263

Mutation Research, 259 (1991) 263-276 © 1991 ElsevierSciencePublishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100063E

MUTGEN 00036

Mechanism of action of food-associated polycyclic aromatic hydrocarbon carcinogens Anthony Dipple and C. Anita H. Bigger N CI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702-1201 ( U.S.A.) (Received6 February 1990) (Accepted 10 August 1990)

Keywords: Polycyclicaromatic hydrocarbons; Mechanism of mutagenesis and carcinogenesis; Mutagenesis, mechanism of; Carcinogenesis, mechanism of

Summary The polycyclic aromatic hydrocarbon carcinogens are formed in the inefficient combustion of organic matter and contaminate foods through direct deposition from the atmosphere or during cooking or smoking of foods. These potent carcinogens and mutagens require metabolism to dihydrodiol epoxide metabolites in order to express their biological activities. In vitro studies show that these reactive metabolites can react with the bases in D N A with different specificities depending upon the hydrocarbon from which they are derived. Thus, the more potent carcinogens react more extensively with adenine residues in D N A than do the less potent carcinogens, with the result that mutation at A . T base pairs is enhanced for the more potent carcinogens. In the past few years, considerable clarification of the mechanisms of metabolic activation have been achieved and the focus for the immediate future is expected to be on how the reactive metabolites actually bring about biological responses.

(1) Introduction As described in the previous paper by Lijinsky, polycyclic aromatic hydrocarbons can contaminate food products either by direct deposition from the atmosphere or by being introduced

Correspondence: Dr. Anthony Dipple, NCI-Frederick Cancer Research and DevelopmentCenter, ABL-Basic Research Program, P.O. Box B, Frederick, MD 21702-1201 (U.S.A.). By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

through the smoking, curing or cooking of food. These toxic chemicals are distributed widely throughout the environment because they are formed through incomplete combustion of organic matter in internal combustion engines, in power generators and in refuse burning and forest fires. (1.1) Identification of specific polycyclic aromatic hydrocarbon carcinogens Early indications of the carcinogenic properties of polycyclic aromatic hydrocarbons arose from observations by Percival Pott in 1775 of the carcinogenic properties of combustion products such as soot, but it was not until 1930 that carcinogenic activity was associated with a specific

264

K-

region

Dlbenz(a,h)anthracene

Fig. 1. First pure chemical recognized to have carcinogenic activity. defined chemical structure. In the 1920s, Kennaway had demonstrated that carcinogenic tars could be produced in the laboratory by pyrolysis of hydrocarbons in a hydrogen atmosphere (Kennaway, 1924, 1925) thereby establishing that the carcinogen in these tars must be a hydrocarbon of some kind. By using the fluorescence of these synthetic tars as a putative marker for their carcinogenic properties, the pioneering researchers in Kennaway's laboratory were able to identify dibenz[a,h]anthracene (Fig. 1) as a chemical carcinogen in 1930 (Kennaway and Hieger, 1930). Subsequently, benzo[a]pyrene was identified as the principal fluorescent carcinogen in various tars (Cook et al., 1933).

(1.2) Polycyclic aromatic hydrocarbons require metabolic activation In these early studies, carcinogenic activity was monitored by painting solutions or suspensions of the hydrocarbons on mouse skin and observing the development of skin tumors. For this reason, the hydrocarbons were thought of as directly acting carcinogens (producing tumors locally at the site of administration) in contrast to the azo dye carcinogens, for example, which did not induce local tumors on the skin. The dyes were administered by mouth and generated liver tumors in rats, indicating that their action might be mediated through some metabolites formed in the liver. The direct-acting qualities of the hydrocarbons suggested that metabolism might not mediate their carcinogenic actions because it was not clear initially that xenobiotic metabolism could occur in organs other than the liver. A number of studies argued that metabolism must occur in the skin, however. In 1951, Miller showed that benzo[a]pyrene became covalently

bonded to epidermal proteins of mouse skin and such binding (which did not occur in the absence of some metabolic activity) was later confirmed for other hydrocarbons using radiotracer techniques (Heidelberger and Moldenhauer, 1956). Perhaps the most influential study in establishing the involvement of metabolism came from Brookes and Lawley (1964) who showed for a series of hydrocarbons, a correlation between extents of covalent binding to DNA in the skin and carcinogenic activity.

(1.3) Identification of the carcinogenic metabolites o] polycyclic aromatic hydrocarbons Although an extensive search for the active metabolites of the polycyclic aromatic hydrocarbons was undertaken in several laboratories, another 10 years passed before Peter Sims and his colleagues (1974) identified vicinal dihydrodio! epoxides (Fig. 2) as the probable ultimate carcinogenic metabolites. For some years it had been widely thought that epoxides formed at the bond with greatest double-bond character (the 'K-region') might be the active forms of the hydrocarbons. However, in 1973 (Baird et al., 1973) it was shown that the DNA-binding metabofite of 7-methylbenz[a]anthracene was not the K-region epoxide and, in the same year, that the 7,8-dihydrodiol of benzo[a]pyrene was bound to DNA in the presence of microsomes some 15-fold more efficiently than benzo[a]pyrene itself (Borgen et al., 1973). These two findings directed Sims away from K-region epoxides and towards an active metabolite formed from the 7,8-dihydrodiol, that he surmised would be a dihydrodiol epoxide (Fig. 2). Support for this concept developed very rapidly and it was soon shown that the metabolites on the dihydrodiol epoxide pathway were very mutagenic and carcinogenic (reviewed in Dipple et al., 1984). Jerina and Daly added an important qualification of the dihydrodiol epoxide concept by showing, 12

I

11

2

10

3

i"

9 7

6

5

~,"

"

~ OH

7,8 - E p o x i d e

7,8 - D i h y d r o d i o l

OH 7,8 - D l h y d r o d l o l 9,10 -

epoxlde

Fig. 2. The dihydrodiolepoxide route of metabolic activation of polycyclicaromatichydrocarbons(Simset al., 1974).

265 from structure-activity considerations, that the key dihydrodiol epoxide metabolites would probably be those where the epoxide was adjacent to a 'bay region' (Jerina and Daly, 1976) (Fig. 3). This generalization has held up quite well over the past 14 years with the active forms of many hydrocarbons having been shown to be bay region dihydrodiol epoxides. The major achievement in detailing the mechanism of action of the polycyclic aromatic hydrocarbons is comprised by the knowledge that has been generated concerning the metabolic activation of these chemicals. However, this is only one small component of their overall mechanism of action. We now need to understand the events involved after these reactive metabolites are formed. How do they react with the cell and how do these reaction products lead to mutagenesis and carcinogenesis? These events are much less well-defined than those involved in metabolism but some interesting relationships are now beginning to emerge.

