The role of metabolism in chemical mutagenesis and chemical carcinogenesis

215

Mutation Research, 75 (1980) 215--241 © Elsevier/North-Holland Biomedical Press

ICPEMC WORKING PAPER 2/2 THE R O L E OF METABOLISM IN CHEMICAL MUTAGENESIS AND CHEMICAL CARCINOGENESIS *

A.S. WRIGHT Shell Research Limited, Shell Toxicology Laboratory (TunstaU), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG (Great Britain)

(Received 24 August 1979) (Accepted 7 September 1979)

Introduction Chemical carcinogens and chemical mutagens m a y conveniently be classified as genotoxic agents. This general term was developed to cover toxic, lethal and heritable effects to karyotic and extrakaryotic genetic material in germinal and somatic cells [1]. In this paper, the use of the term is n o t intended to imply that all chemical carcinogens or indeed all chemical mutagens exert their adverse biological effects by means of direct interactions with DNA. Thus, while clearly appropriate in the case of chemical mutagens, the application o f the term "genotoxic agent" to chemical carcinogens acknowledges the opinion that the essential characteristic o f these latter chemicals resides in their ability to induce heritable changes in cell phenotype. Such phenotypic changes must involve heritable alterations in genetic structure (mutation) and/or transmissible changes in the regulation of genetic information (aberrant differentiation). The nature o f genotoxic agents Genotoxic agents can be considered under t w o main headings. T h i s p a p e r is b a s e d o n a p r e s e n t a t i o n g i v e n a t t h e f i r s t m e e t i n g o f C o m m i t t e e 2 o f t h e I n t e r n a t i o n a l C o m m i s s i o n f o r P r o t e c t i o n a g a i n s t E n v i r o n m e n t a l M u t a g e n s a n d C a r c i n o g e n s ( I C P E M C ) , Versailles, F r a n c e , D e c e m b e r 4 - - 7 , 1 9 7 8 . It h a s b e e n a g r e e d t o p u b l i s h t h i s d o c u m e n t as a w o r k i n g p a p e r f o r C o m m i t t e e 2 o f I C P E M C . T h e v i e w s e x p r e s s e d are t h o s e o f t h e a u t h o r a n d d o n o t n e c e s s a r i l y r e p r e s e n t t h o s e o f t h e C o m m i s s i o n . T h e y a r e p r e s e n t e d t o s t i m u l a t e d i s c u s s i o n a n d c o m m e n t s will be welcomed by the author. I C P E M C is a f f i l i a t e d w i t h t h e I n t e r n a t i o n a l A s s o c i a t i o n o f E n v i r o n m e n t a l M u t a g e n S o c i e t i e s a n d is s p o n s o r e d b y t h e I n s t i t u t d e la Vie. All c o r r e s p o n d e n c e w i t h I C P E M C s h o u l d b e a d d r e s s e d t o t h e secretary: Dr. P.H.M. Lohman, Medical Biological Laboratory TNO, P.O. Box 45, 2280AA RijswiJk ( T h e N e t h e r l a n d s ) , Tel. 1 5 - 1 3 8 7 7 7 , t e l e x 3 1 1 0 1 p m t n o ul. (ICPEMC document: 40A-1979-WP2/2-C2)

216

(1) Precursor genotoxic agents (pre-carcinogens, pre-mutagens) This class of genotoxic agents comprises a large number of chemicals of diverse chemical structures. The precursor agents can be grouped in the following classes. Polynuclear aromatic and heterocyclic hydrocarbons. Aromatic and heterocyclic primary, secondary and tertiary amines and azo dyes. Nitroaryl and nitrofuran compounds. Nitrosamines, nitrosocarbamates. Alkyltriazines, dialkylhydrazines. Acetamide, thioamides, carbamates. Certain chlorinated hydrocarbons, e.g. vinyl chloride monomer. Natural products e.g. cycasin, safrole, certain mycotoxins, pyrrolizidine alkaloids, bracken fern carcinogen. Precursor genotoxic agents possess no genotoxic properties per se hut are converted into ultimate genotoxic agents by metabolism * in susceptible organisms. Consequently, precursor agents are inactive in biological systems that lack the enzymes needed to convert them into their ultimate genotoxic forms. (Such metabolic under-competence m a y not always be the reason for the absence of genotoxic effects in a resistant species.) Thus, certain products formed during the metabolism of pre-mutagens in susceptible species have generally been found to be effective mutagens in test systems that yield negative results with the parent compounds. In the case of pre-carcinogens, such metabolic products have generally been found to be more effective than the parent in terms of carcinogenic p o t e n c y and in the range of tissues affected. Additionally, the incorporation of appropriate enzymes into previously insensitive biological test systems renders the systems susceptible to the genotoxic action of precursor genotoxic agents.

(2) Ultimate genotoxic agents Chemicals in this class, e.g. alkylating agents, possess the intrinsic properties necessary for interaction with critical cellular targets, thereby initiating the genotoxic process. The intrinsically effective, ultimate genotoxic agents generally give positive results in test systems that yield negative results with precursor compounds. In certain instances, the metabolic products or intermediates generated from ultimate genotoxic agents may also be intrinsically effective mutagens or carcinogens. However, this seems to be the exception rather than the rule and the metabolites of ultimate genotoxic agents are usually devoid of genotoxic activity. Consequently, the expectation would be that the metabolism of ultimate genotoxic chemicals would exert a limiting effect on their genotoxic actions.

(a) Ultimate carcinogens Consideration of the structures of ultimate carcinogens has led to the important generalisation that such agents are strong electrophiles, mainly alkylating and arylating agents, although some carcinogenic acylating agents are known

* F o r t h e p u r p o s e s o f t h i s d i s c u s s i o n , p r e c u r s o r a g e n t s t h a t are c o n v e r t e d i n t o u l t i m a t e g e n o t o x i c a g e n t s b y s p o n t a n e o u s c h e m i c a l r e a c t i o n s i n l i v i n g o r g a n i s m s , e.g. c e r t a i n N - m e t h y l - N - n i t r o s o c o m p o u n d s , are c o n s i d e r e d as t h o u g h t h e y w e r e u l t i m a t e g e n o t o x i c a g e n t s .

217 [2]. In certain cases, the instability of the presumed ultimate carcinogen prevents chemical synthesis. In such instances, e.g. the 2,3-epoxide of aflatoxin B1, the nature of the ultimate reactant has been inferred from the structures o f adducts generated b y reaction of the formed products with biomacromolecules in situ [3]. There are a few apparent exceptions to the generalisation that ultimate carcinogens are electrophilic reactants. One such exception, 6-mercaptopurine, has been reported to cause increases of certain turnouts in the haemopoietic system of rats and mice [4].

(b) Ultimate mutagens and the relationships between mutagens and carcinogens As in the case of the ultimate carcinogens, most ultimate mutagens are also electrophilic reactants. These chemical similarities together with evidence that ultimate carcinogens possess mutagenic activity are consistent with the thesis that chemical mutagenesis is causally related to chemical carcinogenesis. However, as Miller and Miller have pointed o u t [2], these shared properties do n o t constitute p r o o f of a causal association. Thus, it is clear that the electrophilic genotoxic agents are capable of reacting with nucleophilic centres in all informational macromolecules, i.e. DNA, R N A and proteins, any or all of which m a y be critical targets of chemical carcinogens. This indiscriminate reactivity militates against the use of such c o m p o u n d s as molecular probes for the identification of the critical targets of genotoxic agents. Thus, while the same chemical entities may induce neoplasia and mutations it is possible that the key primary targets m a y differ in each of these processes. Recent results [ 5--7 ] indicating that a formal correlation may exist between bacterial mutagenicity and mammalian carcinogenicity have led to much speculation and debate concerning the utility of bacterial test systems for the prediction of mammalian hazards. There are some apparent exceptions to the correlation between such mutagenic and carcinogenic activities. Thus, several mutagens have been reported to be non-carcinogenic and, partly dependent u p o n definitions of the term "chemical carcinogen", vice versa. In the case of chemical mutagens, some of the apparent anomalies may arise because the correlation is based on data obtained in different species, e.g. mutagenicity assessed in bacteria; carcinogenicity assessed in mammals. Interestingly, it seems that there is no authenticated example of a chemical that is known to induce mutation in somatic or germ cells in vivo b u t fails to induce a carcinogenic response in the same species. The "non-carcinogenic" mutagens can be divided into 3 main classes: (1) Electrophilic bacterial mutagens that are believed to be non-carcinogenic because their intrinsic electrophilic reactivity is t o o low or because they fail to penetrate to critical targets, e.g. dichlorvos, certain aliphatic epoxides such as a glycidol and 1,2-epoxybutane (for a review see ref. 8) or their precursors such as squalene. (2) Nucleophilic mutagens, e.g. hydroxylamine, and bisulphite. (3) Ultimate mutagens that induce structural changes in DNA by specific mechanisms b u t which do n o t appear to cause structural modifications in R N A or proteins, at least n o t b y a direct mechanism. It would seem that c o m p o u n d s in this third category which include certain

218 simple intercalating agents and DNA-base analogues (deoxyribosides) could be used with advantage to study the relationships between mutagenesis and carcinogenesis. Their utility in this respect would, of course, be subject to the proviso that they are effective mutagens in vivo. The conclusion that the deoxyribose-base analogues are mutagenic is based entirely on results obtained in in vitro test systems, mainly bacterial test systems. It should also be pointed o u t that, in some instances, e.g. 2'-deoxy-5-iodouridine, the experimental procedures employed in the carcinogenicity assays may n o t have been entirely appropriate, i.e. the compounds were administered via the oral route and this may have resulted in scission of the bond linking the deoxyribose moiety to the base in the gut [9].

The nature o f the critical targets of chemical mutagens and carcinogens DNA is established as the ultimate although not obligatory primary target of chemical mutagens [10]. This knowledge facilitates an appreciation of the influence that endogenous, i.e. host-dependent, factors or processes such as DNA-repair or enzyme-mediated biotransformations of chemical mutagens may have on the development of heritable changes in the structure of DNA in exposed animals. Unfortunately, current knowledge of the mechanism of action of chemical carcinogens is, at best, fragmentary. The fact that the critical target(s) of chemical carcinogens has not been defined is a particularly serious encumbrance in considerations of the possible impact that certain hostdependent factors may have on the carcinogenic process. However, largely because of recent advances in the understanding of the nature of chemical carcinogens [2], coupled with the fund of information on the metabolism of foreign compounds, the indefinite nature of the critical targets of chemical carcinogens is possibly of only marginal significance in considerations of the role of metabolism in chemical carcinogenesis. Despite the current limitations in the understanding of the mechanism of action of genotoxic agents, particularly chemical carcinogens, it seems axiomatic that the adverse biological effects of such compounds must ultimately be dependent upon initial interactions between the toxic agents and key cellular targets. In this paper emphasis is placed upon these initial interactions. However, it should be stressed that, while the electrophilic nature of genotoxic agents has caused attention to be focused upon nucleophilic centres in informational macromolecules as prime targets, the nature of the biological counterparts of such interactions is, in general, not defined or not well-defined. Additionally, it should be borne in mind that modulating * factors such as DNArepair, immune competence or cell replication may exert an important, possibly an overriding influence on the realisation of mutagenic or carcinogenic effects. The nature of the initial interaction between a genotoxic agent and its critical cellular target is u n d o u b t l y a key determinant of the nature of the ensuing biological effect and is entirely dependent upon the physico-chemical proper*

N o t e t h a t p r i m a r y i n t e r a c t i o n s w i t h s u c h m o d u l a t i n g f a c t o r s have b e e n p r o p o s e d as k e y i n i t i a l e v e n t s in the i n d u c t i o n o f g e n o t o x i c e f f e c t s .

219 ties of the interacting c o m p o n e n t s under the conditions prevailing in the microenvironment of the target. Because of the complex nature of genotoxic processes and their susceptibility to modulating influences*, the quantitative relationships between key initial events and overt genotoxic effects are also likely to be complex a n d subject to variation between species, individuals and tissues. Nevertheless, it seems reasonable to infer that the magnitude or frequency of such an effect would be a function of the rate of the initial interaction which would, in turn, be a function of the concentration of the genotoxic agent at the target locus. Whereas it had previously been appreciated that the mammalian enzymes catalysing the metabolism of genotoxic agents were among the more important endogenous factors that influence the concentration of a genotoxic agent at its target and, consequently, the magnitude of primary genotoxic effects, it is n o w clear that the operation of some of these enzymes can also determine the structures of ultimate genotoxic agents and thus the chemical nature of the interactions. Such enzymes must therefore be regarded as key determinants of b o t h the magnitude and nature of the adverse biological effects of genotoxic agents.

