Induction of Microsomal Enzymes by Foreign Chemicals and … · the hydrocarbons induced the de...

[CANCER RESEARCH 42, 4875-491 7. December 1982]0008-5472/82/0042-OOOOS02.00

Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesisby Polycyclic Aromatic Hydrocarbons: G. H. A. Clowes Memorial Lecture1

Allan H. Conney

Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc.. Nutley, New Jersey 07110

I feel very honored indeed, and also very humble, to beselected as the 21st G. H. A. Clowes Memorial Lecturer. Theresearch that I have done would not have been possible withoutthe help, encouragement, and dedication of my many associates and collaborators during the past 25 years, and all ofthese individuals share with me in today's honor. I am also

extremely grateful to the Burroughs Wellcome Co. and toHoffmann-La Roche Inc. for their support of my research in

Tuckahoe from 1960 to 1970 and in Nutley from 1970 to thepresent. Looking back at the names of earlier recipients of theClowes Award, I realize that I am the 6th Clowes Lecturer who,at one time or another in his career, has been associated withthe McArdle Laboratory for Cancer Research at the Universityof Wisconsin. I believe that the large number of awardeesassociated with McArdle is telling us something about thegenius of Dr. Harold Rusch who established McArdle with ahandful of outstanding young investigators. Dr. Rusch helpedto develop in his associates an intense devotion to fundamentalcancer research, and he provided his colleagues with an atmosphere that encouraged them to have critical but constructive interactions with each other in ways that stimulated excel

lence in research by all of the members of the group. In 1952,I had the good fortune of coming to McArdle as a graduatestudent of Drs. James and Elizabeth Miller. Although I failed inmy first research assignment, which was to synthesize pure 2-amino-1-naphthol, the Millers had great patience with me, andthey tried very hard to instill their high standards into myresearch. Whatever small accomplishments I may have madeto biomÃ©dical research were in large part the result of theexcellent training and encouragement that I received from Jimand Betty Miller.

An important problem that has been of great interest to mefor many years is individuality in the response of human beingsand other living organisms to foreign chemicals. Why does adrug or an environmental pollutant cause toxicity in one personand not in another person? Why do some cigarette smokersdevelop lung cancer whereas other cigarette smokers do not?One of the causes of variability in the response of humanbeings to a foreign chemical is individuality in the rate ofmetabolism of the chemical. By the mid-1960s, it was recognized by clinical pharmacologists that the plasma half-lives and

steady state plasma concentrations of drugs varied by as muchas 10- to 20-fold among different individuals (42, 115, 204,

318). Because it seemed likely that there were also largedifferences in the metabolism of chemical carcinogens in different individuals, we investigated the metabolism of BP2 by

surgical biopsy samples of human liver obtained for medicalreasons (81, 176), by placentas obtained after normal childbirth (323, 324), and by cultured human foreskin obtained fromnormal neonates after circumcision (6, 188). In all of thesestudies, we found variability in the rate of metabolism of BP tofluorescent phenolic metabolites by tissue samples that wereobtained from different individuals. More recent studies ondifferences in the ability of liver samples from different individuals to metabolize some chemical carcinogens are illustratedin Chart 1. A 6- to 9-fold difference in the metabolism of BP tofluorescent phenols and in the metabolism of BP 7,8-dihydro-

diol and aflatoxin B, to mutagens was observed among the 10liver samples studied (60, 81) (Chart 1).

Genetic and environmental factors each contribute to varia-

' Presented on April 27, 1981. at the 72nd Annual Meeting of the American

Association for Cancer Research. Washington. D C. Some additional materialhas been added to provide a more complete manuscript.

Received September 8. 1982; accepted September 9. 1982.

2 The abbreviations used are: BP. benzo(a)pyrene; B(e)P. benzo(e)pyrene;

BA. benz(a)anthracene; DBA, dibenz(a.h)anthracene; B(c)Ph, benzo(c)-phenanthrene; MC. 3-methylcholanthrene; DB(a.h)P, dibenzo(a,n)pyrene;DB(a./)P. dibenzo(a./)pyrene; BP 7,8-dihydrodiol. frans-7.8-dihydroxy-7.8-di-hydrobenzo(a)pyrene (other dihydrodiols are similarly abbreviated); BP 7,8-diol-9.10-epoxide 1, 7/i.8o-dihydroxy-9/j,10/<-epoxy-7,8.9,10-tetrahydro-benzo(a)pyrene; BP 7,8-diol-9,10-epoxide 2, 7/<.8a-dihydroxy-9n,10u-epoxy-7,8.9,10-tetrahydrobenzo(a) pyrene (other diol-epoxides are sim

ilarly abbreviated so that the benzylic hydroxyl group and the epoxide oxygenare cis for Isomer 1 and frans for Isomer J); BP Hâ€ž-7,8-epoxide. 7.8-epoxy-7.8.9,10-tetrahydrobenzo(a)pyrene; BP H.,-9,10-epoxide, 9.10-epoxy-7.8,9.10-(etrahydrobenzo(a)pyrene; BP H4-7.8-diol. frans-7,8-dihydroxy-7,8.9,10-tetra-hydrobenzo(a)pyrene (other tetrahydroepoxides and tetrahydrodiols are similarlyabbreviated); DDT, p,p'-dichloro-2,2-diphenyl-1,1.1-trichloroethane Unless

stated otherwise, all compounds are racemic mixtures where enantiomers arepossible.

DECEMBER 1982 4875

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Chart I. Interindividual differences in the metabolism of carcinogens by surgical biopsy samples of 10 different human livers (60, 81 ). The metabolism of BPto fluorescent phenols, expressed as 3-hydroxy-BP (3-HOBP), and the metabolism of aflatoxin B, and BP 7,8-dihydrodiol to mutagens were determined byincubation of the substrate with liver microsomes and NADPH. Each liver biopsywas taken for medical reasons, and only histologically normal samples were usedfor the metabolism studies.

bility in the metabolism of foreign chemicals in different individuals (2, 59, 64, 81, 82, 307-309, 323) and to variability in the

biotransformation of foreign chemicals in the same individualwho is examined on several occasions (5, 81, 82). Variabilityin the in vivo metabolism of a prototype drug that occurs whenit is given to an individual on several occasions is an approachthat we have used for assessing the influence of environmentand life style on the metabolism of foreign chemicals in humanbeings. In these studies, we found that the amount of day-to

day variation in the in vivo metabolism of 3 prototype drugs byseveral normal volunteers depended on both the drug and thesubject studied (5, 81, 82). Since genes and environmentinteract at so many different levels, it is not possible to quantifyaccurately the relative contributions of each to the variabilitythat occurs in the metabolism of foreign chemicals in humanbeings. Factors that influence the metabolism of foreign chemicals in humans include age, disease states, dietary and nutritional factors, hormonal changes in the body, and the ingestionof foreign chemicals. The ability of foreign chemicals to inducethe synthesis of microsomal enzymes3 that metabolize the

compound administered as well as other foreign chemicals andendogenous substrates has been of particular interest to meover the years, and I would like to focus a major portion of mypresentation on this topic.

Induction of Microsomal Enzymes by Foreign Chemicals

In 1952, Richardson ef al. (260) reported that the hepato-carcinogenic activity of 3'-methyl-4-dimethylaminoazobenzene

was greatly reduced when this aminoazo dye was fed to ratswith MC, a carcinogenic polycyclic aromatic hydrocarbon.Additional research by the Millers and their associates demonstrated that several polycyclic aromatic hydrocarbons inhibited the carcinogenicity of 3'-methyl-4-dimethylaminoazo-

benzene and 2-acetylaminofluorene (225). An explanation for

the unexpected antagonism of aminoazo dye carcinogenesisby polycyclic hydrocarbons came from studies by RaymondBrown and myself while we were graduate students with the

! Microsomal enzymes derived from endoplasmic reticulum catalyze the me-

lolism of foreign chemicals and endogenous substrates by oxidation, reduction,"iratinn anH roninnatirtn

Millers. In these studies, we found that administration of MCand other polycyclic aromatic hydrocarbons to rats increasedthe levels of liver enzymes that metabolize aminoazo dyes byM-demethylation and azo link reduction to noncarcinogenic

products (35, 78, 226). The marked stimulatory effect of asingle injection of MC on the metabolism of aminoazo dyes byrat liver is shown in Chart 2. Additional studies revealed thatpolycyclic aromatic hydrocarbons could stimulate their ownmetabolism (79). The rapid induction of hepatic BP hydroxylaseactivity that occurs after a single injection of BP is shown inChart 3. During the course of these studies, we demonstratedthat the A/-demethylation of 3-methyl-4-monomethylamino-

azobenzene and the hydroxylation of BP occurred in livermicrosomes and required NADPH and oxygen (61, 79), andwe also identified several hydroxylated products and quiÃ±onesas metabolites of the in vitro biotransformation of BP (79). Inthese early studies, we found that ethionine blocked the induction of hepatic aminoazo dye and BP metabolism, and this

N-DEMETHYLASE

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Chart 2. Induction of hepatic aminoazo dye N-demethylase and reducÃaseactivities by a single i.p. injection of 1 mg of MC into 50-g rats (78). W-Demeth-ylase activity was determined by measuring the metabolism of 3-methyl-4-mon-omethylaminoazobenzene to 3-methyl-4-aminoazobenzene (3-methyl-AB). Re-ductase activity was determined by measuring the reduction of the azo linkageof 4-dimethylaminoazobenzene (DAB). Demethylase activity is expressed as /igof 3-methyl-4-aminoazobenzene formed per 50 mg of liver per 30 min. ReducÃaseactivity is expressed as /ig of 4-dimethylaminoazobenzene reduced per 30 mg ofliver per 30 min.