(2) Chemistry of DNA modification by hydrocarbon dihydrodiol epoxides (2.1) Site selectivity in the reaction of hydrocarbon dihydrodiol epoxides with nucleic acid bases The hydrocarbon dihydrodiol epoxides come under the general heading of aralkylating agents and it is important to note that their chemistry is quite different from that of alkylating agents. The simplest alkylating agents preferentially alkylate the ring nitrogen atoms of the purine and pyrimidine bases in DNA (Lawley, 1966), whereas alkylating agents that act through alkyldiazonium ions are known to be capable of reaction at exoA

B Bay

O

OH

j ..... " ~ CH3 Bay Region

DlhydrodlolEpoxlde

"""~OH

o

SN2

I l---nFvorW

S 1

I

at N2

Soft N-7 reactivity decreases

Os- and/or N2 reactivity increases

Fig. 4. The electroniccharacter of an alkylatingor aralkylating agent determinesits site selectivity(Dipple, 1988). cyclic oxygen atoms in addition to the pyridinetype ring nitrogens (Loveless, 1969). In contrast to this behavior, aralkylating agents, as first shown by Dipple et al. (1971), react almost exclusively with the exocyclic amino groups of nucleic acid bases under aqueous conditions. It is not entirely clear why the alkylating and aralkylating agents exhibit such a pronounced difference in their chemical reactions with DNA constituents but it has been suggested that these differences are largely due to electronic effects (Dipple et al., 1982). A number of model studies of the reactions of benzylating agents (Moschel et al., 1979, 1980, 1986) and styrene oxide (Latif et al., 1988) with guanosine have led to the concepts summarized in Fig. 4. This suggests that site selectivity is determined to a large extent by the ionic character of the alkylating or aralkylating agent. Thus, agents which do not ionize readily react primarily on the ring nitrogen atom of guanine residues (N-7), whereas a fairly extensively ionized substrate can react at the exocyclic N 2 and 0 6 sites. The preference for reaction at either of these latter exocyclic sites is based on the character of the ionic substrate. Thus, if the substrate delocalizes charge readily from the reaction center (an aralkylating agent) reactions occur on the exocyclic N 2 group while, if the substrate cannot delocalize this charge effectively (an alkylating agent), reaction occurs on the exocyclic 0 6 atom (Dipple, 1988).

HO =-" OH Non-BayRegion

DIhyclrodlolEpoxlde

Fig. 3. Bayregiondihydrodiolepoxidesare usuallythe biologicallyactivedihydrodiolepoxides(Jerinaand Daly,1976).

(2.2) Nucleic acid base selectivity in the reactions oJ hydrocarbon dihydrodiol epoxides with DNA The initial studies of nucleic acid aralkylation were undertaken using 7-bromomethylbenz[a]anthracene (Dipple et al., 1971) and 7-bromomethyl-

266

jCH2 HN

/.CH 2 HN

Fig. 5. Aralkylating agents react with the amino groups of nucleic acid bases under aqueous conditions (Dipple et al., 1971; Rayman and Dipple, 1973a,b) R = H for 7-bromomethyibenz[a]anthracene and R = CH 3 for 7-bromomethyl-12-methylbenz[a]anthracene.

12-methylbenz[a]anthracene (Rayman and Dippie, 1973a) as aralkylating agents (Fig. 5). Each of these compounds reacted primarily with the amino group of deoxyguanosine residues in DNA and much lower yields of products were formed at the amino groups of deoxyadenosine and deoxycytidine. However, for the two bromo compounds the

Hydrocarbon

relative extents of reaction with the different bases in D N A were quite different both in vitro and in vivo (Rayman and Dipple, 1973a,b). An analogous situation clearly exists for the dihydrodiol epoxide metabolites of the hydrocarbons. Here, reaction with the amino groups of the bases is the principal reaction and, for the most extensively

Isomer

1.

Principal Reaction Sites in DNA

O,~

Benzo[a]pyrene

Amino group of guaninea

OH

7,12-Dlmelhyibenz[a]anthracene

2.

3.

O~,/O H

Amino group of guanine, amino group of adenineb,c

Q ~.,,,~OH

Aminogroup of adeninec'd

(~~m,~ss .,.,,0 H

Amino group of adenlnee'f

4.

Benzo[~phenenthracene

~T 5.

"oH

O . ~ O H ~-,~-'~~ ' ',,,SOH

Amino group of adenine> amino group of guaninee'f

aJeffrey el ai., 1976; bDIpple el al., 1984;¢Verlcat el al., 1989; dCheng el al., 1988;eDIpple el al., 1987; tAgarwal et al., 1987.

Fig. 6. The principal site of reaction of dihydrodiol epoxides with DNA depends upon the hydrocarbon and the specific isomeric structure. Note that entries 1, 2, and 5 have the same absolute stereochemistry as do entries 3 and 4.

267 studied hydrocarbon, benzo[a]pyrene, the amino group of guanine residues is clearly the major reaction site (Jeffrey et al., 1976; Koreeda et al., 1976; Osborne et al., 1976). However, for the dihydrodiol epoxide metabolites of 7,12-dimethylbenz[a]anthracene (Dipple et al., I983a,b) and benzo[c]phenanthrene (Dipple et al., 1987; Agarwal et al., 1987), the amino group of deoxyadenosine is the principal site of reaction with some optical isomers but not with others (Fig. 6). Thus, other chemical putties remain to be solved. These include developing rationales for the facts that (a) the dihydrodiol epoxides of some hydrocarbons preferentially react with deoxyadenosine whereas others preferentially react with deoxyguanosine; (b) different diastereomers of the same hydrocarbon dihydrodiol epoxide exhibit quite different base selectivities in their reactions with DNA; and (c) different diastereomers exhibit different mutagenic and carcinogenic properties. (2.3) Characterization of hydrocarbon dihydrodiol epoxide-DNA adducts Characterization of hydrocarbon dihydrodiol epoxide-DNA adducts has been quite difficult, primarily because the dihydrodiol epoxides themselves have not always been easy to obtain in reasonable quantities and because the yields of adducts from their reactions with DNA have been quite low. For these reasons, adduct characterizations have often been carried out on the adducts of ribosides formed in reactions with homopolymers such as polyG. The characterized riboside has then been related to the deoxyribonucleoside of interest usually through its respective circular dichroism spectra. More recently (Dipple et al., 1987; Agarwal et al., 1987; Reardon et al., 1987; Cheng et al., 1988a,b, 1989; Chadha et al., 1989; Nalr et al., 1989) reasonable amounts of the deoxyribonucleosides have been prepared by reaction of the dihydrodiol epoxides with deoxyribonucleoside-5'-phosphates and characterizations have then been possible for the deoxyribonucleosides themselves. The reaction of a dihydrodiol epoxide with high concentrations of deoxyribonucleoside-5'-phosphates usually leads to the formation of reasonable amounts of adduct, though the yield varies with each dihydrodiol epoxide. A difficulty arises

"%3

-o.