The activation o f precursor genotoxic agents The genotoxic actions of pre-mutagens and pre-carcinogens are dependent upon the presence of appropriate activating enzymes. The metabolic pathways leading to the generation of the ultimate reactive species are the subjects of continuing and intensive investigations. As stated above, the precursor .genotoxic agents comprise a wide range of structurally unrelated classes of chemicals. Most of these chemicals are lipophilic at physiological pH. The efficient excretion of lipophilic c o m p o u n d s b y mammals necessitates their conversion into hydrophilic products. In most instances this biotransformation is a multi-step process, each stage catalysed b y a different enzyme and leading to a stepwise increase in hydrophilic character. Certain lipophilic c o m p o u n d s lack a functional group and are essentially unreactive. In such cases, e.g. polycyclic aromatic hydrocarbons, an initial oxygenation reaction is required to prime, i.e. introduce a functional group into, the c o m p o u n d for subsequent metabolic transformation. This primary metabolic reaction is usually catalysed b y membrane-bound (microsomal) monooxygenases.

(a) Nature and distribution o f mono-oxygenases The microsomal mono-oxygenases are widely distributed in mammalian tissues. Indeed, mono-oxygenase activity has been detected in all nucleated mammalian cells studied to date. The mono-oxygenases are located in microsomes {derived from the endoplasmic reticulum b y homogenisation) and in the nuclear envelope [12]. They are complex enzymes and have been resolved into 3 essential components: N A D P H - - c y t o c h r o m e P-450 reductase, phosphatidyl choline and a haemoprotein, c y t o c h r o m e P-450, that functions as the terminal * M a t u r e s p e r m m a y b e i n c a p a b l e o f D N A - r e p a i z [ 1 1 ] . H o w e v e r , t h e repair o f d a m a g e i n d u c e d i n p o s t - m e i o t i c stages p r o b a b l y t a k e s p l a c e at fertillsation.

220 oxygenase [13--17]. Substrate specificity appears to reside in the haemoprotein moiety and there is evidence that cytochrome P-450 exists in multiple forms, e.g. cytochromes P-450 and P-448, within any particular tissue. This multiplicity of form together with the fact that the mono-oxygenases are adaptive or inducible enzymes [18,19] provide mammalian tissues with an extremely versatile system capable of catalysing the oxygenation of a diverse range of endogenous and xenobiotic substrates. Reactions catalysed by the mono-oxygenase system include aromatic and aliphatic hydroxylation, arene and alkene oxide formation, oxidative N-, O- and S-dealkylation, sulphoxidation, oxidative deamination, desulphuration and dehalogenation [18,20]. Marked tissue and species differences in the activities and distribution of mono-oxygenases have been reported. Inter- and intra-species comparisons have demonstrated that the liver generally possesses the highest mono-oxygenase activity. However, activity can also be measured in lung, skin, intestine, kidney, adrenals and other organs. Of the lung enzymes studied, that in rabbit possesses the highest activity [21--23]. A comparative study of mono-oxygenases in the liver, lung and kidney of rat, mouse, rabbit, hamster and guinea-pig showed that the liver was the most active organ [23]. Overall, the hamster yielded the highest values although the rabbit displayed the highest activity in lung. In most of the test species, the individual values for kidney and lung ranged from 15 to 40% of the activity in the liver. It should be noted that the results of mono-oxygenase assays vary markedly according to substrate, strain differences, diet, circadian rhythm, etc. Accordingly, the results of such studies should n o t be interpreted t o o broadly. Nevertheless, while mean values of c y t o c h r o m e P-450, NADPH-cytochrome c (-cytochrome P-450} reductase and c y t o c h r o m e bs of human liver were found to be only slightly lower than rabbit [24], human-liver microsomes generally do n o t possess the high capacity for the oxidative metabolism of foreign c o m p o u n d s characteristic of rabbit- or rodent-liver microsomes [25--29]. Certain N-oxidations that do not require c y t o c h r o m e P-450 may be exceptions to this generalisation [30]. In addition, there is evidence that the mono-oxygenases of primate livers may be resistant to induction b y certain microsomal enzyme inducers [31]. Because of the key role of the microsomal mono-oxygenases in the activation of precursor genotoxic agents it is clear that these species differences merit further investigation. In addition to species and tissue differences, the properties of the monooxygenase system have also been found to vary according to age and pre-treatment with inducers or inhibitors of mono-oxygenase activity (For a review see ref. 19}. The well-known sex difference in the activities of the mono-oxygenases of rat liver (male > female} may not be a general phenomenon and is n o t characteristic of all organs in the rat. Thus, an investigation in the rat revealed no sex difference between the activities in lung or intestine [32]. Intra-species comparisons have indicated that adult-liver microsomes are more effective than the corresponding preparations from foetal liver in catalysing mono-oxygenation reactions. This generalisation appears to hold with respect to human liver [33]. The distributions of the terminal oxygenases, c y t o c h r o m e P-450 and P-448, also vary with age. Thus, cytochrome P-448 has been reported to be the predominant form in foetal and neonatal rat liver, whereas P-450 tends to predominate in the adult rat. The specificities of these

221 enzymes differ. For example, 4-hydroxylation of biphenyl is mediated by cytochrome P-450 while 2-hydroxylation of this substrate is catalysed by P-448. Interestingly, it has been reported that 2-hydroxylation is enhanced by in vitro incubation of adult rat-liver microsomes with carcinogenic polycyclic aromatic hydrocarbons and certain other carcinogens [34]. Comparisons of the DNA-adducts generated during the incubation of certain genotoxic polycyclic aromatic hydrocarbons with bacteria or isolated DNA in the presence of rat-liver microsomes with those formed either during in vivo exposure of mouse skin or in vitro incubation with mouse embryo cells have revealed apparent qualitative differences [35--37]. It is, as yet, uncertain whether these differences are a consequence of qualitative differences in the metabolism of polycyclic aromatic hydrocarbons in rats and mice. In vitro studies of the metabolism of 2-acetylaminofluorene revealed no N-hydroxylation in the presence of guinea-pig liver microsomes [38,39]. N-Hydroxylation was, however, detected in the corresponding incubations with ratliver microsomes. This may be a significant species difference. Thus, the guineapig is reputed to be resistant to the hepatocarcinogenic action of 2-acetylaminofluorene [40]. 2-Aminofluorene, i.e. the non-acetylated analogue, is N-hydroxylated by guinea-pig liver microsomes [41].

(b ) Mono.oxygenation reactions It is now clear that a primary mono-oxygenation reaction is the key event in the activation of many precursor genotoxic agents such as the polycyclic aromatic hydrocarbons and aflatoxin B~. A general scheme illustrating the activation and deactivation of aromatic hydrocarbons is shown in Fig. 1. (Deactivation reactions are discussed later) The genotoxic polycyclic aromatic hydrocarbons can undergo oxygen insertion reactions at a number of double bonds. The products of these concurrent reactions vary considerably in carcinogenic/mutagenic potency [42,43]. Although such mono-oxygenase-catalysed biotransformation of polycyclic aromatic hydrocarbons can lead to the generation of powerful carcinogens and mutagens, it has been suggested that single-electron oxidation to radical cations

G

[0] "-- ~ 0 Mono-mogenm , /

/

/

[ Glutathione

Nhydretue

"'i- x ,oo//OH

/.\" O-Sulphltu

OH R

O-Glucuronicles

Fig.1.Majorp a t h w a y s o f m e t a b o l i s m

of aromatic hydrocarbons.

222

may also provide a route for the activation of such compounds [44,45]. Many lipophilic compounds that possess functional groups, e.g. aromatic amines, also undergo oxygenation reactions despite the fact that alternative metabolic routes are available for their conversion into hydrophilic products (Fig. 2). Thus, oxygen addition to the amino-N atom to yield the correponding N-hydroxy compounds has been implicated as an essential step in the activation of the genotoxic aromatic amines [40]. The substitution of the two hydrogen atoms ortho to the amino group by, for example, two methyl groups is believed to block such activation. Thus, benzidine is a powerful mutagen and carcinogen whereas the corresponding 3,3',5,5'-tetramethyl analogue is inactive both as a carcinogen [46] and as a mutagen [47]. Ashby [48] has attributed the inactivity of the latter compound to steric effects and has pointed out that the sterically less hindered 3,3',5,5'-tetrafluorobenzidine is strongly mutagenic [47]. Primary oxygenation reactions have also been implicated in the activation of the genotoxic nitrosamines and related compounds [49]. The activation pathway(s) has not yet been elucidated. However, esterified 1-hydroxyalkyl-alkylnitrosamines have been proposed as the proximate mutagens (Fig. 3). Metabolites of this type may arise via hydroxylation and conjugation at the resulting hydroxy group [50,51]. ( c ) Other primary reactions Although the activation of most classes of precursor genotoxic agents necessitates an initial oxygenation step, there are instances when other types of primary metabolic reactions lead to the generation of proximate or ultimate genotoxic agents. For example, the activation of genotoxic nitro compounds,

R~ N~.~~ ~NHOH

0

\

o.

~

IH

N ~.AC

R Fig. 2. Principal p r i m a r y reactions undergone by aromatic a m i n e s .

223

CH3\ N --N

.IvO

c./

CH3~ =

N --N

''''tO

.oc. CH3 ~ O AcOCH2/~-NN

Fig. 3. P o s s i b l e s t a g e s i n t h e m e t a b o l i c

activation

of

dimethylnitrosamine.

e.g. 4-nitroquinoline-l-oxide (4-NQO), is believed to involve reduction to the corresponding N-hydroxy derivatives. Such reduction may be mediated by a number of different enzymes [52]. Interestingly, Salmonella is capable of reducing 4-NQO into 4-hydroxyaminoquinoline-l-oxide (4-HAQO) [53,54]. Although there is evidence suggesting that 4-HAQO is an ultimate mutagen, this compound can be further metabolished into a more reactive form(s) by the action of certain species of aminoacyl-tRNA synthetases such as serinyl-tRNA synthetase (Fig. 4) [55--57]. Whilst azoreductase-catalysed reduction may lead to the detoxification of certain genotoxic azo dyes, e.g. 4-methylaminoazobenzene [58], in other instances such reaction may be directed towards activation of the azo compound. For example, the action of azoreductase on 2-[4'
NHOH O

O

~iAm

f"

?o

I Aminoacid,ATP,I

i~yl-,~P

©omplex

f 0 IP N-O-C-CH-R NH 2

I OH

O II N-O-C--CH--R

NOH

NH 2

O-C--CH-R II O

F i g . 4. P r o p o s e d m e c h a n i s m f o r t h e a c t i v a t i o n o f 4 - n i t r o q u i n o U n e - l - o x i d e .

--

II O

R

224 /t

XOA©

0

COOH

N'--O

N,%,Ac

I

:H H

Fig. 5. The metabolic activation of 2-acetylaminofluorene.

oN

o

"

~

o. +

HO\~~

,

,

~

Fig. 6. The metabolic activation o¢ benzo[a]py~ene.

225 highly reactive alkylating agent [59]. Similarly, the carcinogenic action of Scarlet Red has been attributed [48] to the azoreductase-catalysed generation of o-aminoazotoluene. At least two azoreductases, one soluble and the other microsomal, exist in rat liver [60]. In other instances, a third type of primary metabolic reaction, esterase- or amidase-catalysed hydrolysis, may lead to the scission of precursor genotoxic agents, e.g. the glycoside cycasin, to yield proximate or ultimate reactants [61]. There is evidence that esterase or amidase-catalysed deacetylation of N-hydroxy-2-acetylaminofluorene leads to the ultimate mutagen in the coupled microsome--microbial mutation assay [62].