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Chart 3. Induction of hepatic BP metabolism by a single i.p. injection of BPinto 50-g rats (79). Disappearance of BP from the incubation mixture wasmeasured. Examination of the products formed revealed a mixture of hydroxylated products and quiÃ±ones.

4876 CANCER RESEARCH VOL. 42



G. H. A. Clowes Memorial Lecture

block was prevented by methionine (78, 79). These observations, coupled with the lack of an in vitro effect of the hydrocarbon inducers on monooxygenase activity, suggested thatthe hydrocarbons induced the de novo synthesis of liver micro-somal enzymes that metabolize aminoazo dyes and polycyclicaromatic hydrocarbons.

In 1957, I went to Dr. Bernard B. Brodie's laboratory at the

NIH to explore the possibility that microsomal enzyme inductionwas a broad phenomenon that was caused by many foreignchemicals. While at the NIH, I worked with Dr. John J. Burnswho asked me to identify metabolites of 2 centrally actingmuscle relaxant drugs, zoxazolamine and chlorzoxazone.These studies resulted in the isolation and identification ofhydroxylated metabolites of zoxazolamine and chlorzoxazonefrom human urine as well as the demonstration of the NADPH-

dependent hydroxylation of zoxazolamine and chlorzoxazoneby rat liver microsomes (63, 67, 84). The structures of zoxazolamine, chlorzoxazone, and their metabolites are shown inChart 4. During the course of these studies, we found thattreatment of rats for 4 days with several structurally unrelatedchemicals stimulated the metabolism of zoxazolamine (65), andthis drug became a useful prototype substrate for studies onthe induction of microsomal monooxygenases. While at theNIH, Dr. Burns and I studied the possible induction of microsomal monooxygenases by barbiturates and several structurally unrelated drugs that were known to stimulate the synthesisof ascorbic acid in the rat. We found that barbiturates andother previously known stimulators of ascorbic acid synthesiswere potent inducers of hepatic aminoazo dye /V-demethylase

activity and that polycyclic aromatic hydrocarbon enzyme inducers were potent stimulators of ascorbic acid synthesis (62).During these studies, we demonstrated that treatment of ratswith several structurally unrelated drugs increased the levelsof liver microsomal enzymes that metabolized the compoundadministered as well as other substrates, and evidence wasalso obtained for induced enzyme synthesis and for enhancedsynthesis of total microsomal protein (65). While we werestudying the effects of drugs and polycyclic aromatic hydrocarbons as inducers of microsomal monooxygenases at theNIH, Drs. Herbert Remmer and Ryuichi Kato working independently also demonstrated a stimulatory effect of barbiturates and other drugs on drug metabolism (161, 163, 256,257). Although the enhanced metabolism of foreign chemicalsthat occurs after exposure of an organism to a chemical isoften an adaptive protective response that enhances the detoxification and elimination of the compound from the organism,some chemicals are metabolized to toxic products, and theinduction of microsomal enzymes may increase the toxicity ofthese chemicals.

ZOXAZOLAMINE

CHLORZOXAZONE

6-HYDROXY-ZOXAZOLAMINE

6-HYDROXY-CHLORZOXAZONE

The induction of monooxygenases by foreign chemicals iswidespread in nature and has been observed to occur inmammals, fish, insects, plants, bacteria, and many other organisms. Several hundred synthetic and naturally occurringcompounds with diverse structures are now known to increasethe levels of microsomal enzymes when administered to experimental animals. Examples of inducers of microsomal monooxygenases include drugs, steroids, industrial chemicals, pesticides, herbicides, food preservatives, certain dyes used ascoloring agents, polycyclic aromatic hydrocarbons, and normally occurring constituents in our diet.

Effect of Enzyme Induction on the in Vivo Metabolism of Chemicals

The magnitude of the change in the metabolism and actionof a chemical that may occur after enzyme induction in animalsis illustrated by data obtained from rats given the musclerelaxant drug zoxazolamine, which is metabolized by livermicrosomal enzymes to the pharmacologically inactive compound, 6-hydroxyzoxazolamine (Chart 4). Administration of BP

to rats rapidly stimulated hepatic zoxazolamine hydroxylaseactivity, and maximum enzyme levels were observed within 24hr (65). Although some increase in zoxazolamine hydroxylaseactivity was found 24 hr after starting phÃ©nobarbital administration, maximum stimulation was not obtained unless the animals were treated for 3 to 4 days (65). The increased amountof zoxazolamine hydroxylase activity in liver microsomes ofrats treated with phÃ©nobarbital or BP was associated with anaccelerated rate of metabolism of zoxazolamine in vivo. Thehalf-life of zoxazolamine was 9 hr in control rats, 48 min inphenobarbital-pretreated rats, and only 10 min in rats pre-treated with BP (Chart 5). These dramatic changes in the half-

life of zoxazolamine were paralleled by changes in the durationof action of the drug. Control rats given a high dose of zoxazolamine were paralyzed for 730 min, rats pretreated for 4

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Chart 4. Metabolism of zoxazolamine and chlorzoxazone to hydroxylatedproducts.

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MINUTESChart 5. In vivo metabolism of zoxazolamine in rats pretreated with phÃ©no

barbital or BP (65) Immature male rats were given i.p. injections of sodiumphÃ©nobarbital (37 mg/kg) twice daily for 4 days or BP (25 mg/kg) 24 hr beforean i.p. injection of zoxazolamine (100 mg/kg). Total body homogenates weremade at various times after the injection of zoxazolamine. and the amount ofzoxazolamine per g of tissue was determined.

DECEMBER 1982 4877



A. H. Conney

days with phÃ©nobarbital were paralyzed for 102 min, and ratspretreated for 1 day with BP were paralyzed for only 17 min(65).

Several studies demonstrated that microsomal enzyme induction enhances the in vivo metabolism of polycyclic aromatichydrocarbons (187, 269, 270, 321). Treatment of rats withMC 24 hr prior to an injection of 7,12-dimethylbenz(a)anthra-cene decreased the tissue concentrations of 7,12-dimethyl-benz(a)anthracene and its 7-hydroxymethyl and 12-hydroxy-

methyl metabolites (187). During the course of these studies,we found that treatment of rats with MC increased the levelsof liver microsomal enzymes that metabolized 7,12-dimethyl-benz(a)anthracene, and we also found that treatment withthe enzyme inducer stimulated the further metabolism of thehydroxymethyl metabolites of 7,12-dimethylbenz(a)anthracene

(187). It was of considerable interest that the further metabolism of primary metabolites of 7,12-dimethylbenz(a)anthracenewas inhibited by high concentrations of 7,12-dimethylbenz-

(a)anthracene in the incubation mixture so that the profile ofmetabolites observed depended on the concentration of substrate (187). The accumulation of primary metabolites underconditions of high substrate concentration and their furthermetabolism under conditions of low substrate concentrationare important general concepts that have also been pointedout in studies with BP (132). The induction of increasedamounts of BP hydroxylase activity in rat liver microsomes isreflected in vivo by an enhanced rate of biliary excretion of BPmetabolites and by decreased blood and tissue concentrationsof BP (269, 270, 321 ). The stimulatory effect of BP on its ownmetabolism is also illustrated by a decreased tissue concentration of this compound when it is administered chronically. At24 hr after a single p.o. dose of 1 mg of [3H]BP to adult rats,

the concentration of this hydrocarbon in dorsal fat was 249ng/g, whereas at 24 hr after 7 daily doses of the tritiatedhydrocarbon, the concentration of BP in dorsal fat was only 24ng/g (270).