%3

syn Olhydrodlol Epoxlde Enantlomers

-o.

anti Dlhydrodiol Epoxlde Enantlomers

Fig. 7. Structures of 4 isomeric dihydrodiol epoxides. Each structure represents a single optically active compound but these materials are most readily synthesized as racemates i.e. mixtures of both enantiomers. because each hydrocarbon dihydrodiol epoxide can exist in 4 isomeric forms (Fig. 7). The 2 diastereomeric forms (called here the syn and anti forms) are available separately but it is not always possible to obtain the pure optical enantiomers of each diastereomer. If a racemic dihydrodiol epoxide is used, 4 products are usually obtained in reactions with a given nucleotide. These result from cis and trans opening of the epoxide ring of each enantiomer by the amino group of the nucleotide (Fig. 8). Of course, if optically pure material is available, the chemistry is simpler and only 2 products are formed, from cis and trans opening of the epoxide. All of these products are usually separable by high-pressure liquid chromatography and they can usually be identified by a combination of spectroscopic methods, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy being the most important. Products which are identical, except that they are formed from different enantiomers of the dihydrodiol epoxide e.g. (i) and ('tii) in Fig. 8, exhibit CD spectra which are exact mirror images of one another. Products which differ only in configuration at the carbon which has undergone reaction, OH OH R'NH'%~ OH R-NH~OH

O•OH

+ Reaction

4,, 4,, °.

with dGMP dAMP dCMP

(.-..~)

(I) Irons

(11)©Is

OH R'NH~

OH '~OH .i R'NH~

OH

H

(111)trans (iv) cls Fig. 8. Rac~mic dihydrodiol epoxide forms 4 products, optically active dihydrodiolepoxideformsonly 2 products.

268 e.g. (i) and (ii) in Fig. 8 (these are cis/trans isomers), have CD spectra which are of opposite signs but are not exact mirror images. These spectral properties allow the inter-relationships of the reaction products to be established. It is usually possible then to assign the cis or trans structures on the basis of N M R measurements. If a racemic mixture was used in the reaction, the only remaining difficulty is in assignment of absolute stereochemistry i.e. determining which product has structure (i) and which has structure (iii) (Fig. 8), for example. If the preparation of one of the optically pure dihydrodiol epoxides is not possible, these assignments can be made from the CD spectra if closely related products have been assigned and their spectra are available for comparison. Alternatively, for the case of 7,12-dimethylbenz[a]anthracene, these assignments were made by treating cells separately with the 2 optically active dihydrodiol precursors of the dihydrodiol epoxide and thereby generating products with known absolute stereochemistry (Vericat e t al., 1989).

(3) Relationship between metabolism, DNA modification, and biological activity

(3.1) Stereospecificity in the metabolic formation of hydrocarbon dihydrodiol epoxides In the chemical synthesis of dihydrodiol epoxides, products obtained are racemic mixtures unless specific procedures are included to resolve the dihydrodiol precursors (Harvey, 1985). However, in the metabolic formation of bay region dihydrodiol epoxides from the parent hydrocarbons in animal tissues, a great deal of stereospecificity or at least stereoselectivity is observed. For example, in the metabolism of benzo[ a]pyrene by liver microsomes from rats pretreated with 3-methylcholanthrene, the 7,8 bond is selectively oxidized to give almost exclusively 1 enantiomer of the 7,8-epoxide i.e. the (7R,8S)-epoxide (S.K. Yang et al., 1977; Levin et al., 1980a). Either enantiomer of the epoxide is hydrated by epoxide hydrolase (Armstrong et al., 1981) through stereospecific attack of water at the 8 position to give a trans-dihydrodiol. Further oxidation of these dihydrodiols to dihydrodiol epoxides again occurs stereoselectively but the actual ratio of syn- to anti-di-

hydrodiol epoxide formed depends upon the source of the enzymatic activity used (reviewed in Dipple et al., 1984). Despite quantitative variations, for most hydrocarbons it seems that the anti-(R, S)-dihydrodiol-(S, R)-epoxide is predominantly formed from the (R,R)-dihydrodiol. A lesser amount of a syn-(S, R)-dihydrodiol-(S, R)epoxide is usually the predominant product from the (S,S)-dihydrodiol. This has been established for several hydrocarbons including benzo[ a]pyrene (Levin et al., 1980a), benzo[c]phenanthrene (Pruess-Schwartz et al., 1987) and 7,12-dimethylbenz[a]anthracene (Vericat et al., 1989). Unfortunately, the dihydrodiol epoxide which predominates as a metabolic product, i.e. the (R,S)dihydrodiol-(S,R)-epoxide, is usually found to be a much more potent carcinogen than the other isomers (reviewed in Jerina et al., 1986). Although only a small fraction of any hydrocarbon exposure is converted metabolically to the dangerous dihydrodiol epoxides, biological systems preferentially generate the most noxious isomer of the four.

(3.2) Comparison of DNA adducts formed from hydrocarbon dihydrodiol epoxides exhibiting different tumorigenic activities It is not yet clear exactly how the dihydrodiol epoxides initiate the carcinogenic process but many workers believe that the formation of DNA adducts constitutes one key event. To evaluate the possible deleterious effects of specific DNA adducts, we compared the adducts generated by 1 extensively studied hydrocarbon carcinogen, benzo[a]pyrene (BaP) with those formed by a much more potent tumorigen, 7,12-dimethylbenz[a]anthracene (DMBA) (Table 1) (Dipple et al., 1983b). This somewhat simplistic comparison inTABLE 1 COMPARISON OF 7,12-DIMETHYLBENZ[a|ANTHRACENE (DMBA) WITH BENZO[a]PYRENE (BaP) Property Tumor-initiatingactivity Binding to DNA in skin Reaction with deoxyadenosinein DNA

DMBA/BaP - 30/1 a - 2/1 b - 30/1 c

a Wislockiet al., 1980; Slaga et al., 1979; Scribner et al., 1980. b Phillips et al., 1979. c Ashurst and Cohen, 1981; Biggeret al., 1983.