The activation of proximate genotoxic agents Proximate genotoxic agents may be defined as intermediates on the metabolic pathways leading to the ultimate genotoxic reactants. The metabolic pathways followed by foreign compounds include a transferase-catalysed reaction of a functional group with endogenous conjugating agents, e.g. glucuronic acid in the form of UDPGA or sulphate in the form of PAPS. Such conjugation reactions lead to dramatic changes in the physicochemical properties of the xenobiotic substrates which limit biotransformation reactions and lead to rapid excretion of the foreign compound via the bile and/ or urine. Consequently, conjugation with such moieties is often the final step in the metabolism of many foreign compounds. Furthermore, the products of such reactions usually lack biological activity. There are, however, certain important exceptions to this latter generalisation. Thus, certain conjugation reactions are believed to play a critical role in the activation of proximate genotoxic agents. Current concepts of the further metabolic activation of the N-hydroxyamines and N-hydroxyamides have developed principally from studies on derivatives of 2-aminofluorene (Fig. 5) [2]. The glucuronidation or sulphation of the N-hydroxyamides affords chemically reactive molecules that are capable of interacting with cellular molecules such as DNA. Although the sulphuric acid ester is considered to be a major ultimate carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in rat liver, other ultimate electrophilic derivatives are probably also involved in the carcinogenicity of the compound (Fig. 5). The sulphuric acid ester is both extremely reactive and polar. These properties may explain why this compound is only weakly carcinogenic when applied to mouse skin and also why the addition of PAPS and rat-liver cytosol reduced the mutagenic activity of N-hydroxy-2-acetylaminofluorene in the Salmonella test [62]. Recent evidence suggests that the deacetylated analogue of this latter compound, i.e. N-hydroxy-2-aminofluorene, is ultimately responsible for the mutagenicity of 2-acetylaminofluorene in the coupled microsome-microbial mutation assay [62]. Although N-hydroxylation is regarded as an essential step in the activation of genotoxic arylamines, there is evidence suggesting that N-acetylation may represent the first step in the route to ultimate carcinogens. The reaction is catalysed by the soluble enzyme, arylamine N-acetyltransferase and is dependent upon the presence of the high energy donor-substrate, acetyl-coenzyme A.

226 The reaction is general for aromatic amines and hydrazino compounds. The transferase is present in the cytosol of various species. However, it is of considerable interest that dog-liver cytosol contains no detectable aryl-N-acetyltransferase and that dog liver is not susceptible to the carcinogenic action of 2-aminofluorene, 4-aminobiphenyl or 2-aminonaphthaiene [63]. It seems clear that hepatic N-acetyl transferase is a determinant of liver carcinogenesis by arylamines [64]. The primary oxidation of the genotoxic polycyclic aromatic hydrocarbons leads directly to the formation of powerful electrophilic reactants, i.e. arene oxides. Thus, the generation of the ultimate genotoxic forms of such hydrocarbons may n o t proceed via proximate agents. The secondary metabolic reactions undergone by arene oxides are directed towards deactivation (see later). However, in the case of certain polycyclic aromatic hydrocarbons, the operation of one of these "deactivating" pathways may give rise to proximate carcinogens (Fig. 6). For example, one of the products of the primary mono-oxygenation of b e n z o [ a ] p y r e n e is the 7,8-oxide. A principal pathway for the deactivation of this metabolite involves hydration, catalysed by epoxide hydratase, an enzyme previously considered to be invariably associated with the detoxification of alkene and arene oxides. The product of this reaction, the corresponding 7,8-trans-dihydrodiol, is incapable of undergoing chemical reaction with cellular macromolecules. However, this dihydrodiol is a substrate of the microsomal mono-oxygenase system and is further metabolised to yield the corresponding 9,10-epoxide. This latter c o m p o u n d is believed to be the ultimate carcinogenic form of b e n z o [ a ] p y r e n e [65,66]. Thus, in this instance, epoxide hydratase appears to play an indirect activating role. The deactivation o f genotoxic agents

(a) Precursor and proximate genotoxic agents In general, proximate genotoxic agents and precursors that possess functional groups may undergo concurrent reactions cataiysed by different enzymes. The operation of a particular pathway, is of course, dependent upon the presence of the appropriate enzyme. Some of these enzyme-mediated reactions are directed towards activation, others towards the destruction of genotoxic potential. For example, the genotoxic aminoazo compounds, e.g. azo dyes, can undergo a variety o f primary metabolic reactions including the oxygenation of aromatic nuclei or of the amino nitrogen atom and reduction of the azo group (Fig. 7). It is generally believed that oxygen addition at the aminonitrogen atom is associated with the activation of such compounds. On the other hand, reduction of the azo group to yield two monocyclie amines may often be an effective detoxification step. However, it should be borne in mind that certain monocyclic amines, e.g. o-toluidine, have been reported to be carcinogenic. Primary and certain secondary amines can undergo conjugation reactions to yield N-glucuronides. Such reaction would have a limiting effect on N-oxygenation. Carbamates and amides also form N-glucuronides. These latter conjugates are more stable than those of the aromatic amines. Although such primary conjugation reactions with UDPGA may generally he directed towards detoxification there may be instances, e.g. 2-acetylaminofluorene (discussed above),

227

0

~ N/H~CH3 "

6

l /N~

"-'X /0 GI~.

CH3

~ Primary mtabolh: reactions of aromatic amines

(of.Figs2and5)

/ OS03H N\CH3

Fig.7. Principalmetabolicreactionsof azo dyes. when glucuronidation reactions lead to the generation of reactive electrophiles. A variety of mechanisms have been proposed for the oxidation of aromatic nuclei in biological systems. For example, oxygen insertion at double bonds of aromatic nuclei (and in alkenes) yields intrinsically reactive electrophiles. Spontaneous rearrangement of the resulting arene oxides to yield non-genotoxic phenols provides a mechanism for the deactivation of such compounds. Recently, however, a direct route (direct insertion reaction of oxygen into C--H bonds) to phenolic metabolites has been shown to operate with certain aromatic c o m p o u n d s e.g. chlorobenzene, nitrobenzene [67,68]. There is no evidence to suggest that the genotoxic polycyclic aromatic hydrocarbons can be h y d r o x y l a t e d directly. However, it is possible that such direct C-hydroxylation may provide an effective detoxication route for some precursor and proximate genotoxic agents that contain an aromatic nucleus. Where there is more than one enzyme competing for one or more centres in the molecule, it is probable that the operation of a particular pathway is strongly influenced b y dosage rate. Thus, d e p e n d e n t upon the Km values and

228

the rate constants for the enzyme catalysed reactions, it is possible that only one route is operative or predominant at low doses [69]. The physico-chemical properties of the compound may contribute to this selectivity by limiting penetration to enzymes capable of catalysing alternative metabolic reactions. In the case of the genotoxic polycyclic aromatic hydrocarbons, it would appear that there is no alternative to the activation pathway(s), i.e. oxygen insertion at double bonds (Figs. 1, 6). However, polycyclic aromatic hydrocarbons possess a variety of double bonds that are capable of undergoing oxygen insertion reactions. Consequently, mono-oxygenation can give rise to a variety of arene oxides and these products display marked variations in their ability to induce carcinogenic or mutagenic effects [42,43]. Thus, at least to some extent, the genotoxic effects of the polycyclic aromatic hydrocarbons may be governed by the specificities of the mono-oxygenases available for such activation and also by the efficacy of secondary enzymes in catalysing the deactivation of the formed ultimate genotoxic agents. However, Sephadex LH20 chromatography of hydrolysates of DNA which had been incubated with benzo [a]pyrene in the presence of hepatic microsomes from each of 3 strains of mice revealed no correlation between the size of any peak formed in vitro and the susceptibility to tumour formation in vivo [70,71]. Similar findings have been reported with respect to in vivo binding of polycyclic hydrocarbons to the DNA of mouse skin and have led to the suggestion that strain susceptibility to hydrocarbon carcinogenesis may be determined by differences in the fidelity of DNA-repair or to different strain responses to promoting agents [72]. The results of recent experiments indicate that the susceptibilities of rat tissues to carcinogenic alkylating agents correlated with an excision-repair deficiency in the target tissues [73,74]. However, assuming that DNA is the critical target of chemical carcinogens, the formation and persistence of presumed pro-mutagenic lesions in DNA do not appear to be the only factors determining malignant transformation [ 75 ].

(b ) The deactivation o f ultimate genotoxic reactants As previously stated, most, ultimate genotoxic agents are electrophilic reactants. Mammalian tissues contain two enzyme systems, the S-glutathione transferases and the epoxide hydratases, that are especially efficient in scavenging electrophiles, the latter specifically epoxides, thereby affording protection to cellular nucleophiles against attack by such agents. The roles of these enzymes in the detoxification of arene oxides are illustrated in Fig. 1. 7 glutathione transferases have been identified [76,77]. The electrophilic centres that are subject to attack by glutathione include those in alkyl halides, aralkyl halides, aryl halides, epoxides, alkenes, chlorotriazines, nitro compounds and compounds containing electrophilic nitrogen or suphur atoms [64]. Glutathione transferase activity has been detected in all species examined to date, including man. The liver is the richest source of these enzymes but activity has been detected in most other tissues. Apparent exceptions, based on the assay of aryltransferase activity in Japanese monkey, are: pancreas, erythrocytes and blood plasma [78]. Measurements in the rat indicate that glutathione transferase activity is low at birth and rises steadily, for about 40 days, to the adult level [79,80]. The glutathione transferases are located mainly in the

229 cytosol [64]. However, Oesch has recently reported the existence of a membrane-bound (microsomal) glutathione transferase [ 81]. It is generally believed that the reactions between the nucleophilic sulphur atom of glutathione and electrophilic centres in genotoxic agents lead directly to a loss of bioactivity and to the rapid excretion of the formed products. However, some examples have appeared to suggest that such conjugation reactions may not always result in detoxification. Thus, certain nucleophilic substitution reactions at sulphur may lead to the generation of hemi-sulphur mustards. For example, Hathway and Bedford [82] have proposed that the efficient production of S-(2-hydroxyethyl)-glutathione from ethylene dibromide in liver preparations depends upon the intrinsic reactivity of the product of the first displacement reaction which is a "sulphur mustard" derivative (Fig. 8). Such a mechanism may explain the dependence of the bacterial mutagenicity of 1,2-dichloroethane and 1,2-dibromoethane on glutathione [83,84]. The epoxide hydratases catalyse the hydrolysis of a carbon--oxygen bond of epoxide rings. This reaction invariably results in the deactivation of genotoxic epoxides (Fig. 1) although in rare instances, e.g. benzo[a]pyrene, the hydrated product may be further biotransformed into another ultimate genotoxic agent (Fig. 6). Formerly, it was considered that epoxide hydratase activity was located exclusively in the endoplasmic reticulum in close functional and topological association with the microsomal mono-oxygenases. Consequently, it was believed that, while such enzymes may be effective in the deactivation of directly administered alkene or arene oxides, their subcellular location was ideally suited for the deactivation of epoxides generated endogenously by the action of the microsomal mono-oxygenases. However, the very recent discovery of a soluble epoxide hydratase may lead to a revision of these views, at least with respect to the capacity of epoxide hydratase to protect against the affects of direct exposure to epoxides [85]. To date, soluble epoxide hydratase has been detected in mouse and rabbit livers. Only very low activity was measured

BrCH2CH2Br

_.,

GSH

~

I GSCH2CH+Br ]

~2CH20 H Fig.8.

Reaction of ethylene dibromide with glutathione.