Occurrence of Multiple Cytochrome P-450s and Specificity in

their Induction

During the course of studies at the NIH, we found thatpolycyclic aromatic hydrocarbons have marked specificity asinducers of microsomal monooxygenases in rat liver, and wesuggested the presence in rat liver of multiple monooxygenasesthat are under separate regulatory control (67). In these studies, it was found that treatment of rats with BP increased thelevels of liver microsomal enzymes that hydroxylate BP andzoxazolamine and that N-demethylate 3-methyl-4-monomethy-

laminoazobenzene, but little or no induction occurred for thehydroxylation of chlorzoxazone or the N-demethylation of Ben-

adryl (67). Although treatment of rats with BP caused a dramatic decrease in the pharmacological action of zoxazolamineby stimulating its in vivo metabolism, treatment with BP did notstimulate the metabolism of hexobarbital or decrease its pharmacological action (65). Studies by Axelrod ef al. (12) and byWeisburger era/. (320) suggested that different animal specieshave monooxygenases with different catalytic properties, andadditional studies indicated that inhibitors of monooxygenaseactivity exert specificity with regard to the reactions inhibited(13). Our observations and those by Axelrod and Weisburgersuggested the presence of multiple monooxygenases in theliver, and studies during the next several years demonstrated

that multiple cytochrome P-450s were terminal oxygenases forthe oxidative metabolism of drugs, carcinogens, and manyother foreign chemicals.

Initial reports that carbon monoxide inhibited monooxygenase activity in liver (61 ) and placenta (267) were followed by aseries of experiments by Klingenberg (170), Garfinkel (108),and Omura and Sato (241) that led to the identification of acarbon monoxide-binding cytochrome in liver microsomes. Thishemoprotein was called cytochrome P-450 by Omura and Sato(241). The role of cytochrome P-450 in the hydroxylation of

drugs was elucidated in a series of elegant experiments byCooper ef al. (92), and the induction of increased levels ofhepatic cytochrome P-450 by phÃ©nobarbital was demonstrated

by Orrenius and Ernster (242) and by Remmer and Merker(258). Subsequent studies showed that treatment of rats withpolycyclic aromatic hydrocarbons caused the appearance of amicrosomal hemoprotein with spectral properties that weredifferent from those of cytochrome P-450 in control rats andfrom those of cytochrome P-450 in rats treated with phÃ©nobar

bital (7, 175, 277). The hemoprotein in liver microsomes fromrats treated with MC was called cytochrome Pi-450 or cytochrome P-448. Studies by Alvares ef al. (8) demonstrated that

induction of the spectral changes by MC was prevented byinhibitors of protein and RNA synthesis suggesting that MChad stimulated the de novo synthesis of a new cytochrome P-

450.Testosterone has been used extensively as a prototype

substrate that undergoes multiple hydroxylation reactions. During the course of studies on the hydroxylation of testosteronein the 6/3-, 7Â«-,and 16a-positions by rat liver microsomes, we

found that the 3 hydroxyle.tion reactions were under separateregulatory control and that one hydroxylation reaction could beselectively stimulated or innibited without influencing the others(74). For instance, chlorthion markedly inhibited the 16Â«-hy-droxylation reaction but had little or no effect on the 6/2- or 7a-

hydroxylation reaction. In addition, different patterns of development with age and specific effects of enzyme inducers onthe 3 hydroxylation reactions were observed. We found thatcarbon monoxide inhibited the microsomal 6/8-, 7u-, and 16a-

hydroxylation of testosterone to different extents. The CO/O2ratios needed for 50% inhibition of the 16a-, 6/8-, and 7a-

hydroxylation of testosterone were 0.93, 1.54, and 2.36, respectively (72). The photochemical action spectrum for the 3hydroxylation reactions revealed that light at about 450 nmwas more effective than light at other wave lengths in reversingthe CO inhibition. These results suggested the presence in livermicrosomes of several cytochrome P-450s that function in the

hydroxylation of testosterone at the various positions and thatthe cytochrome responsible for the 16Â«-hydroxylation reactionwas more sensitive to CO than the cytochromes which functionfor the 6ÃŸ-and 7a-hydroxylation reactions.

Although studies with liver microsomes provided strong evidence that phÃ©nobarbital and MC induced the de novo synthesis of different monooxygenases, a definitive demonstration ofthe induction of different monooxygenases required the solu-bilization and purification of these membrane-bound enzymes

from the tissues of animals treated with phÃ©nobarbital and MC.In 1957, we reported on the use of detergent (deoxycholate)for the solubilization of liver microsomal monooxygenase activity (61), and subsequent studies have used various detergentsfor the solubilization and purification of the monooxygenase





systems. In 1968 and 1969, Drs. Lu and Coon reported on theresolution and reconstitution of a monooxygenase system fromrabbit liver microsomes (206, 207), and they found that cyto-chrome P-450, lipid, cytochrome P-450 reducÃase,and NADPH

were all required for optimal catalytic activity. Following thesestudies, Dr. Lu joined my laboratory and together with Dr.Kuntzman we initiated a program to purify and reconstitute thecytochrome P-450s from control rats and from rats treated withphÃ©nobarbital and MC. We found that the cytochrome P-450s

that were partially purified from liver microsomes of control,phenobarbital-treated, and MC-treated rats had markedly different substrate specificities when they were reconstituted withexcess NADPH-cytochrome P-450 reducÃase and lipid (77,

208, 209). In addition, the different partially purified cytochrome P-450S had different regioselectivities for the metabolism of testosterone in the 6ÃŸ-,la-, and 16a-positions (77,

209). These results provided the first direct demonstration forthe presence in liver microsomes of multiple cytochrome P-

450s with different catalytic activities. Additional studies bynumerous investigators have resulted in the isolation of manyhighly purified forms of cytochrome P-450 from the liver and

extrahepatic tissues of animals (89, 184, 210). In much of theresearch on the identification and purification of the multiplecytochrome P-450s, microsomal enzyme inducers have been

used to increase the concentrations of specific forms of cytochrome P-450, thereby facilitating the identification and puri

fication of these hemoproteins.Levin and his associates have purified to apparent homoge

neity 4 different cytochromes P-450 (P-450a, P-450b, P-450c,and P-450d) from liver microsomes of rats treated with variousinducers (264, 265), and they found remarkably different NH2-and COOH-terminal amino acid sequences for these hemoproteins (24, 25). The large differences in the NH2- and COOH-

terminal amino acid sequences for these 4 forms of cytochromeP-450 in rat liver microsomes indicate that each of the hemoproteins is a separate gene product. Cytochrome P-450b is themajor P-450 in liver microsomes from phenobarbital-treatedrats, cytochrome P-450c (also called cytochrome P-448 or P,-450) is the major P-450 in liver microsomes from rats treatedwith MC, and cytochrome P-450d is the major P-450 in liver

microsomes from rats treated with isosafrole. The availabilityof several pure cytochrome P-450s led to the preparation of

highly specific antibodies for these hemoproteins and to theuse of these antibodies for the quantification of specific cytochrome P-450 isozymes in tissues (299, 300). It was foundthat treatment of rats with phÃ©nobarbital for 4 days caused a50-fold increase in the concentration of cytochrome P-450b inliver microsomes, a 3-fold increase in the concentration ofcytochrome P-450a, and no change in the amount of cytochrome P-450C. Treatment of rats with MC caused a 50-foldincrease in the concentration of cytochrome P-450c in livermicrosomes, a 4-fold increase in cytochrome P-450a, and noincrease in cytochrome P-450b. Treatment of rats with Aroclor1254 caused a 45- to 60-fold increase in the concentrations ofboth cytochromes P-450b and P-450c in liver microsomes aswell as a 5-fold increase in the concentration of cytochrome P-

450a. The ability of Aroclor 1254 to increase greatly the levelsof several cytochrome P-450s in rat liver microsomes may

explain why the 9000 x g liver supernatant fraction obtainedfrom rats treated with Aroclor 1254 is so effective in metabolizing a diverse group of chemicals to mutagens in the Ames

system. It should be pointed out that, although the antibodiesto cytochromes P-450a, P-450b, and P-450c are highly spe

cific, the possibility of reaction of an antibody (even a monoclonal antibody) with more than one cytochrome P-450 is animportant problem. The antibody for cytochrome P-450b described above does cross-react with a newly isolated hemopro-tein (cytochrome P-450e) that is present in small amounts inliver microsomes from rats treated with phÃ©nobarbital or poly-

chlorinated biphenyls (266). Additional studies revealed thatcytochrome P-450e is structurally very similar to cytochromeP-450b (266), but the 2 hemoproteins are encoded by different

mRNAs (314). The recent use of monoclonal antibodies for thecharacterization of the cytochrome P-450 isozymes in cells

opens up a new dimension for future studies on the structure,function, and regulation of the multiple cytochrome P-450s.

Each of the highly purified cytochrome P-450s has its own

characteristic but often overlapping substrate specificity (89,184, 210), suggesting that the rates of detoxification andmetabolic activation of foreign chemicals will depend on therelative amounts of the various cytochrome P-450s that are

present in an organism. The catalytic activities of cytochromesP-450a, P-450b, and P-450c toward 6 substrates are described in Table 1. Cytochrome P-450b has good catalyticactivity for the oxidative A/-demethylation of benzphetamineand for the 16Â«-hydroxylation of testosterone, whereas theother cytochrome P-450s that were studied have poor catalyticactivity for these reactions. Cytochrome P-450c has good

catalytic activity for the hydroxylation of BP and zoxazolamine,and this hemoprotein also has high catalytic activity for thebiotransformation of BP and other polycyclic aromatic hydrocarbons to mutagens and to their proximate and ultimate carcinogenic metabolites (198, 264, 292, 354).