269 dicates that DMBA is some 30-fold more potent as a tumor initiator than is BaP and that, in their reactions with DNA, the only property roughly correlated with this is their different reactivities towards deoxyadenosine residues in DNA. This observation was used to suggest that reactions with deoxyadenosine residues might for some reason be more important events in tumor initiation than reactions with deoxyguanosine residues (Dippie et al., 1983b). Although no proof of this hypothesis has been forthcoming, some interesting observations have been made. For example, in Balmain's studies of the activation of the H-ras oncogene in mouse skin tumors initiated by DMBA (QuintaniUa et al., 1986), activation mostly resulted from mutation at the adenine in the second position of codon 61. Similarly, when the H-ras oncogene is activated by reaction with BaP dihydrodiol epoxide in vitro the efficiency of activation at adenine residues (i.e. activation at adenine residues divided by the number of reactions at adenine) was almost 10 times greater than at guanines (Vousden et al., 1986). Other intriguing relationships have also been described. DMBA is probably the most potent tumor initiator amongst the hydrocarbons and a large fraction of its reactions with DNA (over 50%) can be attributed to reactions with deoxyadenosine residues in DNA (Milner et al., 1985). This contrasts with the trace amounts of reaction with deoxyadenosine found for the much less potent carcinogen, benzo[a]pyrene (Ashurst and Cohen, 1981). Although benzo[c]phenanthrene is a relatively weak carcinogen, the synthetic dihydrodiol epoxides derived from this hydrocarbon are potent tumor initiators (Levin et al., 1980b) and the (R,S)-dihydrodiol-(S,R)-epoxide is regarded as the most potent dihydrodiol epoxide yet examined (Levin et al., 1986). This latter dihydrodiol epoxide again reacts predominantly (> 50%) with deoxyadenosine residues in DNA (Dipple et al., 1987) so some association between ability to initiate tumors and to react with deoxyadenosine residues seems to exist. The only other property which roughly correlates with these properties is distortion from planarity in the parent hydrocarbon structure. Thus, DMBA and benzo[c]phenanthrene are grossly distorted from planarity because of the steric problem of accommodating all

the hydrogen atoms adjacent to the bay region (Hirshfeld, 1963; Zacharias et al., 1984).

(4) Mutagenic specificity of hydrocarbon dihydrodiol epoxides (4.1) Systems for the study of mutagenic specificity of carcinogens The obvious complexity of the relationship between the chemistry of adduct formation and carcinogenic properties has led to several studies of a simpler biological response i.e. mutagenic activity. However, relative mutagenic activities determined using the Ames assay (Ames et al., 1973) or Chinese hamster cells (Arlett, 1977) do not provide a very subtle measure of biological activity and, currently, these assays are being replaced by assays that measure not only mutagenic activity but also the distribution of the mutations in a target sequence and the nature of each mutation (Miller, 1983). Such studies were initially carried out using a bacterial system (Eisenstadt, 1985) but, with the development of suitable shuttle vectors, such' mutagenic specificity studies can utilize mammalian, even human, cells to generate mutations in the vector (Drinkwater and Klinedinst, 1986; DuBridge et al., 1987; Seidman et al., 1985). The system of Seidman and Dixon and their collaborators involves the chemical modification of the vector DNA with a mutagen, transfection of the treated DNA into human cells, recovery of the plasmid DNA and transformation of E. coli, which has a defective LacZ gene, due to an amber mutation, with the treated vector's progeny DNA to allow selection of mutants. Since the E. coli are grown in ampicillin, only those bacteria that have the amp r gene from uptake of the plasmid can grow. If the plasmid is not mutated in the supF gene, expression of this suppressor tRNA gene allows the mutant E. coli to make/~-galactosidase and form blue colonies on X-Gal plates. If the plasmid supF gene is mutated by the chemical treatment, the E. coli mutation will not be suppressed and the colonies will be white or pale blue. These colonies are collected, and the plasmid DNA therein is ultimately sequenced to define the specific mutation present (Seidman et al., 1985).

270

o. C T Begin tRNA

~

T T T T

C

OH ( _ ) anti Benzo[c]phenanthrene Dihydrodiol Epoxlde H

G G G

T G

End tRNA I

A A

G T A G A CA I A 110T 130 A A 150 A AA 170 A i T = A A I A AA I 5' GGTGGGGTTCCCGAGCGGCcAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACTTCGAAGG7-TCGAATCCTTCCCCCACCACCA3' A G T A A T C A G T A A T C A G T A G T C A G T A G T T A A T A O. /0. A A A T r ~ j.OH / .".... ,,OH A A G

"'OH

A A ~ ~

~

~ /

'~OH ~ ~ ~ racemic anti Benzo[a]pyrene ~Dihydrodlol Epoxide

Fig. 9. Comparison of mutagenic hotspots in the supF tRNA DNA sequence for (-)anti-benzo[c]phenanthrene dihydrodiol epoxid~ [(4R,3S)-dihydrodiol-(2S,1R)-epoxide] (Bigger et al., 1989) and racemic anti-benzo[a]pyrene dihydrodiol epoxide [(7S,8R)-dihydrodiol-(9R,10S)-epoxide and (7R,8S)-dihydrodiol-(9S, lOR)-epoxide] (J.L. Yang et al., 1987). 12, deletion; zx, insertion.

(4.2) Comparison of the mutagenic specificities of different hydrocarbon dihydrodiol epoxides Mutations arise more frequently at some sites in the target gene than at others and in Fig. 9 hotspots for mutation by racemic anti-benzo[a]pyrene dihydrodiol epoxide, as reported by Maher and her colleagues (J.L. Yang et al., 1987), and hotspots for mutation with optically active benzo[c]phenanthrene dihydrodiol epoxide are compared (Bigger et al., 1989). For this purpose, we have defined a hotspot as a base pair at which 4 or more mutations were detected. The main points that can be gathered from an examination of Fig. 9 are that there are hotspots which are unique to particular chemical carcinogens. For example, site 106 is a hotspot for benzo[c]phenanthrene but not for benzo[a]pyrene, and site 123 is a hotspot for benzo[a]pyrene but not for benzo[c]-phenanthrene. Some sites are common to both carcinogens but at some of these, e.g. site 112, the mutational change induced by each carcinogen is different. Thus, it is quite clear that the sites and nature of the mutational events are unique to each of these different hydrocarbon carcinogens. All the base substitution mutations reported are included in the quantitative comparison shown in Table 2. The differences in mutagenic specificity between the 2 chemicals are very obvious in this

analysis where it can be seen that the vast majority of the benzo[a]pyrene-induced mutations arise at G- C base pairs (90%) whereas only 49% of the benzo[c]phenanthrene-induced mutations arise at G . C pairs. This correlates very well with the chemical properties of these 2 agents discussed earlier (Section 3.2), and this correlation is strong support for the idea that these mutations are targeted to the DNA adducts produced by the original chemical treatment. TABLE 2 BASE CHANGES INDUCED BY TWO DIFFERENT HYDROCARBON DIHYDRODIOL EPOXIDES

( -- )Anti-benzo[c]phenanthrene dihydrodiol epoxide a

Racemic anti-benzo[ a ]pyrene dihydrodiol epoxide b

Untreated

Treated

Untreated

Treated

Transversions G. C --* T-A G. C --, C. G A - T - , T-A A.T ~ C.G

2 3 1 0

29 (29%) 15 (15%) 35(35%) 7 (7%)

6 1 0 0

45 (64%) 13 (18%) 3 (4%) 0

Transitions G-C---, A . T A-T~G.C

0 0

5 (5%) 9 (9%)

0 0

6 (8%) 4 (6%)

Base changes

a Bigger et al., 1989. b Yang et al., 1987.