230 in rat liver. Results to date indicate that the soluble enzyme is most active on lipophilic, monosubstituted epoxides. Di-, tri- and tetra-substituted epoxides are also hydrated with the hydration rates decreasing in that order. It is not known whether or not this soluble enzyme can influence the genotoxicity of aflatoxin B, or the carcinogenic polycyclic aromatic hydrocarbons. The balance between activation and deactivation Apart from the key role of activating enzymes in conferring electrophilic properties on non-genotoxic precursors, the kinetics of the enzyme-mediated biotransformation of genotoxic agents are important determinants of the magnitude of the primary lesions induced b y these chemicals. In the case of direct exposure to intrinsically reactive genotoxic agents, the efficiencies and capacities of the deactivating enzymes are of paramount importance. In the case of exposure to precursor agents, it is the balance between the activating and deactivating enzymes that is important. These enzymic determinants of genotoxicity operate at the level of the cell and it is clear that individual mammalian cell types display major variations in their capacity to activate precursor genotoxic agents. Variations in the capacities of different mammalian cell types to deactivate genotoxic agents are less well documented. Quantitative information on the effective balance between activation and deactivation is sparse although the results of carcinogenicity studies in rodents indicate that, for many compounds, the balance favours activation. Current approaches to determine the overall balance between factors that influence the cellular concentration of ultimate genotoxic reactants are based on measurements of the extent of covalent interactions between the ultimate electrophilic reactants and cellular macromolecules. In addition to reaction kinetics at the level of the cell, the overall in vivo kinetics of metabolism of a c o m p o u n d may play an important role in determining the Concentration of the genotoxic agent at cellular targets in the intact animal. In this respect, metabolism at the portals of entry, i.e. skin, lungs and intestine, and in the liver is especially important. Compared with other organs, the liver has a high capacity to metabolise foreign compounds. The limiting effect of hepatic metabolism on the exposures received b y other tissues has recently been demonstrated in rats dosed orally with dimethylnitrosamine [86]. Metabolism in vitro (1) In vitro test systems While cultured mammalian cells and bacterial cells can be used to detect genotoxic activity in vitro, such simple models cannot accurately mimic the complex kinetics of foreign c o m p o u n d s in vivo. Although certain species of bacteria have been shown to possess a cytochrome P-450-dependent mono-oxygenase system that bears some resemblance to the mammalian system (for a review see ref. 87) it is clear that the test organisms employed routinely in the coupled liver microsome-microbial reverse mutation test are generally ineffective in activating precursor mutagens into

231

their ultimate genotoxic forms. Consequently, in the absence of mammalian mono-oxygenases, these detector cells fail to detect most of the precursor genotoxic agents. Certain mammalian cell lines that are employed in the mammalian cell transformation assay also display low mono-oxygenase activity. Such metabolic undercompetence has led to the development of test systems in which preparations from mammalian tissues, principally liver, are added to the detector cells in order to mimic the in vivo metabolism of test compounds. The general current trend is to use, as the extra-cellular enzyme source, the postmitochondrial supernatant ($9 fraction) from the livers of Aroclor-treated rats. However, these systems can be adapted to employ enzymes from a wide variety of mammalian tissues and species, including humans. This is a potential advantage particularly in the study of the mechanism of action of genotoxic agents. The objective of the test determines h o w closely the metabolism of the test c o m p o u n d s under the in vitro test conditions should relate to that occurring in vivo. For example, if the objective is to determine whether the enzymes in a particular tissue may be capable of conferring genotoxic properties (as defined by the genetic end-point of the test, i.e. bacterial mutagen or mammalian celltransforming agent) on inactive precursors then it is essential that all of the potential activating enzymes in that tissue are present and operational in the in vitro test system. However, there is evidence to suggest that the procedures employed in the preparation of subcellular fractions, e.g. liver microsomes, can lead to perturbations. Thus, major differences in the profile of metabolites were observed when b e n z o [ a ] p y r e n e was incubated with intact hamster embryo cells or with microsomes derived from these cells [88]. For reasons discussed later, the possibility that some of these observed differences may be attributed to inefficient operation of secondary metabolic pathways is largely irrelevant to the current issues. Such perturbations indicate that it may be incautious to presume that expected activation pathways are being followed in vitro. Nevertheless, in practically all instances, the operation of specific metabolic pathways is presumed rather than estabhshed experimentally. A clear-cut example of the dangers of such presumption is provided by phenobarbitone. Phenobarbitone has been tested in the coupled Salmonella--rat liver $9 test with negative results [89]. In vivo studies have shown that phenobarbitone is efficiently metabolised b y the rat (Fig. 9). The major metabolite is the 4ohyd r o x y derivative and this is excreted in the urine together with the corresponding O-glucuronide [90]. The detection of 5-(3,4-dihydroxy-l,5-cyclohexadien-l-yl)-5-ethyl barbituric acid in the urine of male rats and guinea pigs dosed orally with phenobarbital provides evidence that the oxidative metabolism of this c o m p o u n d may proceed via an arene oxide intermediate [91]. Thus, the results of these in vivo metabolism studies indicate that phenobarbitone is a typical substrate of the rat-liver microsomal mono-oxygenase system. However, in vitro studies in our laboratory [92] and in Peters' laboratory [93] with liver microsomes and 10 000 g supernatants from non-induced and induced rats have failed to detect any metabolism of phenobarbitone. While the reasons for this are n o t understood it is k n o w n that the sustained mono-oxygenation of dieldrin b y rat-liver microsomes is dependent u p o n the operation of the glucuronyl transferases [94] and it is possible that the incorporation of UDPGA into the in vitro system m a y help to drive metabolism of phenobarbitone and other compounds.

232

~

E..,ICON\ C

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o-1" \ Y \c~,,/ "% /9 fl I o

i?

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1 0

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II I E~,/C~N\ ~C

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.,,L,. /.,J

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of phenobarbitone.

(2) The balance between activation and deactivation Although there may be instances when oxidative pathways fail to operate effectively in vitro there is little d o u b t that the balance between the operation of activation and deactivation enzymes in " a p p r o v e d " versions of the coupled microsome-microbial reverse mutation test favours activation. This clearly imposes severe constraints on the extent to which test results can be interpreted. The two most important deactivating enzymes, epoxide hydratase and the S-glutathione transferases, are present in the $9 fractions derived from mammalian liver. Indeed, it is probable that such fractions represent the richest source of these enzymes. Epoxide hydratase has no special co-factor requirements and should be fully operational under the conditions prevailing in the in vitro test systems. However, the results of recent studies have shown that, because of dilution and oxidation of glutathione during the preparation of $9 fractions, the extracellular glutathione transferases would be almost completely inoperative under the conditions normally operating in such in vitro tests [95]. Consequently, it was n o t surprising to find that a normal physiological concentartion (5 mM) of glutathione afforded protection to Salmonella typhimurium TA100 against the mutagenic action of cis-l,3-dichloropropene [95]; a comp o u n d that is efficiently metabolised via the glutathione pathway in vivo [96].

233 Interestingly, this protection was at least as effective in the absence of S9 as in its presence. The addition of glutathione has also been reported to protect against the bacterial mutagenicity of 1,2-dibromo-3-chloropropane [97 ]. These results which indicate that glutathione conjugation inactivates genotoxic agents contrast with recent findings that, in special cases, e.g., 1,2-dibromoethane and 1,2-dichloroethane, such conjugation reactions may activate compounds in the Salmonella test [83,84]. The relevance of these latter, i.e. activation, reactions to the in vivo situation is unknown. Guinea-pig cytosol has been shown to inhibit the mutagenic effects observed when benzo[a]pyrene was incubated with Salmonella typhimurium in the presence of guinea-pig or mouse liver microsomes. The mechanism of this inhibitory effect is unknown [98]. The role of conjugation reactions, e.g. glucuronidation and sulphation, that are dependent upon the presence of high energy co-substrates should not be neglected in any consideration of the relevance of in vitro generated results to the in vivo situation. Such reactions may proceed at sub-optimal rates in $9 fractions. Thus, the concentrations of PAPS and UDPGA are probably ratelimiting in $9 fractions. Indeed, UDPGA is destroyed in subcellular preparations by the action of pyrophosphorylase. In a general sense, it is unlikely that the operation of sulphotransferase or glucuronyl transferases play a major role in the direct deactivation of ultimate genotoxic agents. Nevertheless, such pathways lead to dramatic changes in polarity and rapid secretion of the conjugates from cells, thus limiting alternative biotransformation reactions and should not be neglected. The formation of N, O-sulphates and N,O-glucuronides have been proposed as key reactions in the conversion of certain proximate carcinogens into ultimate carcinogens. Such metabolites are probably effective only when they are generated within cells. Thus, the physical properties (and chemical reactivity) of these conjugates militate against their penetration into cells, and this may provide an explanation why the enzymic formation of the sulphate ester of N-hydroxy-2-acetylaminofluorene reduced the mutagenicity of N-hydroxy-2-acetylamino-fluorene in the Salmonella test [62]. Similarly, the breakdown of the sulphate ester by reaction with extracellular materials has been advanced as the reason why this compound is a relatively ineffective carcinogen when tested either subcutaneously in rats or on the skin of mice or rats [40]. The functional relationships between microsomal mono-oxygenation, epoxide hydration and glucuronidation has recently been investigated in liver microsomes and in isolated hepatocytes [99]. The results provide a good illustration of one of the limitations to the use of subcellular fractions. In the hepatocyte and in microsomes fortified with NADPH and UDPGA, naphthalene is oxygenated to its 1,2-oxide which is cleaved to a dihydrodiol (by epoxide hydratase) which in turn is conjugated with glucuronic acid (Fig. 10). However, microsomes afford the dihydrodiol as the major metabolite; hepatocytes afford the glucuronide. Only by using additional microsomal protein or the allosteric effector of glucuronyl transferase, UDP-N-acetyl-glucosamine, could microsomes be forced to yield reasonable quantities of the glucuronide. These findings should be borne in mind when interpreting the results of in vitro tests of the mutagenicity of compounds containing aromatic moieties.

234

OH ~

OH

Fig. I 0 . Metabolism o f naphthalene in h e p a t o c y t e s .

Epoxide hydratase has been reported to be effective in affording protection to Salmonalla typhimurium against the mutagenic action of b e n z o [ a ] p y r e n e in the presence of microsomal fractions from the livers of untreated mice [100]. There is evidence that epoxide hydratase may be more efficient than the glutathione transferases in the deactivation of arene oxides [101], Using $9 and microsomal fractions from various strains of rats and mice, Oesch and co-workers [102] have shown that species and strain differences in mono-oxygenases and in secondary enzymes, e.g. epoxide hydratase, are reflected in dramatic differences in the mutagenicity of b e n z o [ a ] p y r e n e in the coupled liver microsome-microbial reverse mutation assay. Manipulation of the activities of these enzymes, by the use of inhibitors of epoxide hydratase and by pre-treatment of animals with microsomal enzyme inducers, resulted in expected changes in mutagenicity that provided compelling evidence of a causal link between enzyme profiles and mutagenic effect.

(3) Pre-treatment with microsomal enzyme inducers Because epoxide hydratase is present and, presumably, fully functional in $9 fractions, the balance between this activity and mono-oxygenase activity may be expected to relate closely to the in vivo balance. However, the $9 fractions that are employed in microbial mutagenicity tests are usually derived from the liver of animals that have been pre-treated with microsomal enzyme inducers, e.g. Aroclor 1254. Such pretreatment may lead to metabolic over-competence of certain enzymes, particularly the mono-oxygenases that are key factors in the activation o f many precursor genotoxic agents. Such inbalance has been advanced as the explanation for the large increase in mutation frequency obtained with benzo [a ] pyrene in Salmonella typhimurium when a microsomal fraction from control rat livers was replaced b y the corresponding fraction from Aroclor-treated rats [103]. The nature of the microsomal enzyme inducer employed in such pre-treatment can have a dramatic influence not only upon the overall activity o f mono-oxygenases b u t also on the profile of mono-oxygenases in tissues [103--107] which may in turn lead to a fundamental change in the profile of metabolites. Experiments with microsomes from the livers of untreated mice have shown that, at high concentrations of epoxide hydratase, the mutagenicity of b e n z o [ a ] p y r e n e towards Salmonella typhimurium strains TA1537 and TA98 was reduced b y more than 95% and ca. 75% resp. [108]. Similar results were obtained using microsomes

235 from phenobarbitone-treated mice. However, when microsomes from 3-methylcholanthrene-treated mice were used, epoxide hydratase was found to have a much weaker and more complex (multiphasic) effect [108] suggesting that pretreatment with 3-methylcholanthrene leads to a pattern of mutagenicaUy active benzo [a ] pyrene metabolites that is fundamentally different from that obtained with microsomes from control or phenobarbitone-treated mice. Consequently, the results obtained in tests employing such pre-treatment should be interpreted with extreme caution particularly in cases where the test c o m p o u n d possesses a multiplicity of sites that are susceptible to mono-oxygenase-catalysed reactions. The results obtained with microsomes from the 3-methylcholanthrenetreated mice [108] were probably caused by the preferential induction of c y t o c h r o m e P-448 by this hydrocarbon. This conclusion is in accord with the results of a study showing that the maximal effect of epoxide hydratase on the purified c y t o c h r o m e P-448-mediated mutagenicity of b e n z o [ a ] p y r e n e in Salmonella typhirnurium strains T A 1 5 3 8 and TA98 was to reduce the mutation freuqencies by 50% and 30%, resp. [13,43]. Pre-treatment with microsomal enzyme inducers can give rise to additional complications. For example, the $9 fraction from the livers of Aroclor-treated rats (500 mg kg -1 b o d y weight) contain large quantities of residual Aroclor (ca. 300 ~g g-I liver equivalent) [109]. It is probable that these residues will compete with precursor genotoxic agents for the microsomal mono-oxygenases. Such competition would provide an explanation for the observation that the mutation rates obtained with 2-acetylaminofluorene in the Salmonella test were reduced at concentrations below 15.6 ~g m1-1 but were increased severalfold at concentrations above 31 #g m1-1 when an $9 fraction from the livers of control rats was replaced by the corresponding fraction from Aroclor-treated rats [110].