Wood et al. (354) have utilized the purified cytochrome P-

450s, purified epoxide hydrolase, and short incubation periodsunder carefully defined conditions for studies on the metabolicactivation of chemicals to mutagens. Marked differences in theabilities of cytochromes P-450b and P-450c to metabolize BPto mutagens during a 5-min incubation in liquid medium areshown in Chart 6. Using purified cytochrome P-450c and short

incubation conditions, we found that purified epoxide hydrolasepartially inhibits the accumulation of mutagenic metabolites ofBP (354), and similar results were reported by Bentley ef a/.(18) in studies with liver microsomes, purified epoxide hydrolase, and the usual incubation conditions of Ames. In additionalstudies, we found that epoxide hydrolase and cytochrome P-

Table 1Catalytic activities of purified cytochromes P-450a, P-450b, and P-450c from

rat liver microsomes (184, 264)Catalytic activities of the purified cytochrome P-450s were determined in the

presence of NADPH after reconstitution with cytochrome P-450 reducÃase andphosphatidylcholine.

nmol product/min/nmol cytochrome P-450

SubstrateAnilineBenzphetamineBPZoxazolamine7-EthoxycoumarinTestosterone6/3-Hydroxy-7a-Hydroxy-16o-Hydroxy-P-450a5.13.20.10.90.6<0.14.4<0.1P-450b6.7176.00.52.412.1<0.1<0.12.4P-450C9.35.119.227.948.50.3<0.1<0.1

DECEMBER 1982 4879



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Chart 6. Metabolism of BP to mutagens by cytochromes P-450b and P-450cobtained from rat liver microsomes (354). BP was incubated in liquid mediumwith cytochrome P-450, cytochrome P-450 reducÃase, phosphatidylcholine,NADPH, and S typhimurium strain TA 98 for 5 min. The reaction was stoppedwith menadione. and molten top agar was added. The usual pour plate procedureof Ames was then followed.

450c were both required for the metabolism of BP 7,8-oxide tomutagenic metabolites (354), and this observation provided adirect demonstration that epoxide hydrolase can play a role inthe metabolism of chemicals to toxic products. It seems likelythat studies with the purified cytochromes P-450 and epoxide

hydrolase will have increasingly greater importance for theelucidation of the pathways of metabolic activation and detoxification of chemical carcinogens.

Examples of chemicals that induce different forms or mixtures of forms of cytochrome P-450 in rat liver microsomesinclude MC, phÃ©nobarbital, isosafrole, pregnenolone 16a-car-

bonitrile, ethanol, cholestyramme, clofibrate, and Aroclor 1254(103, 105, 111, 113, 116, 130, 240, 263-265, 313). Furtherresearch is needed to determine the number of cytochrome P-450s in liver and in extrahepatic tissues and to determine thefactors that regulate the synthesis and activity of these enzymes. Studies on the mechanism of microsomal enzyme induction revealed the presence of a cytoplasmic receptor thatbinds 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aro

matic hydrocarbons, and this receptor is important for theinduction of microsomal monooxygenases by these compounds (254). Several recent studies have indicated that administration of MC or phÃ©nobarbital to rats stimulates theformation of specific mRNAs which code for the synthesis ofspecific cytochrome P-450s in cell-free protein-synthesizing

systems (19, 58, 101). Important advances have also beenmade in the cloning of cytochrome P-450 genes (1, 29, 106,

107, 234), and this and other aspects of the molecular biologyof the cytochrome P-450 enzymes are exciting areas for future

research.

Effects of Enzyme Induction on the Toxicity of Chemicals

I would like to consider the effects of high levels of thecytochrome P-450s on the toxicity of chemicals in an organism.

This is a particularly important question because all of us areliving in an environment where we are exposed to variousamounts of potentially toxic chemicals that are metabolized bythe cytochrome P-450 enzymes. The answer to whether theinduction of the microsomal cytochrome P-450s is good, bad,

or unimportant for an organism is complex and depends on theforms of cytochrome P-450 that are induced and also on the

chemical environment of the organism. The effect of inductionby barbiturates or polycyclic aromatic hydrocarbons on theacute toxicity of several chemicals is shown in Table 2. Although pretreatment of rats with barbiturates protects animalsfrom the lethal effects of warfarin, meprobamate, and strychnine by stimulating the metabolism of these compounds tonontoxic products (66, 138, 162), this same treatment increases the toxicity of Schradan, carbon tetrachloride, bro-mobenzene, and acetaminophen by stimulating the metabolismof these compounds to toxic products (109, 162, 229, 366).Although induction of the cytochrome P-450s with phÃ©nobar

bital is associated with an increased toxicity of bromobenzene,the induction of a different profile of cytochrome P-450s with

MC is associated with protection of rats from the hepatotoxicityof bromobenzene (366). The later induction regimen also protects rats from the lethal effect of a high dose of zoxazolaminebut increases the amount of liver necrosis caused by acetaminophen (66, 229).

Suitable inducers of microsomal enzymes inhibit the carci-nogenicity of aminoazo dyes (225, 260), 2-acetylaminofluo-rene (225), BP (317), 7,12-dimethylbenz(a)anthracene (136),aflatoxin B, (221), 4-dimethylaminostilbene (288), urethan

(361), diethylnitrosamine (178), and bracken fern (243), presumably by stimulating the metabolism of the carcinogen viadetoxification pathways to a greater extent than via toxificationpathways. Butylated hydroxyanisole and butylated hydroxytol-uene are examples of compounds that inhibit the carcinogenic-ity of several chemicals in mice (316), and recent studies haveshown that butylated hydroxyanisole is an effective inducer ofaniline hydroxylase, epoxide hydrolase, and glucuronyl trans-ferase activities in liver microsomes (48, 49). In addition, treatment of mice with butylated hydroxyanisole increases the levelsof glutathione S-transferase, glucose-6-phosphate dehydro-genase, and UDP-glucose dehydrogenase in the liver cytosol(17, 48). Butylated hydroxyanisole also increases the concentration of free sulfhydryl groups in tissues (16), and the increased levels of sulfhydryl groups may protect animals byreacting with and inactivating ultimate carcinogens. The induction profile of butylated hydroxyanisole is very different fromthat which has been reported for phÃ©nobarbital, polycyclichydrocarbons, and several other inducers of microsomal enzymes.

In most instances studied, pretreatment of animals with amicrosomal enzyme inducer or the combined administration ofthe inducer and a carcinogen inhibits the tumorigenic effect ofthe chemical administered. However, this is not always thecase. The combined administration of phÃ©nobarbital and saf-

Table 2

Effect of pretreatment of rats with enzyme inducers on the acute toxicity ofchemicals

Inducer Effect

PhÃ©nobarbital or 1. Protection from lethal effects of warfarin, meprobamate,thiopental and strychnine (66, 138, 162)

2. Increased lethal effects of Schradan and carbon tetrachloride (109, 162)

3. Increased liver necrosis caused by bromobenzene andacetaminophen (229, 366)

MC 1. Protection from lethal effect of zoxazolamine (66)2. Decreased liver necrosis caused by bromobenzene

(366)3. Increased liver necrosis caused by acetaminophen

(229)





role causes more hepatic tumors than safrole alone (337). It isof considerable interest that several inducers of liver micro-

soma! enzymes are promoters of liver tumors (252). For example, phÃ©nobarbital inhibits the carcinogenicity of 2-acetyl-

aminofluorene when the 2 compounds are fed together, butphÃ©nobarbitalcauses the appearance of additional liver tumorswhen the barbiturate is administered chronically after the cessation of a low initiating dose of 2-acetylaminofluorene (252,

253). Although several microsomal enzyme inducers are promoters of liver tumors, it is not known whether the induction ofmicrosomal enzymes and tumor promotion are causally related.