271

The major fraction of the mutations could be accounted for if DNA polymerase inserts an adenine opposite chemically modified adenine and guanine residues (Table 2).

Agents such as benzo[c]phenanthrene, which react extensively with deoxyadenosine residues in DNA, open up new avenues for research since little is known about the biological consequences of bulky damage on adenine residues. Analysis of these mutagenic properties suggests that each specific isomer of each hydrocarbon dihydrodiol epoxide can exhibit a potentially unique biological activity related to its unique chemical properties.

(5) Effects of hydrocarbon-DNA adducts on DNA

polymerase activity In 1979, Strauss and his colleagues (Moore and Strauss, 1979) showed that DNA adducts gener-

Codon 12

Codon 61 Antisense BaPDEs

Sense BcPhDEs 12"

Sense BcPhDEs BaPDEs

t-

® 2 :=

o :D

CGTA+-+-~

c

12

2

CGTA+- + -

T

A '+ - '

D

-8 38

-3 -2

-7

1'

Gq G A

-1

Fig. 10. Autoradiographs showing sites of arrest of T7-DNA polymerase (Sequenase) caused by chemical modification of ras DNA sequences by dihydrodiol epoxide derivatives of benzo[c]phenanthrene (BcPhDEs) and benzo[a]pyrene (BaPDEs). Panels A and B show sequences adjacent to codon 61 in the sense and antisense strands, while panels C and D show sequences adjacent to codon 12 in the sense strand. The hydrocarbon derivatives used to treat the DNA are indicated above each lane by 1 (syn) or 2 (anti). The absolute stereochemistries are as follows: + 1 BcPhDE, (4S,3R)-dihydrodiol-(2S,1R)-epoxide; - 1 BcPhDE, (4R,3S)-dihydrodiol(2R,1S)-epoxide; + 2 BcPhDE, (4S,3R)-dihydrodiol-(2R,1S)-epoxJde; - 2 BePhDE, (4R,3S)-dihydrodiol-(2S,1R)-epoxJde; + 2 BaPDE, (TR,8S)-dihydrodiol-(gS,lOR)-epoxJde; - 2 BaPD~ (7$,8R)-dihydrodiol-(9R,lOS)-epoxide. The symbols C, G, T and A indicate the sequence of the template strand of unmodified ras DNA. Lanes labeled solvent refer to polymerase action on ras DNA treated with solvent alone.

272

ated from the dihydrodiol epoxide of benzo[a]pyrene could inhibit the action of DNA polymerase. This phenomenon has been utilized to look at the sequence specificity of the interaction of several dihydrodiol epoxides with the ras oncogenes in vitro (Reardon et al., 1989). Although it was not possible to interpret these experiments with great precision, it could be seen (Fig. 10) that individual optical isomers of dihydrodiol epoxides exhibit subtly different sequence specificities in their reactions with DNA. For example, at the guanine labeled 1, the syn-(4R,3S)dihydrodiol-(2R,1S)-epoxide of benzo[c]phenanthrene gives the most intense band, while at the adenine labeled 2, anti-(4S,3R)-dihydrodiol(2R,1S)-epoxide inhibits the polymerase most effectively. At other sites, all isomers are similarly effective, i.e., the bands labeled 3, and there are sites, e.g., the bands labeled 4, where the syn isomers are more effective than the anti isomers. Another point of interest is that the benzo[c]phenanthrene derivatives generate arrest sites associated with the purines in the template strand but where the template contains runs of pyrimidines (e.g., at regions marked 5 and 6), polymerase arrest is not seen. In contrast, the arrest sites formed with anti-benzo[a]pyrene dihydrodiol epoxides, although showing some subtle differences between enantiomers (as indicated by 7 and 8), demonstrate a clear preference for reaction at guanines only, as indicated by the absence of bands in the region labeled 9, where the template sequence is devoid of guanines. These findings are consistent with previous chemical studies (see Section 3.2) demonstrating the base-binding preferences of the dihydrodiol epoxides of benzo[c]phenanthrene and benzo[a]pyrene. A third point is that dihydrodiol epoxides with the same absolute stereochemistries, but derived from different hydrocarbons, show dramatic differences in their sequence specificities. For example, the anti(7R,8S)-dihydrodiol-(9S,lOR)-epoxide of benzo[a]pyrene shows more selectivity in binding to deoxyribonucleotides of codon 12 (Fig. 10D) than the benzo[c]phenanthrene derivative [anti-(4R, 3S)-dihydrodiol-(2S,1R)-epoxide] with the same absolute stereochemistry (Fig. 10C). These findings show that not only is the specific base modification in DNA determined by the differences in

3'--A

3'

5 '32p

G

,,,-, A

G

5'

3'

3' 5 '~p

A

G--5'

A

G

5'

o- D. D.

A*--

--G*

Fig. 11. An oligonucleotide (5'-TsGT~0ATsC2T4CT3CT-3') modified with 7-bromomethylbenz[a]anthraceneeither at the single adenine (A*) or singleguanine(G * ) was incubatedwith 32p-5'-end-labeled primer (5'-AGA3GA4G2-3'), appropriate deoxyribonucleoside-5'-tripbosphates and DNA polymerase (Sequenase) for 30 sec at room temperature.The products were purified by column chromatography.Aliquots of the primeroligonucleotide complex were incubated with individual deoxyribonucleoside-5'-triphosphates or H20 as indicated and Sequenase for 3 min at 37 ° C. The autoradiographs at the bottom of the figure show the polyacrylamide gel electrophoretic separation of products which terminate opposite the hydrocarbon adduct. . \

chemistry of these reactive metabolites but even the distribution over specific bases in a sequence is mandated by the complex interaction of the chemistry and stereochemistry of the dihydrodiol epoxides. In other approaches using DNA polymerases in vitro, it is becoming possible to measure which specific bases are incorporated opposite various chemical adducts in D N A (Kuchino et al., 1987; O'Connor and Stohrer, 1985; Michaels et al., 1987; Boosalis et al., 1987). There is no evidence on base incorporation opposite hydrocarbon dihydrodiol epoxide adducts so far, but Reardon et al. (1990) have investigated incorporation opposite bromomethylbenz[a]anthracene-DNA adducts. These adducts (Fig. 5), like dihydrodiol epoxide adducts, place a hydrocarbon moiety on the amino groups of the bases. As shown in Fig. 11, for both modified adenine and guanine residues, TT-DNA polymerase preferentially incorporated adenine residues opposite these adducts. It is of interest to note that if the same situation obtains for dihydrodiol epoxide adducts, this incorporation would

273

account for the major mutagenic events recorded in Table 2.

