH + OH" + O2 M )

On the other hand, even the significance of ^02 in the

DNA damage and mutagenicity should not be underestimated, in

view of the role played by this oxygen radical species in the

Benzo [a] pyrene induced DNA damage and carcinogenicity (Wei

et a?., 1989). The existence of all active oxygen and other

toxic species can be envisaged in the system presumably due

reactions have been suggested to occur in several systems

under a variety of genotoxic conditions (Marx, 1987; De-Flora

et a7., 1989; Wei et al., 1989; Halliwell and Gutteridge,

1990; Touati and Farr, 1990; Dizdaroglu et al., 1991).

A possible source of the active oxygen species in case

of oxathiolane steroid is shown in (Fig.6, Chapter-IV). A

similar mechanism has also been postulated for aziridinyl

derivative by our group (Islam and Ahmad, 1990).

The results obtained with the E. coli, phage and

Salmonella suggest that the test steroids bring about the DNA

damage and initiate SOS-response with the concomitant

induction of mutation. The Ames tester strains carry pKMIOI

plasmid which is believed to enhance the error prone repair

process (Walker, 1984, Little et aT. , 1989). The induction

of SOS response with the test steroids (Figs.1-4, Chap-V)

was supported by the high sensitivity of recA, lexA and rer

mutants of E. coTi strains. The role of recA'^ and lexA"*" genes

in initiating the error-prone repair in E. coli is well

documented (Walker, 1985). The role of rer gene has also

been implicated in initiating the SOS response Aziridnyl

steroids have been shown to induce SOS responses in E. coli

(Islam and Ahmad, 1991). A significant amount of induction

of prophage in X lysogen and requirements of de novo

protein synthesis for this process (Table-1, Chapter-V)

further substantiate our postulation. Moreover, the

remarkable degree of enhanced mutagenesis in the amp and tet

loci of PBR322 after treatment of both plasmid DNA as well as

E. coli cells (Weigle mutagenesis) further supports our idea

regarding the initiation of SOS response in the w.t. E. coli

cells (Tables-3, Chapter-V). Furthermore, the results of

Chapter-V (Table-3) also indicate that the steroid induced

mutagenesis may also be due to misreplication in addition to

misrepair. Steroid induced mutagenesis was recA dependent

(Chapter-V). The role of recA protein in diminishing the

proof reading activity of DNA poly me rase III is an

established fact (Lu et al. , 1986). Moreover, the damaged

DNA could induce an inhibitor which might reduce the proof

reading 3'-5' exonucieolytic activity of DNA polymerases

(Vi Hani et a1. , 1978).

The considerable reduction in the transformation

frequency in case of recA mutant of E. coli on steroid

treatment would be the result of DNA modification which goes

unrepaired (Table-2, Chapter-V). It can be presumed that the

reduction of transformation in steroid treated pBR322 as well

as bacterial cells is the result of DNA damage caused by

these treatments (Bucala et a7., 1984; Palu et a1., 1987).

Moreover, the in vitro inactivation rate for double-stranded

pBR322 allows one to predict the frequency of steroid damage

that may be occurring in the larger genome.

The results presented in Chapter-V (Figs.5-11)

demonstrated that the test steroids inactive the phage to

a significant extent particularly when the phage was

complexed with SOS defective E. coli strains (Figs.5-11,

Chapter V). In addition the reduction in PFU of red was

remarkably significant on steroid treatment (Figs. 9 and 10,

Chapter V) indicating the role of red gene in the steroid

induced lesions of A -DNA. The red^ gene function of A is

considered to be complementary to that of recA^ gene of E.

coli (Srivastava,1973, Musarrat and Ahmad, 1991). These

results confirm the active role of SOS system in the steroid

induced lesions in the DNA.

A plausible explanation can be envisaged in view of the

induction of single and double strand breaks and the

destabi1ization in the secondary structure of DNA on

treatment with steroids (Chapter-VI). This is evident by the

increased level of single strandedness in duplex DNA as

observed by S^ nuclease hydrolysis of treated calf thymus DNA

(Fig.1, Chapter VI), decrease in the melting temperature

(Fig.2) and the decrease in the mol. wt. of calf thymus DNA

on steroid treatment as observed by alkaline and neutral

sucrose gradient centrifugation (Figs.3, 5, 6; Chapter VI). A

significant amount of single strandedness in calf thymus DNA

was earlier reported in case of aziridinyl steroids by our

group (Islam,S, Ph.D. Thesis, 1991). The single strandedness

of DNA as well as the steroid induced DNA degradation was

significantly enhanced in the presence of transition metal

ion Cu(II) (Figs.1b, Table-4, Chapter-VI). Moreover, a

significant inhibition of S^ hydrolysis by active oxygen

radical scavengers clearly indicate the role of these active

oxygen species in the strand breakage (Table-2, Chapter VI).