Stimulatory Effect of Drugs and Environmental Chemicals onDrug Metabolism in Humans

The induction of microsomal enzymes observed in animalshas significance in humans, and there are many examples ofdrugs that stimulate their own metabolism or the metabolism ofother chemicals in humans (59, 64). An early example of astimulatory effect of one drug on the metabolism of a seconddrug in humans is the stimulatory effect of barbiturates on themetabolism of coumarin anticoagulants. We found that treatment of rats with phÃ©nobarbital for several days increased theactivity of liver microsomal enzymes that metabolize the anticoagulant drugs bishydroxycoumarin and warfarin (94, 138),and we also found that administration of 65 mg of phÃ©nobarbitalper day lowered the plasma levels and decreased the anticoagulant activity of bishydroxycoumarin in humans (94). Thepotential hazards of this interaction were investigated in acanine model (322), and the results of a representative studyare shown in Chart 7. In this study, a dog was given 1 mg ofbishydroxycoumarin per kg of body weight every 48 hr forseveral weeks until a constant concentration of drug in theplasma and a constant anticoagulant effect (prolonged pro-

thrombin time) were achieved. When phÃ©nobarbital was administered to the dog without changing the bishydroxycoumarinregimen, the concentration of bishydroxycoumarin in theplasma decreased, and the anticoagulant effect of the drugwas abolished. Even when there was a 5-fold increase in the

dose of bishydroxycoumarin, adequate anticoagulant therapyand an adequate concentration of bishydroxycoumarin in theplasma were not achieved while phÃ©nobarbital treatment wascontinued. When phÃ©nobarbital administration was discontinued, the concentration of bishydroxycoumarin in the plasmaand the anticoagulant effect of this drug increased during the

DAYS OF TREATMENT

BISHYDROXVCOUMARIN(MG/KG PER 48 HR) 13

PHENOBARBITAL â€¢¿� â€¢¿�I 10 MG/KG PER DAY |

Chart 7. Effect of phÃ©nobarbital treatment on the plasma concentration andanticoagulant action (prothrombin time) of bishydroxycoumarin in the dog (322).

next few days to such an extent that the dog bled severely andrequired vitamin K to save its life. These results indicate thatcombined therapy with a coumarin anticoagulant and a stimulator of drug metabolism can be hazardous if the enzymeinducer is withdrawn and therapy with the anticoagulant iscontinued without an appropriate decrease in the dose. Theinteraction between phÃ©nobarbital and bishydroxycoumarin isan example of a drug interaction that has led to serious consequences and to some deaths in humans. Since patients areoften treated with combinations of drugs, the possibility of druginteractions is an important consideration during human drugtherapy.

Chemicals in the workplace can stimulate the oxidative metabolism of drugs in humans. People exposed to large amountsof DDT (255), polychlorinated biphenyls (4, 173), Kepone(114), or combinations of DDT and lindane (171) in chemicalfactories have an enhanced rate of oxidative drug metabolism.Similarly, anesthesiologists who are exposed to large amountsof volatile anesthetic chemicals in the operating room (102,118) and gasoline station attendants who are exposed to highlevels of petroleum (117) also have an enhanced rate of drugmetabolism. These results indicate that people who are occu-pationally exposed to enzyme-inducing chemicals in their environment or who are treated with enzyme-inducing drugs willhave an enhanced rate of metabolism of some drugs, and it islikely that these individuals will also have an enhanced rate ofmetabolism of some environmental chemicals that are metabolized by the same enzymes that metabolize drugs.

Stimulatory Effect of Cigarette Smoking on the Metabolism ofChemicals

Since BP and many other polycyclic aromatic hydrocarbonsare liver microsomal enzyme inducers that are present in cigarette smoke, Dr. Richard Welch and I studied the effects ofcigarette smoke on monooxygenase activity in both rats andhumans. We found that exposure of pregnant rats to cigarettesmoke stimulated the hydroxylation of BP in the placenta andin the maternal liver, lung, and intestine (326). Increased activity was also found in fetal liver, indicating that enzyme inducersin cigarette smoke pass through the placenta and reach thefetus. Additional studies revealed that several polycyclic aromatic hydrocarbons present in cigarette smoke are potentinducers of BP hydroxylase activity in rat placenta (321, 324).Examples of these inducers include BP, DBA, BA, chrysene,3,4-benzofluorene, anthracene, pyrene, and fluoranthene

(324). Since cigarette smoke and hydrocarbons in cigarettesmoke induced monooxygenase activity in rat placenta, wecollaborated with Drs. Paul Poppers and Michael Finster atColumbia University to investigate the effects of cigarette smoking on the oxidative metabolism of several drugs and carcinogens by human placentas that were obtained after normalchildbirth. We found that placentas from cigarette smokers hadmarkedly higher levels of BP hydroxylase (323, 324), aminoazodye A/-demethylase (324), and zoxazolamine hydroxylase

(154) activities than did placentas from nonsmokers (Table 3).The average BP hydroxylase activity in the placentas of smokers was about 60-fold higher than in the placentas of non-

smokers. Similar studies indicated that the average zoxazolamine hydroxylase activity in the placentas from smokers wasabout 15-fold higher than in the placentas from nonsmokers.

DECEMBER 1982 4881



A. H. Conney

Cigarette smoking caused a much smaller stimulatory effect onthe O-dealkylation of 7-ethoxycoumarin (141) and did notchange the oxidative aromatization of A"-androstene-3,17-

dione to estradiol and estrone (85). Our results suggested thepresence in human placenta of several monooxygenases thatare under separate regulatory control.

Large interindividual differences occurred for the amount ofBP hydroxylase activity in placentas that were obtained fromsmokers and nonsmokers (85, 141, 154, 323, 324). BP hydroxylase activity varied about 6-fold among the placentasfrom nonsmokers and about 80-fold among the placentas from

smokers. The highest BP hydroxylase activity in the placentasfrom the smoking group was at least 400-fold higher than the

lowest BP hydroxylase activity in the placentas from the nonsmoking group. It was also of interest that placenta! BP hydroxylase activity varied over a 70-fold range among subjects

who smoked about the same number of cigarettes (Table 4),and we suggested that variability among different cigarettesmokers in the induction of enzymes that metabolize chemicalcarcinogens in tobacco smoke may help explain why somepeople develop cancer when exposed to cigarette smoke andothers do not (323, 324). A few years later, we suggested the

Table 3Effect of cigarette smoking on the metabolism of chemicals by human placenta

Placentas from nonsmokers and from women who smoked 7-40 cigarettes/day during pregnancy were examined for ability to metabolize several chemicals.

Enzyme activity inplacentaReaction

measuredBP

hydroxylation (85. 141, 154, 323. 324)3-Methyl-4-monomethylaminc-

azobenzene N-demethylation (324)Zoxazolamine hydroxylation (154)7-Ethoxycoumarin O-dealkylation (141)Estrogen biosynthesis (85)Nonsmokers0.5

Â±0.Ia*<4C0.4

Â±0.1d29 Â±3e44 Â±9'Cigarette

smokers31

Â±426 Â±56

Â±246 Â±547 Â±5

" Average Â±S.E.6 nmol fluorescent phenolic metabolites equivalent to 3-hydroxy-BP formed

per g placenta per hr.nmol 3-methyl-4-aminoazobenzene formed per g placenta per hr.

0 nmol tritiated water formed per g placenta per hr.9 nmol 7-hydroxycoumarin formed per g placenta per hr.' nmol estradiol and estrone formed per g placenta per hr from A4-androstene-

3,17-dione.

Table 4Interindividual differences in BP hydroxylase activity in placentas from cigarette

smokers (85, 324)Placentas from womenwho smoked 15 to 20 cigarettes/day during pregnancy

were examined for ability to metabolize BP to fluorescent phenolic metabolites.Data are expressed as 3-hydroxy-BP formed. Variability in BP hydroxylaseactivity was not related to medication taken during or prior to delivery.

SubjectABCDEFGH1JKLM3-Hydroxy-BPformed

(nmol/g placenta/hr)0.91.02.22.65.05.27.417.017.420.960.265.667.8

use of tissue culture systems for the determination of basalactivity and inducibility of carcinogen-metabolizing enzymes in

human tissues as an approach for detecting differences in theabilities of different individuals to metabolize carcinogens andother toxic chemicals. Our initial studies to determine thefeasibility of this approach were encouraging and revealed thatBA caused the induction of BP hydroxylase activity in culturedhuman foreskin (6, 188). A 3-fold variation in basal BP hydrox

ylase activity in foreskins from different individuals was observed as well as marked individuality in the degree of inductionin the different foreskins studied. The amount of inductionvaried from about 70 to 500% in the 13 foreskins examined (6,188), and similar results were obtained by others in studieswith cultured human lymphocytes and monocytes (15, 44,164). In more recent studies, Dr. C. Harris and his associateshave demonstrated marked interindividual differences in themetabolic activation of several carcinogens to DNA-bound ad-

ducts by several human tissue culture systems (119). It ispossible that appropriate metabolic studies on the bioactivationand detoxification of environmental carcinogens by culturedhuman cells from different individuals may help in the identification of individuals who have a high cancer risk.

Since the metabolism of phenacetin to its major metabolite,/V-acetyl-p-aminophenol (Chart 8), occurs by a polycyclic hy-drocarbon-inducible enzyme system in rat liver (83), we utilized

phenacetin as a prototype drug to determine whether or notcigarette smoking stimulates the in vivo metabolism of somechemicals in humans. Collaborative studies with Dr. EugenePantuck at Columbia University and with Dr. Ronald Kuntzmanrevealed that cigarette smoking lowered the plasma levels ofphenacetin (Chart 9) administered p.o. without changing itsplasma half-life or the plasma levels of total /V-acetyl-p-aminophenol (247, 248). The ratio of the concentration of total N-acetyl-p-aminophenol to that of phenacetin in plasma was

markedly increased in the cigarette smokers. These resultssuggested that cigarette smoking stimulated the in vivo metabolism of phenacetin in the gastrointestinal tract and/or during

H5C20 -\â€”/

PHENACETIN N-ACETYL-p-AMINOPHENOL

Chart 8. Metabolism of phenacetin to N-acetyl-p-aminophenol.