(6) Conclusion It is not yet possible to define in detail the mechanism of action of polycyclic aromatic hydrocarbon carcinogens. However, a tremendous clarification of the mechanisms through which they are metabolically activated has developed over the last 10-20 years. These developments include some very careful studies of the stereochemistry of metabolism and of the biological properties of individual optically active metabolites. As progress in the area continues, it is becoming clear that each polycyclic aromatic hydrocarbon-reactive metabolite is unique in its chemistry and therefore, in its interactions with the cell. These differences can be very subtle but are sufficient to shift the main site of reaction with DNA from the amino group of guanine residues to the amino group of adenine residues. Much less is known about adenine adducts than about guanine adducts but since it is now possible to generate both of them and to examine the sequence contexts in which they preferentially occur and the way in which these substrates are operated on by DNA polymerases in in vitro systems, there is hope that the mechanism through which such adducts generate mutations will be clarified in the future.

Acknowledgement Research sponsored by the National Cancer Institute, DHHS, under contract No. NO1-CO74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

References Agarwal, S.K., J.M. Sayer, H.J.C. Yeh, L.K. Pannell, B.D. Hilton, M.A. Pigott, A. Dipple, H. Yagi and D.M. Jerina (1987) Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[c]phenanthrene3,4-diol-l,2-epoxides, J. Am. Chem. Soc., 109, 2497-2504. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973)

Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. Nat. Acad. Sci. (U.S.A.), 70, 2281-2285. Arlett, C.F. (1977) Mutagenicity in cultured mammalian cells, in: D. Scott, B.A. Bridges and F.H. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier/North-HoUand, Amsterdam, pp. 141-154. Armstrong, R.N., B. Kedzierski, W. Levin and D.M. Jerina (1981) Enantioselectivity of microsomal epoxide hydrolase toward arene oxide substrates, J. Biol. Chem., 256, 47264733. Ashurst, S.W., and G.M. Cohen (1981) In vivo formation of benzo[ a]pyrene diol epoxide-deoxyadenosine adducts in the skin of mice susceptible to benzo[a]pyrerte-induced carcinogenesis, Int. J. Cancer, 27, 357-364. Baird, W.M., A. Dipple, P.L. Grover, P. Sims and P. Brooke,s (1973) Studies on the formation of hydrocarbon-deoxyribonucleoside products by the binding of derivatives of 7,methylbenz[a]anthracene to DNA in aqueous solution and in mouse embryo cells in culture, Cancer Res., 33, 2386-2392. Bigger, C.A.H., J.T. Sawicki, D.M. Blake, L.G. Raymond and A. Dipple (1983) Products of binding of 7,12dimethylbenz[a]anthracene to DNA in mouse skin, Cancer Res., 43, 5647-5651. Bigger, C.A.H., J. Strandberg, H. Yagi, D.M. Jerina and A. Dipple (1989) Mutagenic specificity of a potent carcinogen, benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)epoxide, which reacts with adenine and guanine in DNA, Proc. Natl. Acad. Sci. (U.S.A.), 86, 2291-2295. Boosalis, M.S., J. Petrnska and M.F. Goodman (1987) DNA polymerase insertion fidelity, Gel assay for site-specific kinetics, J. Biol. Chem., 262, 14689-14696. Borgen, A., H. Darvey, N. Castagnofi, T.T. Crocker, R.E. Rasmussen and I.Y. Wang (1973) Metabolic conversion of benzo[alpyrene by Syrian hamster liver microsomes and binding of metabolites to deoxyribonucleic acid, J. Med. Chem., 16, 502-506. Brooke.s, P., and P.D. Lawley (1964) Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin: Relation between carcinogenic power of hydrocarbons and their binding to deoxyribonucleic acid, Nature (London), 202, 781-784. Chadlm, A., J.M. Sayer, H.J.C. Yeh, H. Yagi, A.M. Chela, L.K. Pannell and D.M. Jerina (1989) Structures of covalent nucleoside adducts formed from adenine, guanine, and cytosine bases of DNA and the optically active bay-region 3,4-diol, 1,2-epoxides of dibenz[a,j]anthracene, J. Am. Chem. Soc., 111, 5456-5463. Cheng, S.C., A.S. Prakaslh M.A. Pigott, B.D. Hilton, H. Lee, R.G. Harvey and A. Dipple (1988a) A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracene that reacts predominantly with adenine residues in DNA, Carcinogenesis, 9, 1721-1723. Cheng, S.C., A.S. Prakash, M.A. Pigott, B.D. Hilton, J.M. Roman, H. Lee, R.G. Harvey and A. Dipple (198813) Characterization of 7,12-dimethylbenz[a]anthracene-adenine nucleoside adducts, Chem. Res. Toxicol., 1, 216-221.