(4) Species differences The results of comparative microbial assays showed that the $9 fraction derived from the livers o f c o t t o n rats was more effective than the sg,~raction from rat liver in the mutagenic activation o f 2-acetylaminofluorene [111]. This finding was unexpected because the c o t t o n rat is considered to be resistant to this hepatocarcinogen [112]. It is uncertain whether the positive results obtained in the Salmonella test with cotton-rat $9 fraction arose because of differences in the metabolism of 2-acetylaminofluorene in vitro and in vivo or because the in vivo metabolism of 2-acetylaminofluorene leads to the generation of a bacterial mutagen(s) that is not an effective carcinogen. The results of in vivo studies which indicated that metabolite binding to DNA was lower in the livers of c o t t o n rats than in rats are consistent with both explanations [112]. Very recent results support the latter explanation, i.e. the metabolite responsible for the microbial mutagenicity of 2-acetylaminofluorene may differ from the metabolite(s) responsible for the carcinogenic action of this comp o u n d [62]. Ashby and Styles [113] have also drawn attention to certain chemicals, specifically b e n z o [ a ] p y r e n e and 7,12-dimethylbenzanthracene, that m a y produce mutation in vitro through the formation of metabolites that are n o t

236 believed to initiate carcinogenisis in vivo. As evinced by a strong positive results in Sal.monella [100,102], liver microsomes from untreated mice are effective in catalysing the conversion of b e n z o [ a ] p y r e n e into mutagenic forms. The finding that microsome-derived epoxide hydratase inhibited (ca. 95%) the mutagenicity of b e n z o [ a ] p y r e n e in this test system [100,114] indicated that the metabolite(s) chiefly responsible for the mutagenic effect was different from the presumed ultimate carcinogenic form of benzo[a]pyrene, i.e. the 9,10-oxo-7,8-dihydrodiol (Fig. 6). Thus, the operation of epoxide hydratase is essential for the conversion of b e n z o [ a ] p y r e n e into this diol epoxide that is, itself, a poor substrate for epoxide hydratase [115]. Similarly, the incubation of 7-methylbenz[a]anthracene [36], 7,12-dimethylbenz(a)anthracene [37] and benzo(a)pyrene [35] with Salmonella or isolated preparations of DNA in the presence of S9 from the livers of rats, variously pretreated with microsomal enzyme inducers, gave rise to DNA adducts that do n o t occur in the skin of mice after in vivo exposure to these c o m p o u n d s or in mouse e m b r y o cell treated in vitro. While it seems unlikely that the metabolites giving rise to the former adducts contribute to the carcinogenicity of these hydrocarbons in mice it is n o t yet known whether these metabolites are formed in vivo in the rat or contribute to the carcinogenic action of the hydrocarbons in species other than the mouse. Nevertheless, these findings emphasise that the appropriateness of various in vitro metabolic activation systems should be experimentally established, n o t simply assumed. Conclusions In view of the imbalance between activation and deactivation enzymes, major species and tissue differences in the activities and specificities of such enzymes, differential enhancing and inhibitory effects caused by pre-treatment with microsomal enzyme inducers and differences in metabolic pathways in vivo and vitro, it is clear that the results of in vitro tests for genotoxic agents must be interpreted within the boundaries imposed by the test conditions. Despite such restrictions, the results of such in vitro tests are valuable in that they provide a preliminary indication that a particular c o m p o u n d may pose a genotoxic hazard in vivo. If the definition of the hazard is to be improved, then it would seem important to ensure that the metabolism of the test c o m p o u n d in the in vitro test system relates as closely as possible to that occurring in vivo. The practicability of this is partly dependent upon the enzyme profiles of the detector cells. Apart from the apparent absence of effective mono-oxygenases and the presence of an enzyme(s) that can effect the reduction of nitro groups [53,54], there is little published information relating to the ability of Salmonella typhimurium to metabolise genotoxic agents. However, ongoing studies in our laboratory have failed to detect significant epoxide hydratase or glutathione transferase (aryl-, arakyl- or epoxide-) activities in Salmonella typhimurium TA100. Information on the metabolism of genotoxic agents in cultures of mammalian cells that are employed in the mammalian cell transformation assay is t o o scant to warrant any firm conclusions as to their ability to mimic metabolism in vivo. The general opinion is that certain lines are deficient in mono-oxy-

237

genase activity. It is uncertain whether such deficiencies are balanced by parallel decreases in the activities of secondary enzyme systems. Primary cultures of mammalian cells are not normally employed in the cell transformation assay. Comparative studies [ 3 7 , 6 6 , 1 1 6 ] have demonstrated that the hydrocarbondeoxyriboside products from the D N A of mouse skin exposed in vivo to certain genotoxic polycyclic aromatic hydrocarbons are chromatographically the same as the products formed in mouse-embryo cell cultures. Such results suggest that primary cell cultures may effectively mimic in vivo metabolism, at least in qualitative terms, and may therefore provide a useful tool for the detection of potential mammalian genotoxic agents. However, the fortification of cultured mammalian cells with $9 fractions would introduce similar problems to those observed in the coupled liver microsome-microbial reverse mutation test. Probably the most obvious anomalous feature of the latter test is the generation of positive results with many non-hepatocarcinogens. Thus, the $9 fraction derived from rat liver converts precursor genotoxic agents into bacterial mutagens that are apparently not carcinogenic to rat liver. Such results indicate that the test cannot predict a carcinogenic hazard for any particular tissue nor for any particular species. The reasons for this may reside in qualitative or quantitative differences in metabolism (including DNA-binding of metabolites) in vivo and in vitro. Alternatively, the genetic end-point of this test may not be appropriate for the prediction of carcinogenic hazards posed by chemicals to mammals. References 1 Ehrenberg, L., P. Brookes, H. Druckrey, B. Lageri~rf, J. Li t w i n and G. Williams, The relation of cancer i n d u c t i o n and genetic damage, in: C. Ramel (Ed.), Evaluation of Genetic Risks of E n v i r o n m e n t a l Chemicals, Ambio, Special R e p o r t No. S, Royal Swedish A c a d e m y of Sciences/Universitetsforlarget, 1973, pp. 15--16. 2 Miller, E.C., and J.A. Miller, The m e t a b o l i s m of chemical carcinogens to reactive electrophiles and their possible mech anisms of action in carcinogenesis, in: C.S. Searle (Ed.), Chemical Carcinogens, ACS Monograph 173, Am. Chem. Soc., Washington, 1976, pp. 737--762. 3 Swenson, D.H., E.C. Miller and J.A. Miller, A f l a t o x i n Bi-2,S-oxide: Evidence for its f o r m a t i o n in rat liver in vivo and b y h u m a n liver microsomes in vitro, Biochem. Biophys. Res. Commun., 60 (1974) 1036--1043. 4 Prejean, J.D., D.P. Griswold, A.E. Casey, J.C. Peckham, J.H. Weisburger, E.K. Weisburger and H.B. Wood Jr., Careinogenicity studies on clinically used anticancer agents, Proc. Am. Assoc. Cancer Res., 13 (1972) 112. 5 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: A simple test system co mb ining liver h o m o g e n a t e s for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 2281--2285. 6 Ames, B.N., J. McCann and E. Yamasaki, Methods for de t e c t i ng carcinogens and mut a ge ns w i t h the S a l m o n e l l a / m a m m a l i a n microsome m u t a g e n i c i t y tests, Mut a t i on Res., 31 (1975) 347--364. 7 Ames, B.N., Carcinogenicity tests, Science, 191 (1976) 241--245. 8 Lawley, P.D., Carcinogenesis by alkylating agents, in: C.S. Searle (Ed.), Chemical Carcinogens, ACS M o n o g r a p h 173, Am. Chem. Soc., Washington, 1976, pp. 83--244. 9 Hadidian, Z., T.N. Frederickson, E.K. Weisburger, J.H. Weisburger, R.M. Glass and N. Mantel, Tests for chemical carcinogens, R e p o r t on the activity of derivatives of aromatic amines, nitrosamines, quinolines, nitroso-alkanes, amides, epoxides, aziridines and purine a nt i me t a bol i t e s , J. Natl. Cancer Inst., 4 1 (1968) 985---1036. 10 Drake, J.W., and R.H. Baltz, The b i o c h e m i s t r y of mutagenesis, Annu. Rev. Biochem., 45 (1976) 11-37. 11 Sotomay or, R.E., G.A. Sega, and R.B. Cumming, Unscheduled DNA synthesis in spermatogenic cells o f mice treated in vivo with the direct alkylating agents c y c l o p h o s p h a m i d e and m i t o m e n , M u t a t i o n Res., 50 (1978) 229--240.