The induction of DNA strand breaks by active oxygen species

in presence of Cu(II) has been well documented (Dizdaroglu et

a?., 1991). Moreover, the nick formation in plasmid and

phage DNA (Figs.7, 8) and the significant decline in the

survival of polA and Tig mutants as compared to wild type E.

coli further support this idea.

Ionizing radiation which is one of the sources for 'OH

induces direct strand breaks in addition to other lesions

such as sugar damage, base damage and cross linking (Lliaks,

1991). Complex lesions like double strand breaks cleave the

DNA molecule and reduce its molecular weight. The complex

lesions can represent a more serious problem since they may

either be irreparable or require for their repair complicated

mechanisms such as recombination. These lesions may also be

mis-repaired (Tobias et al., 1980) with relatively high

probability giving rise the exchange type chromosome

aberration (Dewey et al, 197i; Joshi et a1., 1982) and

deletion type mutations (Thacker, 1985; Breimer, 1987).

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List of Publications and Communications

1. Qadri, S.A., Islam, S. and Ahmad, M. (1992) "Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli". Mutation Research (The Netherlands), 298, 53-60.

2. Qadri, S.A. and Ahmad, M. (1992). "Steroid induced single strand breaks in DNA mediated by active oxygen radicals and its biological consequences", (in press) Biochemistry Int. (Australia).

3. Qadri, S.A. and Ahmad, M. (1993). "Production of activated oxygen species by oxathiolane steroidal derivative and its role in mutagenicity", communicated to Mutation Research (The Netherlands)

4. Qadri, S.A., Islam, S. and Ahmad, M. (1993). "Steroid induced lesions in DNA", communicated to Mutagenesis (U.K.).

5. Qadri, S.A. and Ahmad, M. (1993). "Steroid induced damage and mutagenesis in E. coli and Phage lambda", communicated to Anti Cancer Drug Design (U.K.).

SUMMARY

Both naturally occuring and synthetic steroids have

wide application in industries and medicine (LaBarecque,

1961; Knipling, 1962; Oboshi et a1., 1967; Fishbein, 1984;

Barnes, 1987 ; Zbigniew and Krystyma, 1988; Henry et al. ,

1989; Nakaaki, 1989). Moreover, oxizolines and aziridines

have shown to possess diversified types of biological

activities (Brokovec and Wood, 1969; Levett, 1970a, b;

Lusthof et a1., 1988). Some steroids have been derivatized

in order to widen their spectrum in pharmacological use

(Shafiullah, et al., 1988; 1989; 1990). Before expecting

any further development in this field of pharmaceutical

chemistry, the hazardous effect of these steroids in terms of

genotoxicity were tested in light of genotoxic effect of

certain other steroids (Allen et al., 1981; Metzler, 1984;

Heywood, 1986; Ochs et al. , 1986; Sadek and Abdelmguid,

1986). Our group has also highlighted the genotoxic effects

of some synthetic steroidal derivatives (Islam et al., 1991;

Islam and Ahmad, 1991). The short-term testing along with

the DNA damage and repair assays would be the first step

forward in the direction of minimizing the exposure to the

potentially toxic steroids.

The significant findings and possible explanations of

the ste roid induced mutagenesis and generation of active

oxygen species as well as its interaction with DNA are

summarized as under:

I. Ames testing of test steroids

1. All the test steroids resulted in the enhanced

mutagenicity and responded significantly even in the

absence of post mitochondrial fSg) fraction, however,

the addition of Sg fraction further enhanced the

rr.utagenic activity of the steroids.

2. The steroids preferential ly acted on the AT base pair

transition mutants, TA102 and TA104.

3. The parent steroids exhibited less mutagenicity

compared with their oxygenated derivatives.

4. Among the oxathiolane series of steroids, the acetoxy

derivative was more mutagenic in presence of Sg fraction

as compared to other derivatives.