274 Cheng, S.C., B.D. Hilton, J.M. Roman and A. Dipple (1989) DNA adducts from carcinogenic and non-carcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide, Chem. Res. Toxicol., 2, 334-340. Cook, J.W., C.L. Hewett and I. Hieger (1933) The isolation of a cancer-producing hydrocarbon from coal tar, J. Chem. Soc., 395-405. Dipple, A. (1988) Reactive metabolites of carcinogens and their interactions with DNA, in: P. Politzer and F.J. Martin Jr. (Eds.), Chemical Carcinogens, Elsevier, Amsterdam, pp. 32-62. Dipple, A., P. Brookes, D.S. Mackintosh and M.P. Rayman (1971) Reaction of 7-bromomethylbenz[a]anthracene with nucleic acids, polynucleotides and nucleotides, Biochemistry, 10, 4323-4330. Dipple, A., R.C. Moschel and W.R. Hudgins (1982) Selectivity o f alkylation and aralkylation of nucleic acid components, Drug Metab. Rev., 13, 249-268. Dipple, A., M.A. Pigott, R.C. Moschel and N. Costantino (1983a) Evidence that binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues, Cancer Res., 43, 4132-4135. Dipple, A., J.T. Sawicki, R.C. Moschel and C.A.H. Bigger (1983b) 7,12-Dimethylbenz[a]anthracene-DNA interactions in mouse embryo cell cultures and mouse skin, in: J. Rydstrtim, J. Montelius and M. Bengtsson (Eds.), Extrahepatic Drug Metabolism and Chemical Carcinogenesis, Elsevier, Amsterdam, pp. 439-448. Dipple, A., R.C. Moschel and C.A.H. Bigger (1984) Polynuclear aromatic carcinogens, in: C.E. Searle (Ed.), Chemical Carcinogens, Vol. 1, 2nd Edn., Am. Chem. Soc., Washington, DC, pp. 41-163. Dipple, A., M.A. Pigott, S.K. Agarwal, H. Yagi, J.M. Sayer and D.M. Jerina (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA, Nature (London), 327, 535-536. Drinkwater, N.R., and D.K. Klinedinst (1986) Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency, Proc. Natl. Acad. So. (U,S.A.), 83, 3402-3406. DuBridge, R.B., P. Tang, H.C. Hsia, P.-M. Leong, JoH. Miller and M.P. Calos (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system, Mol. Cell. Biol., 7, 379-387. Eisenstadt, E. (1985) The mutational consequences of DNA damage induced by benzo[a]pyrene, in: R. Harvey (Ed.), Polycyclic Hydrocarbons and Carcinogenesis, Am. Chem. Soc., Washington, DC, pp. 327-340. Harvey, R.G. (1985) Polycyclic hydrocarbons and carcinogenesis, in: R. Harvey (Ed.), Polycyclic Hydrocarbons and Carcinogenesis, Am. Chem. Soc., Washington, DC, pp. 35-62. Heidelberger, C., and M.G. Moldenhauer (1956) The interaction of carcinogenic hydrocarbons with tissue constituents, IV. A quantitative study of the binding to skin proteins of several C14-1abeled hydrocarbons, Cancer Res., 16, 442-449. Hirshfeld, F.L. (1963) The structure of overcrowded aromatic compounds, Part VII. Out-of plane deformation in

benzo[c]phenanthrene and 1,12-dimethylbenzo[c]phenanthrene, J. Chem. Soc., 2126-2135. Jeffrey, A.M., K.W. Jennette, S.H. Blobstein, I.B. Weinstein, F.A. Beland, R.G. Harvey, H. Kasai, I. Miura and K. Nakanishi (1976) Benzo[a]pyrene-nucleic acid derivative found in vivo: Structure of a benzo[a]pyrene tetrahydrodiol epoxide-guanosine adduct, J. Am. Chem. Soc., 98, 57145715. Jerina, D.M., and J.W. Daly (1976) Oxidation at carbon, in: D.V. Parke and R.L. Smith (Eds.), Drug Metabolism, Taylor and Francis, London, pp. 13-32. Jerina, D.M.J.M. Sayer, S.K. Agarwal, H. Yagi, W. Levin, A.W. Wood, A.H. Conney, D. Pruess-Schwartz, W.M. Baird, M.A. Pigott and A. Dipple (1986) Reactivity and tumorigenicity of bay region diol epoxides derived from polycyclic aromatic hydrocarbons, in: J.J. Kocsis, D.J. Jollow, C.M. Witmer, J.O. Nelson and R. Snyder (Eds.), Biological Reactive Intermediates III, Plenum, New York, pp. 11-30. Kennaway, E.L. (1924) The formation of a cancer-producing substance from isoprene (2-methylbutadiene), J. Pathol. Bacteriol., 27, 233-238. Kennaway, E.L. (1925) Experiments on cancer-producing substances, Br. Med. J., 2, 1-4. Kennaway, E.L., and I. Hieger (1930) Carcinogenic substances and their fluorescence spectra, Br. Meal. J., 1, 1044-1046. Koreeda, M., P.D. Moore, H. Yagi, H.J.C. Yeh and D.M. Jerina (1976) Alkylation of polyguanylic acid at the 2-amino group and phosphate by the potent mutagen (±)-7fl,Sa-dihydroxy-9fl,10fl-epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene, J. Am. Chem. Soc., 98, 6720-6722. Kuchino, Y., F. Moil, H. Kasal, H. Inoue, S. Iwai, K. Miura, E. Ohtsuka and S. Nishimura (1987) Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues, Nature (London), 327, 77-79. Latif, F., R.C. Moschel, K. Hemminki and A. Dipple (1988) Styrene oxide as a stereochemical probe for the mechanism of aralkylation at different sites on guanosine, Chem. Res. Toxicol., 1, 364-369. Lawley, P.D. (1966) Effects of some chemical mutagens and carcinogens on nucleic acids, Progr. Nucleic Acid Res. Mol. Biol., 5, 89-131. Levin, W., M.K. Buening, A. Wood, R.L. Chang, B. Kedzierski, D.H. Thakker, D.R. Boyd, G.S. Gadaginamath, R.N. Armstrong, H. Yagi, J.M. Karle, T.J. Slaga, D.M. Jerina and A.H. Conney (1980a) J. Biol. Chem., 255, 9067-9074. Levin, W., A.W. Wood, R.L. Chang, Y. Ittah, M. Croisy-Delcey, H. Yagi, D.M. Jerina and A.H. Conney (1980b) Exceptionally high tumor-initiating activity of benzo[c]phenanthrene bay-region diol-epoxides on mouse skin, Cancer Res., 40, 3910-3914. Levin, W., R.L. Chang, A.W. Wood, D.R. Thakker, H. Yagi, D.M. Jerina and A.H. Conney (1986) Tumorigenicity of optical isomers of the diastereomeric bay-region 3,4-diol1,2-epoxides of benzo[c]phenanthrene in muilne tumor models, Cancer Res., 46, 2257-2261. Loveless, A. (1969) Possible relevance of 0-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of