238 1 2 D e P i e r r e , J . W . , a n d L. E r n s t a r , T h e m e t a b o l i s m o f p o l y c y c l i c h y d r o c a r b o n s a n d its r e l a t i o n s h i p t o cancer, Binchim. Biophys Acta, 473 (1978) 149--186. 1 3 W o o d , A . W . , W. L e v i n , A . Y . H . L u , H . Yagi, O. H e r n a n d e z , D.M. J e r i n a a n d A . H . C o n n e y , M e t a b o l i s m of benzo[a]pyrene and benzo[a]pyrene derivatives to mutagenlc products by highly purified hepatic m i c r o s o m a l e n z y m e s , J. Biol. C h e m . , 2 5 1 ( 1 9 7 6 ) 4 8 8 2 - - 4 8 9 0 . 1 4 L e v i n , W., D. R y a n , S. West a n d A . Y . H . L u , P r e p a r a t i o n o f p a r t i a l l y p u r i f i e d , l i p i d - d e p l e t e d c y t o c h r o m e P - 4 5 0 a n d r e d u c e d N A D P - - - c y t o c h r o m e c r e d u c t a s e f r o m r a t liver m i c r o s o m e s , J . Biol. C h e m . , 249 (1974) 1747--1754. 15 Van der Hoeven, T.A., and M.J. Coon, Preps.ration and properties of partially purified cytochrome P - 4 5 0 a n d r e d u c e d N A D P - - - c y t o c h r o m e P - 4 5 0 r e d u c t a s e f r o m r a b b i t liver m i c r o s o m e s , J. Biol. C h e m . , 249 (1974) 6302---6310. 16 Guengerieh, F.P., Preparation and properties of highly purified cytochrome P-450 and NADPH-cytoc h r o m e P - 4 5 0 r e d u c t a s e f r o m p u l m o n a r y m i c r o s o m e s o f u n t r e a t e d r a b b i t s , Mol. P h a r m a c o l . , 1 3 (1977) 911--923. 1 7 I m a i , J., T h e u s e o f 8 - a m i n o - o c t y i s e p h a r o s e f o r t h e s e p a r a t i o n o f s o m e c o m p o n e n t s o f t h e h e p a t i c microsomal electron transport system, J. Binchem. (Tokyo), 80 (1976) 267--276. 18 Conney, A.H., Pharmacological implications of microsomal enzyme induction, Pharmacol. Rev., 19 (1967) 317--366. 1 9 U]h~ch, V., a n d P. K r e m e r s , M u l t i p l e f o r m s o f c y t o c h r o m e P - 4 5 0 in t h e m i c r o s o m a l m o n o - o x y g e n a s e system, Arch. Toxicol., 39 (1977) 41--50. 2 0 G i l l e t t e , J . R . , D . C . Davis a n d H . A . S a s a m e , C y t o c h r o m e P - 4 5 0 a n d its r o l e i n d r u g m e t a b o l i s m , A n n u . Rev. Pharmacol., 12 (1972) 57--84. 21 Gram, T.E., C.L. Litterst and E.G. Mimnaugh, Enzymatic conjugation of foreign chemical compounds b y r a b b i t l u n g a n d liver, D r u g M e t a b . D i s p o s i t i o n , 2 ( 1 9 7 4 ) 2 5 4 - - 2 5 8 . 2 2 M a t s u b a r a , T., Y. N a k a m u r a a n d Y. T o c h i n o , E l e c t r o n t r a n s p o r t s y s t e m s o f l u n g m i c r o s o m e s a n d t h e i r p h y s i o l o g i c a l f u n c t i o n s , En~.yme h y d r o x y l a t i o n o f a n i l i n e a n d s t e r o i d s , X e n o b i o t i c a , 5 ( 1 9 7 5 ) 2 0 5 - 212. 2 3 L i t t e r s t , C . L . , E . G . M i m n a u g h , R . L . R e a g a n a n d T . E . G r a m , C o m p a r i s o n o f in v i t r o d r u g m e t a b o l i s m b y l u n g , liver a n d k i d n e y o f several c o m m o n l a b o r a t o r y species, D r u g M e t a b . D i s p o s i t i o n , 3 ( 1 9 7 6 ) 259--265. 2 4 P e l k o n e n , O., E . H . K a l t i a l a , T . K . I . L a r n i a n d N . T . K~/rki, C y t o c h r o m e P - 4 5 0 l i n k e d m o n o - o x y g e n a s e s y s t e m a n d d r u g i n d u c e d s p e c t r a l i n t e r a c t i o n s in h u m a n liver m i c r o s o m e s , C h e m . - B i o l . I n t e r a c t . , 9 (1974) 205--216. 2 5 A l v a r e s , A . P . , G. S c h i l l i n g , W. L e v i n , R . K u n t z m a n , L. B r a n d a n d L.C. M a r k , C y t o c h r o m e s P - 4 5 0 a n d b 5 i n h u m a n liver m i c r o s o m e s , Clin. P h a r m a c o l . T h e r a p . , 1 0 ( 1 9 6 9 ) 6 5 5 - - 6 5 9 . 2 6 A c k e r m a n , E., Die D e m e t h y l i e r u n g y o n A m i n o p h e n a z o n u n d C o d e i n i n d e r L e b e r d e s M e n s c h e n , Biochem. Pharmacol., 19 (1970) 1955--1973. 2 7 A c k e ~ n a n , E., A n d I. H e i n r i c h , D i e Aktivit~/t d e r N- u n d O - D e m e t h y l a s e i n d e r L e b e r d e s M e n s c h e n , Binchem. PharmacoL, 19 (1970) 327--342. 2 8 N e l s o n , E . B . , P.P. R a j , K . J . Belfi a n d B.S.S. M a s t e r s , O x i d a t i v e d r u g m e t a b o l i s m In h u m a n liver m i c r o s o m e s , J. P h a r m a c o l . E x p . T h e r a p . , 1 7 8 ( 1 9 7 1 ) 5 8 0 - - 5 8 8 . 29 Holilngworth, R.M., Comparative metabolism and selectivity of organophosphate and carbamate i n s e c t i c i d e s , Bull. W H O , 4 4 ( 1 9 7 1 ) 1 5 5 - - 1 7 0 . 3 0 Z i e g i e r , D . M . , a n d M.S. G o l d , O x i d a t i v e m e t a b o l i s m o f t e r t i a r y a m i n e d r u g s b y h u m a n liver tissue, Xenobiotica, 1 (1971) 325--326. 31 W r i g h t , A . S . , C. D o n n i n g e r , R . D . G r e e n l a n d , K . L . S t e m m e r a n d M . R . Z a v o n , T h e e f f e c t s o f p r o l o n g e d i n g e s t i o n o f d i e l d r i n o n t h e livers o f m a l e r h e s u s m o n k e y s , E c o t o x i c o l . E n v i r o n . S a f e t y , 1 ( 1 9 7 8 ) 477--502. 32 Chhabra, R.S., and J.R. Fouts, Sex differences in the metabolism of xenobiotics by extrahepatic tissue i n r a t s , D r u g M e t a b . D i s p o s i t i o n , 2 ( 1 9 7 4 ) 3 7 5 - - 3 7 9 . 3 3 v o n B a h r , C., A . R a n e , S. O r r e n i u s a n d F. S j o q v i s t , M e t a b o l i s m o f d e s m e t h y l i m i p r a m i n e in h u m a n f o e t a l a n d a d u l t liver m i c r o s o m e s , A c t a P h a r m a c o l . T o x i c o l . , 3 4 ( 1 9 7 4 ) 58---64. 34 McPherson, F.J., J.W. Bridges and D.V. Parke, The enhancement of biphenyl-2-hydroxylation by carcinogens in vitro, Biochem. Soc. Trans., 2 (1974) 618--619. 3 5 K i n g , H . W . S . , M . H . T h o m p s o n a n d P. B r o o k e s , T h e b e n z o [ a ] p y r e n c d e o x y r i b o n u c l e o s i d e p r o d u c t s i s o l a t e d f r o m D N A a f t e r t h e m e t a b o l i s m o f b e n z o [ a ] p y r e n e b y r a t liver m i c r o s o m e s in t h e p r e s e n c e o f DNA, Cancer Res., 35 (1975) 1263--1269. 3 6 T h o m p s o n , M . H . , M . R . O s b o r n e , H.W.S. K i n g a n d P. B r o o k e s , T h e 7 - m e t h y l b e n z [ a ] a n t h r a c e n e d e o x y r i b o n u c l e o s i d e p r o d u c t s i s o l a t e d f r o m D N A a f t e r m e t a b o l i s m o f t h e c a r c i n o g e n b y r a t liver micxosomes in the presence of DNA, Chem.-BioL Interact., 14 (1976) 13--19. 3 7 Bigger, C . A . H . , J . E . T o m a s z e w a k i a n d A. D i p p l e , D i f f e r e n c e s b e t w e e n p r o d u c t s o f b i n d i n g o f 7 , 1 2 d i m e t h y l b e n z [ a ] a n t h r a c e n e t o D N A in m o u s e s k i n a n d in a r a t liver m i c r o s o m a l s y s t e m , B i o c h e m . Biophys. Res. Commun., 80 (1978) 229--235.

239 3 8 B e n k e r t , K., W. Fries, M. Kiese a n d W. L e n k , N - ( 9 - H y d r o x y - 9 H - f l u o r e n - 2 - y D - a c e t a m i d ea n d N - ( 9 - o x o 9 H - f l u o r e n - 2 - y l ) - a c e t a m i d e , M e t a b o l i t e s o f N - ( 9 - H - f l u o r e n - 2 - y l ) - a c e t a m i d e , B i o c h e m . P h a r m a c n i . , 24 (1975) 1375--1380. 39 Irving, C.C., N - H y d r o x y l a t i o n o f t h e c a r c i n o g e n 2 - a c e t y l a m i n o f l u o r e n e b y r a b b i t liver m i c r o s o m e s , Biochim. Biophys., Acta, 65 (1962) 564--566. 4 0 C l a y s o n , D.B., a n d R.C. G a r n e r , C a r c i n o g e n i c a r o m a t i c a m i n e s a n d r e l a t e d c o m p o u n d s , in: C.S. Searle (Ed.), C h e m i c a l C a r c i n o g e n s , ACS M o n o g r a p h 1 7 3 , A m . C h e m . Soc., W a s h i n g t o n , 1 9 7 6 , p p . 3 6 6 - 461. 41 Kiese, M., G. R e n n e t a n d I. W i e d m a n n , N - H y d r o x y l a t i o n o f 2 - a m l n o f i u o r e n e in the g u i n e a pig a n d b y g u i n e a pig liver m i e r o s o m e s in vitro, A r c h . E x p . P a t h o l . P h a r m a c o l . , 2 5 2 ( 1 9 6 6 ) 4 1 8 - - 4 2 3 . 42 J a r i n a , D.M., R.E. Lehr, H. Yagi, O. H e r n a n d e z , P.M. D a n s e t t e , P.G. Wislocki, A.W. W o o d , R . L . C h a n g , W. Levin a n d A.H. C o n n e y , M u t a g e n i c i t y o f b e n z o [ a ] p y r e n e derivatives a n d the d e s c r i p t i o n o f a q u a n t u m m e c h a n i c a l m o d e l w h i c h p r e d i c t s t h e ease o f c a r b o n i u m i o n f o r m a t i o n f r o m diol e p o x i d e s , in: F.J. de Serres, J . R . F o u t s , J . R . B e n d a n d R.M. P h i l p o t (Eds.), In vitro Metabolic A c t i v a t i o n in Mutagenesis Testing, E l s e v i e r / N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 6 , p p . 1 5 9 - - 1 7 7 . 43 W o o d , A.W., W. Levin, A . Y . H . L u , A . H . C o n n e y , H. Yagi, O. H e r n a n d e z a n d D.M, J e r i n a , Use o f a highly p u r i f i e d h e p a t i c m i e r o s o m a l m o n o - o x y g e n a s e s y s t e m a n d e p o x i d e h y d r a s e to m e t a b o l i c a l l y activate a n d d e a c t i v a t e b e n z o [ a ] p y r e n e a n d b e n z o [ a ] p y r e n e derivatives, in: F.J. de Serres, J . R . F o u r s , J . R . Bend a n d R.M. P h i l p o t (Eds.), In vitro M e t a b o l i c A c t i v a t i o n in Mutagenesis Testing, Elsevier/ North-Holland, Amsterdam, 1976, pp. 179--185. 4 4 Cavalieri, E., R. R o t h , C. G r a n d j e a n , J. A l t h o f f , K. Patil, S. L i a k u s a n d S. March, C a r c i n o g e n i c i t y a n d m e t a b o l i c profiles o f 6 - s u b s t l t u t e d b e n z o [ a ] p y r e n e derivatives o n m o u s e skin, Chem.-Biol. I n t e r a c t . , 22 ( 1 9 7 8 ) 5 3 - - 6 7 . 4 5 R o g a n , E., R. R o t h , P. K a t o m s k i , J. B e n d e r s o n a n d E. Cavalieri, Binding o f b e n z o [ a ] p y r e n e a t t h e 1 , 3 , 6 - p o s i t i o n s to nucleic acids in vivo o n m o u s e skin a n d in vitro w i t h r a t liver m i c r o s o m e s a n d nuclei, Chem.-Biol. I n t e r a c t . , 22 ( 1 9 7 8 ) 3 5 - - 5 1 . 4 6 H o l l a n d , V.R., B.C. S a u n d e r s , F . L . Rose a n d A.L. Walpole, A safer s u b s t i t u t e f o r b e n z i d i n e in the detection of blood, Tetrahedron, 30 (1974) 3 2 9 9 - - 3 3 0 2 . 47 G a r n e r , R.C,, A.L. Walpole a n d F.L. Rose, Testing o f s o m e b e n z i d i n e a n a l o g u e s f o r m i c r o s o m a l activ a t i o n to b a c t e r i a l m u t a g c n s , C a n c e r L e t t . , 1 ( 1 9 7 5 ) 3 9 - - 4 2 . 48 A s h b y , J., A p p e n d i x 1, i n : I . F . H . P u r c h a s e , E. L o n g s t a f f , J. A s h b y , J . A . Styles, D. A n d e r s o n , P.A. Lefevre a n d F . R . W e s t w o o d , A n e v a l u a t i o n o f 6 s h o r t - t e r m tests f o r d e t e c t i n g o r g a n i c c h e m i c a l carcinogens, Br. J. Cancer, 37 ( 1 9 7 8 ) 8 7 3 - - 9 5 9 . 49 D r u c k r e y , H., A. S c h i l d b a c h , D. Schahl, R. P r e u s s m a n n a n d S. Ivankovic, Q u a n t i t a t i v e analysis o f the c a r c i n o g e n i c a c t i o n o f d i e t h y i n i t r o s a m i n e , A r z n e i m . - F o r s c h . , 13 ( 1 9 6 3 ) 8 4 1 - - 8 5 1 . 50 F a h m y , O.G., M.J. F a h m y a n d M. Wiessler, a - A c e t o x y d i m e t h y l n i t r o s a m i n e , A p r o x i m a t e m e t a b o l i t e of t h e c a r c i n o g e n i c a m i n e , B i o c h e m . P h a r m a c o l . , 24 ( 1 9 7 5 ) 1145---1148. 51 F a h m y , O.G., M.J. F a h m y a n d M. Wiessler, N - a - A c e t o x y e t h y l - N - e t h y l n i t r o s a m i n e .A p r e c u r s o r o f t h e biologically effective m e t a b o l i t e o f N , N - d i e t h y l n i t r o s a m i n e , B i o c h e m . P h a r m a c o l . , 24 ( 1 9 7 5 ) 2 0 0 9 - 2012. 52 Wang, C.Y., B.C. Behrens, MI I c h i k a w a a n d G.T. B r y a n , N i t r o r e d u c t i o n o f 5 - n i t r o f u r a n derivatives b y r a t liver x a n t h i n e oxidase a n d r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e - c y t o c h r o m e c r e d u c t a s e , B i o c h e m . P h a r m a c o l . , 23 ( 1 9 7 4 ) 3 3 9 5 - - 3 4 0 4 . 53 O k a b a y a s h i , T., a n d A. Y o s h i m o t o , R e d u c t i o n o f 4 - n i t r o q u i n o l i n e 1-oxide b y m i c r o - o r g a n l s m s , C h e m . P h a r m . Bull. ( T o k y o ) , 10 ( 1 9 6 2 ) 1 2 2 1 - - 1 2 2 6 . 54 Y a m a m o t o , N., S. F u l u d a a n d H. T a k a b e , E f f e c t o f a p o t e n t c a r c i n o g e n 4 - n i t r o q n i n o l i n e 1-oxide, o n b a c t e r i a a n d b a c t e r i o p h a g e g e n o m e s , C a n c e r Res., 3 0 ( 1 9 7 0 ) 2 5 3 2 - - 2 5 3 7 . 55 Tada, M., a n d M. Tada, M e t a b o l i c a c t i v a t i o n o f 4 - n i t r o q u i n o l i n e 1-oxide a n d its b i n d i n g t o nuclei acid, P.N. Magee et al. (Eds.), in: F u n d a m e n t a l s in C a n c e r P r e v e n t i o n , Univ. o f T o k y o Press, T o k y o / U n i v . P a r k Press, B a l t i m o r e , 1 9 7 6 , p p . 2 1 7 - - 2 1 8 . 56 N a g a o , M., a n d T. S u g i m u r a , M o l e c u l a r b i o l o g y o f t h e c a r c i n o g e n 4 - n i t r o q u i n o l i n e 1-oxide, Adv. Cancer Res., 2 3 ( 1 9 7 6 ) 1 3 1 - - 1 6 9 . 57 G o t o h , O., A. Wada, M. T a d a a n d M. T a d a , Base a n d base s e q u e n c e specificity o f t h e b i n d i n g o f 4-hyd r o x y a m i n o q u i n o l i n e 1-oxide t o D N A , G a n n , 6 9 ( 1 9 7 8 ) 6 1 - - 6 6 . 58 R u k z k i , E., P a t t e r n o f h y p e r s e n s i t i v i t y to a r o m a t i c amines, C o n t a c t D e r m a t i t i s , 1 ( 1 9 7 5 ) 2 4 8 - - 2 4 9 . 59 H u t s o n , D.H., M e c h a n i s m s o f b i o t r a n s f o r m a t i o n , D.E. H a t h w a y (Ed.), in: F o r e i g n C o m p o u n d M e t a b o lism in M a m m a l s , Vol. 4, C h e m . Soc., L o n d o n , 1 9 7 7 , p p . 2 5 9 - - 3 4 6 . 6 0 A u t r u p , H., a n d G.P. W a r w i c k , S o m e c h a r a c t e r i s t i c s o f t w o a z o r e d u c t a s e s y s t e m s in r a t liver, Relevance t o the a c t i v i t y o f 2-[4'-di(2"-bromopropyl)-aminophenylazo]benzoic acid ( C B I O - 2 5 2 ) a c o m p o u n d possessing l a t e n t c y t o t o x i c a c t i v i t y , Chem.-Biol. I n t e r a c t . , 11 ( 1 9 7 5 ) 3 2 9 - - 3 4 2 . 61 Miller, J . A . , C o m m e n t s o n c h e m i s t r y o f c y c a d s , F e d . P r o c . , 23 ( 1 9 6 4 ) 1 3 6 1 - - 1 3 6 2 . 6 2 T h o r g e i r s s o n , S.S., H . A . J . S c h u t a n d P.J. Wirth, M u t a g e n i c i t y a n d c a r c i n o g e n i c i t y o f a r o m a t i c a m i n e s , 1 7 6 t h ACS N a t i o n a l Meeting, Miami Beach, Florida, Sep, 1 1 - - 1 7 . A b s t r a c t s , Envr. 1 2 8 , 1 9 7 8 .