3OOO

Â¿ 2000

3 Z

p5U 1000

CONTROL

Chart 9. Plasma levels of phenacetin in cigarette smokers and nonsmokers atvarious times after the p.o. administration of 900 mg of phenacetin (247, 248).Each value represents the mean > S.E. from 9 subjects.





its first pass through the liver. Subsequent studies revealedthat cigarette smokers have shorter plasma half-lives of anti-

pyrine (121), theophylline (144, 152), and caffeine (251) thando nonsmokers, but cigarette smokers do not have an enhanced metabolism of phenytoin, meperidine, or nortriptyline(219,238,262).

Effect of Diet on the Metabolism of Chemicals

In 1950, Drs. G. C. Mueller and J. A. Miller reported thatriboflavin adenine dinucleotide was required for the enzymaticreductive cleavage of a carcinogenic aminoazo dye to noncar-

cinogenic metabolites in the liver (232). This observation provided an explanation for the protective effect of dietary riboflavin on aminoazo dye hepatocarcinogenesis (166, 228) andfor the observation that the overall rate of destruction of aminoazo dyes by rat liver slices was dependent on the concentration of riboflavin in the liver (165). Subsequent studies in thelaboratory of the Millers demonstrated enhanced hepatic metabolism of an aminoazo dye when rodents were switched froma semisynthetic diet to a chow diet (35). These investigatorsfound that hepatic aminoazo dye A/-demethylase activity was

markedly stimulated by certain peroxides and sterols presentin the diet. The pioneering studies described above on theinfluence of dietary factors on the metabolism of aminoazodyes revealed that changes in nutrition can have dramaticeffects on the metabolism and action of foreign chemicals.These studies have been extended by many investigators, andthe effects of dietary factors on the metabolism of foreignchemicals in experimental animals have been reviewed recently(45, 123). Substances in food that may influence the metabolism of drugs include macronutrients (carbohydrate, protein,fat), trace substances important to human health (vitamins,trace metals, etc.), and numerous nonnutrient, lipid-soluble

foreign chemicals. Although the latter diverse group of substances has not been well characterized, the presence of thesechemicals in our diet is receiving increasingly more attention(93, 227, 306). Since human beings receive a large part oftheir exposure to exogeneous chemicals through the diet, it isimportant to determine the effects of different diets on themetabolism and action of drugs, environmental carcinogens,and other environmental chemicals. Because of these considerations, Dr. Attallah Kappas and I initiated a research programon the effects of dietary factors on the metabolism of prototypedrugs in humans. An important feature of this clinical research,which was done in collaboration with Dr. Eugene Pantuck atColumbia University and with Drs. Alvito Alvares and KarlAnderson at the Rockefeller University, was the use of normalvolunteers as their own controls. In these studies, we examinedthe metabolism of prototype drugs before, during, and after aspecific dietary change.

Stimulatory Effect of Dietary Charcoal-broiled Beef onHuman Drug Metabolism. Charcoal-broiled beef, a food which

contains relatively high concentrations of polycyclic aromatichydrocarbons, is eaten by large numbers of people. Becauseof the possible influence of this food on the metabolism offoreign chemicals, we studied the effect of feeding charcoal-

broiled beef on the metabolism of foreign chemicals in rats andin humans. The in vitro metabolism of phenacetin by intestine(245) and the metabolism of BP by liver and placenta (120)were stimulated severalfold when rats were fed a diet which

contained charcoal-broiled beef. Additional studies in humansrevealed that feeding charcoal-broiled beef (14 oz/day) for 4

days markedly lowered the plasma concentrations of subsequently administered phenacetin (Chart 10) without changingits plasma half-life or the plasma levels of total /V-acetyl-p-

aminophenol (80, 244). Thus, the ratio of the concentration oftotal A/-acetyl-p-aminophenol to that of phenacetin in plasmawas markedly higher after the subjects were fed charcoal-

broiled beef, and the ratio decreased when the subjects returned to the control diet. Our results indicated that feedingcharcoal-broiled beef greatly increased the metabolism of

phenacetin during its first pass through the liver and/or in thegastrointestinal tract. Additional studies revealed that feedingcharcoal-broiled beef for 4 to 5 days increased the metabolic

clearance rates of antipyrine and theophylline by 38 and 30%,respectively (159). It was of interest that an occasional individual failed to respond to the effects of charcoal-broiled beef

feeding. The reason(s) for individuality in responsiveness tocharcoal-broiled beef feeding is not known.

Stimulatory Effect of Dietary Cabbage and BrusselsSprouts on Human Drug Metabolism. Dr. L. Wattenberg andhis associates have shown that intestinal BP hydroxylase activity in rodents is very sensitive to changes in the content ofcruciferous vegetables in the diet (315). Feeding rats a dietcontaining brussels sprouts or cabbage markedly stimulatedthe intestinal metabolism of BP, 7-ethoxycoumarin, and phenacetin (246, 315). lndole-3-acetonitrile, indole-3-carbinol, and3,3'-diindolylmethane have been identified as examples of

strong inducers of monooxygenase activity that are present inbrussels sprouts and cabbage (205).

The effects of feeding high levels of cabbage and brusselssprouts for several days on the metabolism of antipyrine andphenacetin were studied in 10 normal volunteers (249). Feeding the cabbage- and brussels sprouts-containing diet for 3days decreased the mean plasma half-life of antipyrine by 13%

and increased its metabolic clearance rate by 11%. Returningthe subjects to the control diet for 6 days resulted in a 17%increase in the plasma half-life of antipyrine and a 13% de

crease in its metabolic clearance rate. Although these effectsof feeding cabbage and brussels sprouts are small, they arestatistically significant. Feeding the cabbage- and brusselssprouts-containing diet for 6 days resulted in mean plasma

CONTROL PERIOD No I CHARCOAL BROILED BEEF T CONTROL PERIOD No 2

57! 357I 357

HOURS

Chart 10. Effect of a diet containing charcoal-broiled beef on the plasmaconcentrations of phenacetin in humans (SO, 244). Nine subjects were given aP.O. dose of 900 mg of phenacetin after 7 days on control diet. 4 days on acharcoal-broiled beef diet, and 7 days on the control diet a second time. Eachvalue represents the mean Â±S.E.

DECEMBER 1982 4883



A. H. Conney

concentrations of phenacetin that were decreased 34 to 67%at the various times after the P.O. administration of this drug(Table 5). The mean ratio of the plasma concentration of totalA/-acetyl-p-aminophenol to the plasma concentration of phenacetin was increased when the subjects were fed cabbage andbrussels sprouts, and the ratio returned towards control valueswhen the subjects were again fed the control diet for severaldays. These results suggest that feeding a diet that containscabbage and brussels sprouts stimulates the metabolism ofphenacetin in the gastrointestinal tract and/or during its firstpass through the liver. In addition to the above observations,the ratio of the plasma concentration of conjugated A/-acetyl-p-aminophenol to the concentration of unconjugated N-acetyl-p-

aminophenol was increased 40 to 50% when the subjects werefed the cabbage- and brussels sprouts-containing diet, suggesting that this diet enhances the conjugation of A/-acetyl-p-

aminophenol (249).Regulation of Human Drug Metabolism by Dietary Carbo

hydrate and Protein. Changes in the protein and carbohydratecontent of the diet markedly influence human drug metabolism(Table 6) (3, 160). For these studies, 6 normal volunteers werefed their customary home diet for 2 weeks, a high-protein-low-carbohydrate diet for 2 weeks, a high-carbohydrate-low-pro-

tein diet for 2 weeks, and their customary home diet again for2 weeks. The plasma half-life of antipyrine was determined onDay 10 of each of the 4 study periods, and the plasma half-life

of theophylline was determined on the last day of each of the4 study periods. The average half-lives of antipyrine and the

ophylline increased 63 and 46%, respectively, when the 6subjects were shifted from the high-protein-low-carbohydratediet to the isocaloric high-carbohydrate-low-protein diet, and it

was of interest that each individual had his own characteristicresponse to this alteration in diet. The increase in antipyrinehalf-lives among the 6 subjects ranged from no change in onesubject to 111 % in another subject. The increase in theophylline half-lives among the 6 subjects when they were shifted

from the high-protein-low-carbohydrate diet to the high-carbohydrate-low-protein diet ranged from 14 to 71 %. The above

dietary changes also caused significant changes in the metabolic clearance rates of antipyrine and theophylline, and asimilar stimulation of antipyrine and theophylline metabolismwas observed when the amount of dietary protein was increased at the expense of fat (10). An increase in the amountof either saturated or unsaturated fat in the diet at the expenseof carbohydrate, however, had little or no effect on the metabolism of antipyrine or theophylline (10). Recent studies haveshown that increasing the ratio of protein to carbohydrate inthe diet can have clinical importance since this dietary changewas found to decrease the effectiveness of theophylline inchildren with asthma (104). The results of studies with charcoal-broiled beef, with cabbage and brussels sprouts, and with

changes in the ratio of protein to carbohydrate in the dietindicate that dietary changes can significantly influence theoxidative metabolism of prototype drugs in humans, and it islikely that dietary factors can also influence the metabolismand activity of chemical carcinogens in humans.