275 nitrosamines and nitrosamides, Nature (London), 223, 206-207. Michaels, M.L., D.L. Johnson, T.M. Reid, C.M. King and L.J. Romano (1987) Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct, J. Biol. Chem., 262, 14648-14654. Miller, E.C. (1951) Studies on the formation of protein-bound derivatives of 3:4-benzpyrene in the epidermal fraction of mouse skin, Cancer Res., 11, 100-108. Miller, J.H. (1983) Mutational specificity in bacteria, Annu. Rev. Genet., 17, 215-238. Milner, J.A., M.A. Pigott and A. Dipple (1985) Selective effects of selenium on 7,12-dimethylbenz[a]anthracene-DNA binding in fetal mouse cell cultures, Cancer Res., 45, 63476354. Moore, P., and B.S. Strauss (1979) Sites of inhibition of in vitro DNA synthesis in carcinogen- and uv-treated OX174 DNA, Nature (London), 278, 664-666. Moschel, R.C., W.R. Hudgins and A. Dipple (1979) Selectivity in nncleoside alkylation and aralkylation in relation to chemical carcinogenesis, J. Org. Chem., 44, 3324-3328. Moschel, R.C., W.R. Hudgins and A. Dipple (1980) Aralkylation of guanosine by the carcinogen N-nitroso,N-benzylurea, J. Org. Chem., 45, 533-535. Moschel, R.C., W.R. Hudgins and A. Dipple (19,86) Reactivity effects on site selectivity in nucleoside aralkylation: A model for the factors influencing the site~ of ~a~rcinogennucleic acid interactions, J. Org. Chem., ~1, 4180-4185. Nair, R.V., R.D. Gill, C. Cortez, R.G. H .~vey and J. DiGiovanni (1989) Characterization of DNA adducts derived from (+)-trans-3,4-cfihydroxy-anti-l,2-epoxy-l,2,3,4-tetrahydrodibenz[a, j]anthracene and (±)-7-methyl-trans-3,4-

dihydroxy-anti-l,2-epoxy- l,2,3,4-tetrahydrodibenz[ (;, j ]anthracene, Chem. Res. Toxicol., 2, 341-348. O'Connor, D., and G. Stohrer (1985) Site-specifically ~¢~dified oligodeoxyribonucleotides as templates for Esche~chia coli DNA polymerase I, Proc. Natl. Acad. Sci. (U:~.A.), 82, 2325-2329. Osborne, M.R., F.A. Beland, R.G. Harvey and P. Brookes (1976) The reaction of (+)-7a,Sfl-dihydroxy-9fl,10fl-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene with DNA, Int. J. Cancer, 18, 362-368. Phillips, D.H., P.L. Grover and P. Sims (1979) A quantitative determination of the covalent binding of a series of polycyclic hydrocarbons to DNA in mouse skin, Int. J. Cancer, 23, 201-208. Pott, P. (1775) Chirur~cal Observations, Reprinted in 1963 in Natl. Cancer Inst. Monogr., 10, 7-13. Pruess-Schwartz, D;., W.M. Baird, H. Yagi, D.M. Jerina, M.A. Pigott and A. Dl"pple (1987) Stereochemical specificity in the metabolic activation of benzo[c]phenanthrene to metabolites that covalently bind to DNA in rodent embryo cell cultures, Cancer Res., 47, 4032-4037. Qnintanilla, M., K. Brown, M. Ramsden and A. Balmain (1986) Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis, Nature (London), 322, 78-80.

Rayman, M.P., and A. Dipple (1973a) Structure and activity in chemical caroinogenesis, Comparison of the reaction of 7-bromomethylbenz[a]anthracene and 7-bromomethyl-12methylbenz[a]anthracene with deoxyribonucleic acid in vitro, Biochemistry, 12, 1202-1207. Rayman, M.P., and A. Dipple (1973b) Structure and activity in chemical carcinogenesis, Comparison of the reactions of 7-bromomethylbenz[a]anthracene and 7-bromomethyl-12methylbenz[a]anthr~cene with mouse skin deoxyribonucleic acid in vivo, Biochemistry, 12, 1538-1542. Reardon, D.B., A.S. Pr/~lgash, B.D. Hilton, J.M. Roman, J. Pataki, R.G, Harvey a,!ld A. Dipple (1987) Characterization of 5-methylGhrysene, l,2-dihydrodiol-3,4-epoxide-DNA adducts, Carcino~tmesis, 8, 1317-1322. Reardon, D.B., C,A,I-I, Bigger, J. Strandberg, H. Yagi, D.M. Jerina and A. Dipp|e (1989) Sequence selectivity in the reaction of opti~cally active hydrocarbon dihydrodiol epoxides with rat [-t-ras DNA, Chem. Res. Toxicol., 2, 12-14. Reardon, D.B., C.A,IrI, Bigger and A. Dipple (1990) DNA polymerase actiorl on bulky deoxyguanosine and deoxyadenosine addicts, (~arcinogenesis, 11, 165-168. Scribner, N.K., B, Woodworth, G.P. Ford and J.D. Scribner (1980) The t~fluence of molecular size and partition coeffic~¢nts on the predictability of tumor initiation in mouse skin from mutagenicity in Salmonella typhimurium, Carcinogenesis, 1, 715-719. Seidman, M.M., K. Dixon, A. RaTzaque, R.J. Zagursky and M.L. Berman (1985) A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells, Gcne, 38, 233-237. Sims, P., P.L. G-rover, A. Swaisland, K. Pal and A. Hewer (1974) Metabolic activation of benzo[a]pyrene proceeds by a diol epoxide, Nature (London), 252, 326-328. Slaga, T.J., W.J. Bracken, G. Gleason, W. Levin, H. Yagi, D.M. Jerina and A.H. Conney (1979) Marked differences in the skin tumor-initiating activities of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10epoxide, Cancer Res., 39, 67-71. Vericat, J.A., S.C. Cheng and A. Dipple (1989) Absolute stereochemistry of the major 7,12-dimethylbenz[a]anthraceneDNA adducts formed in mouse cells, Carcinogenesis, 10, 567-570. Vousden, K.H., J.L. Bos, C.J. Marshall and D.H. Phillips (1986) Mutations activating human c-Ha-raM proto oncogene (HRAS1) induced by chemical carcinogens and depurination, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1222-1226. Wislocki, P.G., K.M. Gadek, M.W. Chou, S.K. Yang and A.Y.H. Lu (1980) Carcinogenicity and mutagenicity of the 3,4-dihydrodiols and'other metabolites of 7,12-dimethylbenz[a]anthracene and its hydroxymethyl derivatives, Cancer Res., 40, 3661-3664. Yang, J.L., V.M. Maher and J.J. McCormick (1987) Kinds of mutations formed when a shuttle vector containing adducts of (+)-7fl,8a-dihydroxy-9tt,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]I?yrene replicates in human cells, Proc. Natl. Acad. Sci. (U.S.A.), 84, 3787-3791.

276 Yang, S.K., D.W. McCourt, J.C. Leutz and H.V. Gelboin (1977) Benzo[a]pyrene diol epoxides: Mechanism of enzymatic formation of optically active intermediates, Science, 196, 1199-1201.

Zacharias, D.E., S. Kashimo, J.B. Glusker, R.G. Harvey, S. Amin and S.S. Hecht (1984) The bay-region geometry ot some 5-methylchrysenes: Steric effects in 5,6- and 5,12-dimethylchrysenes, Carcinogenesis, 5, 1421-1430.