240 63 L o w e r , G.M., a n d G.T. B r y a n , E n z y m a t i c N - a c e t y l a t i o n o f c a r c i n o g e n i c a r o m a t i c a m i n e s b y liver c y t o sol o f species d i s p l a y i n g d i f f e r e n t o r g a n susceptibilities, B i o c h e m , P h a r m a c o l . , 22 ( 1 9 7 3 ) 1 5 8 1 - - 1 5 8 8 . 6 4 C r a w f o r d , M.J., a n d D.H. H u t s o n , The use o f a n i m a l s u b c e l l u l a r f r a c t i o n s t o s t u d y t y p e II m e t a b o l i s m o f x e n o b i o t i c s , in: G.D. Paulson, D.S. F r e a r a n d E.P. Marks (Eds.), X e n o b i o t i c M e t a b o l i s m : in vitro M e t h o d s , ACS S y m p o s i u m Series, No. 9 7 , A m . C h e m . Soe., W a s h i n g t o n , 1 9 7 9 , p p . 1 8 1 - - 2 2 9 . 6 5 Sims, P., P.L. Grover, A. Swalsland, K. Pal a n d A. H e w e r , M e t a b o l i c a c t i v a t i o n o f b e n z o [ a ] p y r e n e proceeds by a diol-epoxide, Nature (London), 252 (1974) 326--328. 6 6 K a p i t u l n i k , J., P.G. Wislocki, W. Levin, H. Yagi, D.M. J e r i n a a n d A . H . C o n n e y , T u m o u r g e n i c i t y studies w i t h d i o l e p o x i d e s o f b e n z o [ a ] p y r e n e w h i c h i n d i c a t e t h a t (±)-trans-7,3-8a-dihydroxy-9~,lO~e p o x y - 7 , 8 , 9 , 1 0 - t e t r a h y d r o b e n z o[a] p y r e n e is a n u l t i m a t e c a r c i n o g e n in n e w b o r n mice, C a n c e r Res., 3 8 (1978) 354--358. 67 S e l a n d e r , H . G . , D.M. J e r i n a a n d J.W. Daly, M e t a b o l i s m o f c h l o r o b e n z e n e w i t h h e p a t i c m i c r o s o m e s a n d solubilised c y t o c h r o m e P - 4 5 0 s y s t e m s , A r c h . B i o c h e m . B i o p h y s . , 1 6 8 ( 1 9 7 5 ) 3 0 9 - - 3 2 1 . 6 8 T o m a s z e w s k i , J.E., D,M. J e r i n a a n d J.W. D a l y , D e u t e r i u m i s o t o p e effects d u r i n g f o r m a t i o n o f p h e n o l s b y h e p a t i c m o n o o x y g e n a s e s , Evidence f o r a n alternative to t h e a r e n e oxide p a t h w a y , B i o c h e m i s t r y , 14 ( 1 9 7 5 ) 2 0 2 4 - - 2 0 3 1 . 69 W o o d e r , M.F., A.S. Wright a n d L.J. King, In vivo a l k y l a t i o n studies w i t h d i e h l o r v o s a t p r a c t i c a l use c o n c e n t r a t i o n s , Chem.-BioL I n t e r a c t . , 19 ( 1 9 7 7 ) 2 5 - - 4 6 . 7 0 N e b e r t , D.W., A . R . Boobis, H. Yagi, D.M. J e r i n a a n d R.A. K o u r i , Genetic d i f f e r e n c e s in b e n z o [ a ] p y rene c a r c i n o g e n i c i n d e x in vivo a n d in m o u s e c y t o c h r o m e P i - 4 5 0 - m e d i a t e d b e n z o [ a ] p y r e n e m e t a b o lite b i n d i n g to D N A in vitro, in: D.J. J o l l o w , K.K. Koesis, R. S n y d e r a n d H. V a i n o (Eds.), Biological l~eactive I n t e r m e d i a t e s , P l e n u m , H e w Y o r k , 1 9 7 7 , p p . 1 2 5 - - 1 4 5 . 71 Boobis, A . R . , a n d D.W. N e b e r t , Genetic d i f f e r e n c e s in t h e m e t a b o l i s m o f c a r c i n o g e n s a n d the b i n d i n g o f b e n z o [ a ] p y r e n c m e t a b o l i t e s t o D N A , Adv. E n z y m e Reg., 15 ( 1 9 7 7 ) 3 3 9 - - 3 6 2 . 72 Phillips, D.H., P.L. G r o v e r a n d P. Sims, The c o v a l e n t b i n d i n g o f p o l y c y e l i c h y d r o c a r b o n s to D N A in the skin o f mice o f d i f f e r e n t strains, Int. J. Cancer, 22 ( 1 9 7 8 ) 4 8 7 - - 4 9 4 . 73 Nicholl, J.N., P.F. S w a r m a n d A.E. Pegg, E f f e c t o f d i m e t h y i n i t r o s a m i n e o n persistence o f m e t h y l a t e d g u a n i n e s in r a t liver a n d k i d n e y D N A , N a t u r e ( L o n d o n ) , 2 5 4 ( 1 9 7 5 ) 2 6 1 - - 2 6 2 , 74 Kleihues, P., H.K. C o o p e r a n d J. Buecheler, I n v o l v e m e n t o f D N A r e p a i r in t h e o r g a n specific carcinogenieity o f a l k y l a t i n g agents, in: H. R e m m e r , H.M. Bolt, P. B a n n a s c h a n d H. P o p p e r (Eds.), P r i m a r y Liver T u m o u r s , F a l k S y m p o s i u m 2 5 , MTP Press, 1 9 7 8 , p p . 3 1 9 - - 3 2 6 . 75 Buecheler, J., a n d P. Kleihues, E x c i s i o n o f O 6 - m e t h y l g u a n i n e f r o m D N A of v a r i o u s m o u s e tissues foll o w i n g a single i n j e c t i o n o f N - m e t h y l - N - n i t r o s o u r e a , Chem.-Biol. I n t e r a c t . , 16 ( 1 9 7 7 ) 3 2 5 - - 3 3 3 . 76 H a b i g , W.H., M.J. P a b s t a n d W.B. J a c o b y , G l u t a t h i o n e S-transferases, T h e first e n z y m a t i c step in mere a p t u r i c acid f o r m a t i o n , J . Biol. C h e m . , 2 4 9 ( 1 9 7 4 ) 7 1 3 0 - - 7 1 3 9 . 77 J a c o b y , W.B., The g l u t a t h i o n e S-transferases, A g r o u p o f m u l t i f u n c t i o n a l d e t o x i f i c a t i o n p r o t e i n s , Adv. Enzymol., 46 (1978) 383--414. 78 A s a o k a , K., H. Ito a n d K ~ . T a k a h a s h i , M o n k e y g l u t a t h i o n e S-aryltransferases, II. P r o p e r t i e s o f t h e m a j o r e n z y m e p u r i f i e d f r o m the liver, J. B i o c h e m , ( T o k y o ) , 82 ( 1 9 7 7 ) 1 3 1 3 - - 1 3 2 3 . 79 Baines, P.J., H . G . Bray a n d S.P. J a m e s , M e r e a p t u r i c acid f o r m a t i o n in t h e developing r a t , X e n o b i o t i c a , 7 (1977) 653--663. 8 0 Hales, B.F., a n d A.H. Neims, D e v e l o p m e n t a l aspects o f g l u t a t h i o n e S-transferase B (ligandin) in r a t liver, B i o c h e m . J., 1 6 0 ( 1 9 7 6 ) 2 3 1 - - 2 3 6 . 81 Oesch, F., P a p e r p r e s e n t e d a t the s y m p o s i u m , I n f l u e n c e o f Metabolic A c t i v a t i o n s a n d I n t e r a c t i o n s o n Toxic Effects, 1 8 t h S p r i n g Meeting o f the D e u t s c h e P h a r m a c o l o g i s c h e Gesellschaft, Section Toxicolo g y , Mainz, M a r c h 1 5 , 1 9 7 7 . 8 2 H a t h w a y , D.E. a n d C~T. B e d f o r d , B i o t r a n s f o r m a t i o n s , In: D.E. H a t h w a y (Ed.), " F o r e i g n c o m p o u n d m e t a b o l i s m in m a m m a l s " Vol. 3, C h e m . Soc., L o n d o n , 1 9 7 5 , p p . 2 0 1 - - 4 4 8 . 83 R a n n u g , U., a n d C. R a m e l , The m u t a g e n i c i t y a n d m e t a b o l i s m o f 1 , 2 - d i c h l o r o e t h a n e , 2 n d Int. Conf. E n v i r o n . M u t a g e n s , A b s t r a c t s , 1 9 7 7 , p. 80. 8 4 R a n n u g , U., A. Sandvall a n d C. R a m e l , The m u t a g e n i c e f f e c t o f 1 , 2 - d i c h l o r o e t h a n e o n Salmonella typhimurium, 1. A c t i v a t i o n t h r o u g h c o n j u g a t i o n w i t h g l u t a t h i o n e in vitro, Chem.-Biol. I n t e r a c t . , 20 (1978) 1--16. 85 M u m b y , S.M., S.S. Gill a n d B.D. H a m m o c k , S u b s t r a t e specificity o f soluble m a m m a l i a n e p o x i d e h y drase, 1 7 6 t h ACS Meeting, Miami Beach, F l o r i d a , Sep. 1 1 - - 1 7 , A b s t r a c t s , Envr. 1 3 0 ( 1 9 7 8 ) . 86 Diaz G o m e z , M.I., P.F. S w a n n a n d P.N. Magee, The a b s o r p t i o n a n d m e t a b o l i s m in r a t s o f small oral doses o f d i m e t h y i n i t r o s a m i n e , I m p l i c a t i o n f o r t h e possible h a z a r d o f d i m e t h y l n i t r o s a m i n e in h u m a n f o o d , B i o c h e m . J., 1 6 4 ( 1 9 7 7 ) 4 9 7 - - 5 0 0 . 87 Calien, D . F . , A review of t h e m e t a b o l i s m o f x e n o b i o t i c s b y m i c r o o r g a n i s m s w i t h r e l a t i o n t o s h o r t t e r m test s y s t e m s f o r e n v i r o n m e n t a l m u t a g e n s , M u t a t i o n Res., 55 ( 1 9 7 8 ) 1 5 3 ~ 1 6 3 . 8 8 Selkirk, J . K . , Divergence o f m e t a b o l i c a c t i v a t i o n s y s t e m s f o r s h o r t - t e r m m u t a g e n e s i s assays, N a t u r e ( L o n d o n ) , 2 7 0 ( 1 9 7 7 ) 604---607. 89 M c C a n n , J., E. Choi, E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e Sal-