Activation of Monooxygenases by Flavonoids. Severalyears ago, Wiebel and Gelboin showed that the addition of 7,8-benzoflavone to liver microsomes from MC-treated rats mark

edly inhibited BP hydroxylase activity (328, 329). These investigators also found that 7,8-benzoflavone caused an age-dependent stimulation of BP hydroxylase activity when the flavo-noid was added to liver microsomes from untreated rats (328,329). Dr. J. Kapitulnik and I studied the possible use of 7,8-benzoflavone as a probe for the properties of monooxygenasesin human liver, and we found that the addition of 7,8-benzoflavone to homogenates or microsomes from human liver causeda manyfold stimulation of BP hydroxylation (156). The markedactivating effect (sometimes greater than 10-fold) of 7,8-ben

zoflavone on BP hydroxylation that we observed with humanliver prompted us to study the effects of several flavonoids onmonooxygenase activity in this tissue. The structures of some

Table 5

Effect of a diet containing brussels sprouts and cabbage on the plasma concentration of phenacetin in humans (249)Ten subjects were given a dose of phenacetin (900 mg p.o.) after they had ingested a control diet for 9 days, a brussels sprouts- and

cabbage-containing diet for 6 days, and the control diet for 9 additional days. Each value represents the mean Â±S.E.

Plasma concentration of phenacetin (ng/ml)

DietControl

(1st time)Brussels sprouts

and cabbageControl (2nd time)0.5

hr2316

Â± 1150982 Â±4021570Â±

9461

hr2396

Â± 9601576 Â±6631843

Â± 8912hr1430

Â± 473662 Â±170975

Â± 2803hr497

Â± 137228 Â±49776

Â± 3044hr202

Â± 5690 Â±20277

Â± 975hr111

Â±2936Â±8113

Â± 377hr32

Â±1011Â±325

Â± 8

Table 6

Effect of carbohydrate and protein content of the diet on human drug metabolism (3, 160)

Antipyrine (18 mg/kg) was administered p.o. on Day 10 and theophylline (5 mg/kg) was administeredp.o. on Day 14 of each 14-day dietary regimen. Each value represents the mean Â±S.E. for 6 subjects.

AntipyrineDietHome

diet44% Protein. 35% carbohydrate. 21% fat10% Protein. 70% carbohydrate. 20% fatHome dietHalf-life

(hr)16.2

Â±1.69.6 Â±0.4

15.6 Â±1.714.2 Â±0.6Metabolic

clearancerate (ml/min)37

Â±158 Â±439 Â±440 Â±3TheophyllineHalf-life

(hr)8.1

Â±1.05.2 Â±0.47.6 Â±0.77.5 Â±0.7Metabolic

clearancerate (ml/min)49

Â±476 Â±552 Â±452 Â±2




G. H. A. Clowes Memorai Lecture

of the flavonoids that were studied are shown in Chart 11.Except for the synthetic flavonoid, 7,8-benzoflavone, all of the

compounds shown in this chart are normal constituents ofherbaceous plants commonly found in man's environment, and

many of these flavonoids are present in our diet. The results ofour studies demonstrated that the addition of quercetin, kaemp-ferol, morin, or chrysin to human liver microsomes inhibited BPhydroxylation, but the addition of 7,8-benzoflavone, flavone,

tangeretin, or nobiletin to human liver microsomes caused aseveralfold activation in the hydroxylation of BP (36). Theactivating effect of the latter 4 flavonoids is shown in Chart 12.Similar studies demonstrated that the activator flavonoids alsoincreased the metabolism of aflatoxin Bi to mutagens and toaflatoxin B, 2,3-dihydrodiol (36, 37). In other studies, we foundthat the in vitro addition of 7,8-benzoflavone to human liver

OCH,.OCH,

H,CO

H,CO

7,8-BENZOFLavONE

OCH,

OCH,OCH,

NOBILETIN

'OH

HO O HO O

MORIN CHRYSIN

Chart 11. Structures of some flavonoids.

Â§

I "

ojjj 200

7,8-BENZOFLAVONE TANGERETIN

01 10 100 1000 01 10 IDOloco oÃ i to too xxÂ» oi i io too woo

FLAVONOID (//M)

Chart 12. Activation of BP hydroxylation by the addition of 7,8-benzoflavone,flavone, tangeretin, and nobiletin to human liver microsomes (36,60). Fluorescentphenolic metabolites were measured.

microsomes stimulated the metabolism of BP 7,8-dihydrodiol,B(e)P 9,10-dihydrodiol, antipyrine, and zoxazolamine, but theoxidative metabolism of hexobarbital, coumarin, and 7-ethoxy-coumarin was not appreciably influenced (36, 81, 156, 289).Specificity in the activating effect of 7,8-benzoflavone on the

metabolism of some substrates but not on the metabolism ofother substrates suggested the presence of multiple monoox-ygenases in human liver microsomes.

The effects of flavone and 7,8-benzoflavone on the metabolism of BP by 5 highly purified cytochrome P-450 isozymes

obtained from rabbit liver microsomes were examined, and wefound that the responses to flavone and 7,8-benzoflavone weredependent on the cytochrome P-450 isozyme studied (134).BP metabolism was stimulated more than 5-fold by the addition

of flavone to a reconstituted monooxygenase system consistingof NADPH, cytochrome P-450 reductase, dilauroylphosphati-dylcholine, and cytochrome P-450LM3c or cytochrome P-

450LM4 (Chart 13). In contrast to these observations, an inhibitory effect of flavone on BP metabolism was observed whencytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LMe was used as the source of hemoprotein in

the reconstituted monooxygenase system (Chart 13). Studieson the mechanism of the activating effects of flavone and 7,8-benzoflavone on BP hydroxylation in cholate-solubilized liver

microsomes from rabbits and humans suggested that theseflavonoids stimulated BP metabolism at least in part by enhancing the interaction between cytochrome P-450 and cytochrome P-450 reductase, thereby facilitating the flow of electrons to cytochrome P-450 (133).

Recent studies indicate that the activation of microsomalmonooxygenases may have significance in vivo. The additionof flavone to liver microsomes obtained from neonatal ratscaused a stimulation in the metabolism of [4,6-3H]zoxazolamine

to 6-hydroxyzoxazolamine as measured by the formation of

tritiated water, and we investigated whether flavone couldstimulate the hydroxylation of zoxazolamine in vivo (181). Inthis study, 740 nmol of [4,6-3H]zoxazolamine were injected i.p.

either alone or together with 5 /imol of flavone into neonatalrats. Total-body homogenates were made at various times after

0 200 400 600 0 200 400 600

FLAVONE CONCENTRATION (,/M)

Chart 13. Effect of flavone on the hydroxylation of BP by purified cytochromeP-450 isozymes from rabbit liver microsomes (134). Cytochrome P-450, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and NADPH were incubated with BP in the presence and absence of flavone. Fluorescent phenolicmetabolites were measured, and the data are expressed as 3-hydroxy-BP formed.

DECEMBER 1982 4885



A. H. Conney

the injection, and tritiated water was measured and expressedas 6-hydroxyzoxazolamine. As shown in Chart 14, the i.p.

injection of flavone caused an immediate stimulation in the invivo metabolism of zoxazolamine. Animals treated with flavoneformed severalfold more 6-hydroxyzoxazolamine than did con

trol animals during the first 15 to 120 min after the injection.The ability of an in vitro activator of microsomal monooxygen-

ases to immediately stimulate the oxidative metabolism of achemical in vivo is a new finding, and further studies areneeded to determine the pharmacological and toxicologicalimplications of this observation.