5. The mutagenicity of steroids was further enhanced with

the increase in the number of oxygen atoms in

derivatives. Moreover, the remarkable mutagenic

response with TA102 and TA104 strains suggested for the

oxidative nature of mutagenic species.

These findings are clear indicative of the highly

mutagenic nature of the test steroids with the preferential

induction of AT — ^ GC transition mutation.

II. Role of oxygen radicals in steroid induced mutagenesis

1. The mutagenicity of steroids probably involved the

generation of active oxygen species in the system. Test

steroids when subjected to in vitro generation of oxy

radicals, a significant production of almost every type

of active oxygen species f02'~, "OH, H2O2, was

produced.

2. The production of active oxygen species were

significantly inhibited by the free radical quenchers.

3. A remarkable amount of inhibition in the mutation

frequency of TA102 and TA104 with steroid I in the

presence of oxygen radical scavengers was observed.

Thus indicating the active involvement of oxygen

radicals in the test system.

III. Steroids as inducers of SOS response

1. DNA damage repair mutants of E. coli were highly

sensitive towards the test steroids compared with their

wild type isogenic strain. Only uvrA mutant was

relatively less sensitive towards steroid treatment.

2. A fraction of lysogenic population of ^cI857/AB1157

exhibited the induction of Ibytic cycle during liquid

holding in nutrient broth at 32* 0 even after 3h steroid

I treatment to lysogen. The prophage induction also

required the de novo protein biosynthesis.

3. The test steroid I and VIII as well as benzota]pyrene

treatment induced a s,ignificant amount of mutation in

ampicillin and tetracycline locii of E. coli

transforments after treating pBR322. plasmid DNA.

4. The extent of mutation was high when both E. coli cells

as well as pBR322 was treated resulting in Weigle

mutagenesis,

5. A considerable reduction in the transformation frequency

was observed when the E. coli AB1157 fw.t.) cells were

transformed with steroid treated pBR322 DNA. However,

this reduction was much more pronounced with E. coli

AB2463 (recA).

6. The phage showed a significant reduction in PFU on

steroid I and VIII treatments. The phage inactivation

was enhanced when phage was complexed with DNA repair

defective mutants of E. coli K12 like recA and polA

strains. In case of Tired the inactivation was

remarkable.

7. An enhanced level of c mutation was observed when

treated phage was allowed to infect on E. coTi

host. Induction of c mutation was also noticed when

untreated phage "Xc^ infected the steroid treated E.

CO77 cells. However, the mutation frequency remarkably

increased when the treated phage was adsorbed on treated

E. coli (Weigle mutagenesis).

These findings strongly support for the initiation of

inducible error-prone SOS response with in the steroid-

treated E. coli cells. Our observations further suggest for

the involvement of both mis-repair as well as mis-replication

in the steroid induced mutagenesis.

IV. Steroid induced DNA lesions: in vivo and in vitro studies

1. Steroid treatment at low doses induced S^ susceptible

sites in the calf thymus DNA, although high dose

treatment itself could also degrade the DNA without the

requirement of S^ nuclease.

2. The S^ hydrolysis was significantly enhanced in the

presence of transition metal ions like Cufll).

3. The S^ hydrolysis was, however, inhibited to a

significant level in the presence of oxygen radical

scavengers.

4. The steroid I treatment induced nicks in the super

coiled pBR322 and M13 phage DNA. The phage DNA was

totally degraded at higher concentrations.

5. The nicks in DNA were also induced under in vivo

conditions as observed by the reduction in the survival

of polA and Tig mutants of E. coTi on steroid treatment.

6. The single strand break formation was supported by the

results of alkaline sucrose gradient of steroid treated

calf thymus DNA.

7. Steroid treatment also induced double strand breaks in

calf thymus DNA as was evidenced by neutral sucrose

gradient studies.

8. The thermal melting profile data indicated a reduced Tm

upon treatment of DNA with steroid I. The Tm was

further reduced when DNA was treated with higher steroid

DNA bp/molar ratio.

In view of the above studies, it is evident that at

lower concentrations, ^the steroid primarily causes the

formation of single and double strand breaks, but at higher

doses the treated DNA seems to have undergone degradation.

genotoxrc effec ot f certain steroidal drugs · qadri for his ph.d work. . i am fully satisfied...

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