241

m o n e L l a / m i c r o s o m e test assay o f 3 0 0 c h e m i c a l s , Proc. Natl. A c a d . Sci. (U.S.A.), 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 9 0 Butler, T.C., The m e t a b o l i c h y d r o x y l a t i o n o f p h e n o b a r b i t a l , J. P h a r m . E x p . T h e r a p . , 1 1 6 ( 1 9 5 6 ) 326--336. 91 Harvey, D.J., L. Glazener, C. Stratton, J. Nowlin, R.M. Hilland M.G. Homing, Detection of a 5-(3,4d/hydroxy-l,5-cyclohexadien-l-yl)-metabolite of phenobarbital and mephobarbital in rat, guinea pig and human, Res. C o m m u n . C h e m . PathoL Pharmacol., 3 (1972) 557--565. 92 Crayford, J.V., and D.H. Hutson, Comparative metabolism of phenobarbitone in the rat (CFE) and m o u s e (CFI), Fd. Cosmet. Toxicol., in press. 93 Peters, R.A., M. Shorthouse, C.J.R. Thorne, P.F.V. Ward and N.S. Huskisson, Is phenobarbital a "gratuitous" inducer in male rats? Biochem. Pharmacol, 22 (1973) 2633--2634. 94 Hutson, D.H., Comparative metabolism of dieldrinin the rat (CFE) and in two strainsof m o u s e (CF1 and L A C G ) , Fd. Cosmet. Toxicol., 14 (1976) 577--591. 95 Dean, B.J.,D.H. Hutson, A.S. Wright and D.E. Stevenson, The protective action of Elutathione against the bacterial mutagenicity of cis-l,3-dichloropropene,The Pharmacologist, Vol. 20 (No. 3), Abstract No. 50 (1978) p. 154. 96 Climie, I.J.G., D.H. Hutson, B.J. Mon~ison and G. Stoydin, Glutathione conjugation in the detoxification of (Z)-l,2-dichloropropenc (a component of the nematocide D--D) in the rat, Xenobiotica, 9 (1979) 149--156. 97 Biles,R., T. Connor, N. Trleff and M. Legator, Charaeterisation of S-9 activation of D B C P in the Salmonella test system, The Pharmacologist, Vol. 20 (No. 3), Abstract No. 53, (1978) p. 155. 98 Ashby, J., and J.A. Styles, Does carcinogenic Potency correlatewith mutagenic potency in the A m e s assay? Nature (London), 271 (1978) 452--455. 99 Bock, K.W., G. van Ackeren, F. Lorch and F.W. Birke, Metabolism of naphthalene to naphthalene dihydrodiol Elucuronide in isolated hepatocytes and liver microsomes, Biochem. Pharmacol, 25 (1976) 2351--2356. 100 Ocseh, F.0 P. Bentley and H. Glatt, Prevention of benzo[a]pyrene-indueed mutagenicity by h o m o geneous e p o x i d e h y d r a t a s e , Int. J. C a n c e r , 18 ( 1 9 7 5 ) 4 4 8 - - 4 5 2 . 101 G l a t t , H . R . , a n d Oeseh, F., In a c t i v a t i o n o f e l e c t r o p h i l i c m e t a b o l i t e s b y g l u t a t h i o n e S-transferases a n d l i m i t a t i o n o f the s y s t e m d u e t o s u b c e l i u l a r l o c a l i z a t i o n , A r c h . T o x i c o l . , 39 ( 1 9 7 7 ) 8 7 - - 9 6 . 1 0 2 Oesch, F., D. R a p h a e l , H. S c h w i n d a n d H . R . Glatt, Species d i f f e r e n c e s in a c t i v a t i n g a n d i n a c t i v a t i n g e n z y m e s r e l a t e d t o t h e c o n t r o l o f m u t a g e n i c m e t a b o l i t e s , A r c h . T o x i c o l . , 39 ( 1 9 7 7 ) 9 7 - - 1 0 8 . 1 0 3 R a s m u s s e n , R.E., a n d I.Y. Wang, D e p e n d e n c e o f specific m e t a b o l i s m o f b e n z o ( a ) p y r e n e o n t h e i n d u c e r o f h y d r o x y l a s e a c t i v i t y , C a n c e r Res., 3 4 ( 1 9 7 4 ) 2 2 9 0 - - 2 2 9 5 . 1 0 4 H a u g e n , D.A., T.A. van d e r H o e v e n a n d M.J. C o o n , Purified liver m i c r o s o m a l c y t o c h r o m e P-450, S e p a r a t i o n a n d c h a r a c t e r i s a t i o n o f m u l t i p l e f o r m s , J. Biol. C h e m . , 2 5 0 ( 1 9 7 5 ) 3 5 6 7 - - 3 5 7 0 . 1 0 5 Wiebel, F.J., J . K . Scikirk, H.V. Gelboin, D.A. H a u g e n , T.A. van d e r H o e v e n a d n M.J. C o o n , P o s i t i o n specific o x y g e n a t i o n o f b e n z o [ u ] p y r e n e b y d i f f e r e n t f o r m s o f p u r i f i e d c y t o c h r o m e P - 4 5 0 f r o m r a b b i t liver, Proc. Natl. A e a d . Sci, (U.S.A.), 72 ( 1 9 7 5 ) 3 9 1 7 - - 3 9 2 0 . 1 0 6 C o n n e y , A.H., A . H . Y . L u , W. Levin, A. S o m o g y i , S. West, M. J a c o h s o n , D. R y a n a n d R. K u n t z m a n , E f f e c t o f e n z y m e i n d u c e r s o n s u b s t r a t e specificity o f t h e c y t o c h r o m e P - 4 5 0 ' s , D r u g Metab. Disposition, 1 (1973) 199--210. 1 0 7 Ulirich, V., P. Weber a n d P. W o l e n b e r g , The O - d e a l k y l a t i o n o f 7 - e t h o x y c o u m a r i n b y liver m i c r o somes, A d i r e c t f l u o r o m e t r i c test, H o p p e - S e y l a r s Z. Physiol. C h e m . , 3 5 3 ( 1 9 7 5 ) 1 1 7 1 - - 1 1 7 7 . 1 0 8 B e n t l e y , P., F. Oesch a n d H . R . G l a t t , Dual role o f e p o x i d e h y d r a t a s e in b o t h a c t i v a t i o n a n d inactivat i o n o f b e n z o [ u ] p y r e n e , A r c h . T o x i c o l . , 39 ( 1 9 7 7 ) 6 5 - - 7 5 . 1 0 9 B a l d w i n , M.K., u n p u b l i s h e d w o r k . 1 1 0 T h o m p s o n , C.Z., G.W. W h i t a k e r , J.C. Cline a n d R.E. M c M a h o n , Studies o n t h e m i c r o s o m a l activat i o n o f m u t a g e n e s i s in S a l m o n e l l a , E f f e c t o f i n c r e a s i n g doses o f A r o c i n r 1 2 5 4 , The P h a r m a c o l o g i s t , Vol. 2 0 (No. 3), A b s t r a c t No. 51 ( 1 9 7 8 ) p. 1 5 5 . 111 M c G r e g o r , D.B., The r e l a t i o n s h i p o f 2 - a c e t a m i d o f l u o r e n e m u t a g e n i c i t y in plate tests w i t h its in vivo liver cell c o m p o n e n t d i s t r i b u t i o n a n d its c a r c i n o g e n i c p o t e n t i a l , M u t a t i o n Res., 3 0 ( 1 9 7 5 ) 3 0 5 - - 3 1 6 . 1 1 2 Miller, E.C., a n d J . A . Miller, B i o c h e m i c a l i n v e s t i g a t i o n s o n h e p a t i c carcinogenesis, J. Natl. C a n c e r Inst., 15 ( 1 9 5 5 ) 1 5 7 1 - - 1 5 9 0 . 1 1 3 A s h b y , J., a n d J . A . Styles, F a c t o r s i n f l u e n c i n g m u t a g e n i c p o t e n c y in vitro, N a t u r e ( L o n d o n ) , 2 7 4 (1978) 20--22. 1 1 4 Glatt, H . R . , F. Oeseh, A. Frigerio a n d S. G a r a t t i n i , E p o x i d e s m e t a b o l i c a l l y p r o d u c e d f r o m s o m e k n o w n c a r c i n o g e n s a n d f r o m s o m e clinically u s e d drugs, 1. Differences in m u t a g e n i c i t y , Int. J. Cancer, 16 ( 1 9 7 5 ) 7 8 7 - - 7 9 7 . 1 1 5 Oesch, F., M a m m a l i a n e p o x i d e h y d r a s e , I n d u c i b l e e n z y m e s c a t a l y s i n g the i n a c t i v a t i o n o f c a r c i n o genic a n d c y t o t o x i c m e t a b o l i t e s derived f r o m a r o m a t i c a n d olefinic c o m p o u n d s , X e n o b i o t l c a , 3 (1973) 305--340. 1 1 6 Grover, P.L., A. Hever, K. Pal a n d P. Sims, The i n v o l v e m e n t o f a d i o l - e p o x i d e in the m e t a b o l i c activ a t i o n o f b e n z o [ a ] p y r e n e in h u m a n b r o n c h i a l m u c o s a a n d in m o u s e skin, Int. J. C a n c e r , 18 ( 1 9 7 6 ) 1--6.