Effect of Enzyme Induction on the Metabolism of Steroids andOther Endogenous Substrates

Factors that influence the hydroxylation of foreign chemicalsby liver microsomes also influence the liver microsomal hydroxylation of steroid hormones (59, 70, 174). This researchpointed out that steroid hormones are normal body substratesfor the microsomal monooxygenases and that phÃ©nobarbitaland other microsomal enzyme inducers can stimulate the metabolism of steroid hormones. Treatment of rats with phÃ©nobarbital for 4 days increased the hydroxylation of androgens,estrogens, progestational steroids, and adrenocortical steroidsby enzymes in liver microsomes (Chart 15), and this effect wasparalleled by a decreased action and an enhanced in vivometabolism of exogenously administered steroids (73). Pretreatment of rats with phÃ©nobarbital and other inducers of livermicrosomal enzymes decreased the estrogenic action of estradici and estrone (192, 194, 325), decreased the androgenicaction of testosterone (195, 196), and decreased the anesthetic action of progesterone and deoxycorticosterone (68,177). The marked inhibitory effect of treating rats with phÃ©nobarbital or the insecticide chlordane on the uterotropic action

300

250

z(EO 200

ISO

_ON<XOM>XOtro

50

FLAVONE

CONTROL

30 60 90

TIME (MIN)

IZO

Chart 14. In vivo activation of zoxazolamine metabolism by flavone (181).Neonatal rats were given i.p. injections of 740 nmol of [4,6-3H]zoxazolamine

alone or together with 5 /nmol of flavone. Total body homogenates were made atvarious times after the injection, and 3H?O was measured and expressed as 6-

hydroxyzoxazolamine formed. Points, mean; bars. S.E.

of estrone is shown in Table 7. Pretreatment of rats withphÃ©nobarbital also decreased the uterotropic action of synthetic estrogens and progestational steroids used in oral contraceptives and stimulated the liver microsomal metabolism ofsome of these compounds (189,193). PhÃ©nobarbitaland otherenzyme-inducing drugs have been shown to stimulate the 6/8-

hydroxylation of cortisol in guinea pig liver microsomes (69), toenhance the urinary excretion of 6/8-hydroxycortisol in humans(43, 73, 250, 261), to increase the urinary excretion of polarmetabolites of testosterone in humans (286), to decrease theeffectiveness of a synthetic glucocorticoid in steroid-dependentasthmatics (33), and to stimulate the metabolism of syntheticestrogens and progestational steroids contained in oral contraceptives (28). Anticonvulsants and rifampicin are examples ofdrugs that stimulate the metabolism of the oral contraceptive,ethinyl estradiol, in humans (22, 27, 28), and unwanted pregnancies have occurred in women taking oral contraceptivessimultaneously with anticonvulsants or rifampicin (cf. RÃ©f.189).Some examples of the stimulatory effect of enzyme-inducing

aui

600

400

Â¡Ã•â€ž¿�200

Sui 0

300

200

100

0

Â¿4-ANDROSTENE-3,17-DIONE

PROGESTERONETESTOSTERONE

J \DEOXY

CORTICOSTERONE

ESTRADIOL-1 7ÃŸ

wo,Cm . ESTRONE

â€¢¿� â€¢¿� ESTRADICI

J J JI I CONTROL

â€¢¿�IPHENOBARBITAL TREATED

Chart 15. Stimulatory effect of phÃ©nobarbital treatment on steroid hydroxylation by rat liver microsomes (68, 70, 194). Immature rats were given i.p.injections of 37 mg of sodium phÃ©nobarbital per kg of body weight twice daily for4 days. The animals were killed the following day and liver microsomes wereprepared and incubated with '4C-labeled steroid in the presence of NADPH.

Formation of polar metabolites with the Chromatographie mobility of hydroxylatedsubstrate was measured.

Table 7

Effecf of stimulators of hepatic estrogen hydroxylation on the uterine responseto estrone in the immature female rat (1 94, 325)

In Experiment 1, immature female rats were given i.p. injections of sodiumphÃ©nobarbital (37 mg/kg) twice daily for 4 days. On the fifth day, 0.3 fig ofestrone was injected i.p., and the rats were killed 4 hr later. In Experiment 2,immature female rats were given Â¡.p.injections of chlordane (25 mg/kg) oncedaily for 7 days. Twenty-four hr after the last injection, 0.2 fig of estrone wasinjected i.p., and the rats were killed 4 hr later. Each value represents the meanÂ±S.E. for 6-8 rats.

Experiment12TreatmentControlPhÃ©nobarbitalControlChlordaneUterineEstrone wet wt(mg)19.5

Â±0.6+30.8 Â±1.818.7

Â±0.2-1-19.7 Â±0.527.3

Â±0.9+40.2 Â±1.727.4

Â±1.1+28.3 Â±1.5Inhibition

of response9193





drugs on the urinary excretion of 6/J-hydroxycortisol in humans

are shown in Chart 16. In this study, treatment of humans withW-phenylbarbital or phenylbutazone for several weeks stimulated the urinary excretion of 6/?-hydroxycortisol. Additional

studies pointed out that people who are exposed to high levelsof DDT in a pesticide factory have an enhanced urinary excretion of 6yS-hydroxycortisol (255). Microsomal enzyme inducers

stimulate the metabolism of many endogenous substrates suchas steroid hormones, sterols, bile acids, thyroid hormone,prostaglandins, fatty acids, lipid-soluble vitamins, and bilirubin

(71, 179), but the physiological importance of these effects isstill not very well understood. Newborn infants from womentreated with phÃ©nobarbital before delivery have a lower serumconcentration of unconjugated bilirubin during the first fewdays of life than do infants from untreated mothers (220), andadults who are ingesting large amounts of enzyme-inducing

drugs for prolonged periods of time have a lower plasmaconcentration of unconjugated bilirubin (301 ), an altered profileof steroid metabolites (described above), an enhanced turnoverof thyroid hormone (180, 239), and a lower plasma concentration of 25-hydroxycholecalciferol (137, 287) which is associ

ated with a high incidence of osteomalacia in this population.

Carcinogenesis by Polycyclic Aromatic Hydrocarbons

At this point in my lecture, I would like to change topics andtalk about our studies on carcinogenesis by polycyclic aromatichydrocarbons. It was in about 1972 that I developed a stronginterest in doing more detailed studies on the metabolism ofpolycyclic aromatic hydrocarbons and in identifying proximateand ultimate carcinogenic metabolites of these compounds. Itwas apparent that research of this type would require a majorchemical synthetic program, and I started asking chemistsabout the possibility of synthesizing arene oxides of BP so thatwe could study the metabolism and biological activity of thesecompounds. All the chemists that I asked, except for one, saidthis would be terribly difficult if not impossible. When I askedDr. Donald Jerina at the NIH if it might be possible to synthesizesome of the arene oxides of BP and about the possibility of acollaboration, Dr. Jerina replied that he was interested in acollaboration and that he could easily provide 4 of the areneoxides of BP, and, "by the way," he said, "I can also give you

several dihydrodiols and quiÃ±onesand all twelve of the isomerie

x _ 1000

800

600

400

200

O CONTROLâ€¢¿�DRUG TREATED

PHENYLBUTAZONE(600 MG/OAY.I4DAYS)

N-PHENYLBARBITAL( 300-I2OO MG/DAY .33 DAYS)

SUBJECT â€”¿�Â»A B C O EFGH

Chart 16. Stimulatory effect of phenylbutazone and W-phenylbarbital on theurinary excretion of 6/8-hydroxycortisol in humans (73). Six hundred mg ofphenylbutazone per day were administered for 14 days, or gradually increasingdoses of N-phenylbarbital (300 to 1200 mg per day) were administered for 33days. Urinary 6/?-hydroxycortisol was measured.

phenols of BP. " This was the start of an intensive collaborationwith Dr. Jerina's laboratory which is still continuing today. The

magnitude of the synthetic program initiated by Dr. Jerina hasbeen enormous. During the course of this research, Dr. Jerinaand his associates have synthesized more than 200 potentialmetabolites and reference compounds derived from the polycyclic aromatic hydrocarbon class of carcinogens. Many of thecompounds that were synthesized are highly reactive, andsome are optically active enantiomers. Almost all of the compounds have been of the type that are extremely difficult tomake. I am fortunate to be collaborating with Dr. Jerina who isnot only an outstanding synthetic chemist but also an outstanding biochemist and biologist. Dr. Jerina and the members ofhis laboratory share equally with the members of my laboratoryin whatever accomplishments we may have made to the identification of proximate and ultimate carcinogenic metabolites ofthe polycyclic aromatic hydrocarbons. Drs. Haruhiko Yagi,Dhiren Thakker, and Roland Lehr (now at the University ofOklahoma) from Dr. Jerina's laboratory and Wayne Levin, Dr.

Alexander Wood, and Richard Chang from my laboratory havebeen the principal contributors to this program.

Evidence for the metabolic activation of a polycyclic aromatichydrocarbon to reactive intermediates was first reported in1951 by E. C. Miller. She applied BP to mouse skin and foundcovalent binding of metabolites of this hydrocarbon to skinprotein (224). This study was followed by many other investigations indicating that application of carcinogenic polycyclichydrocarbons to mouse skin resulted in the covalent binding ofmetabolites to DNA, RNA, and protein. Dr. Charles Heidelberger, who was an earlier Clowes Lecturer, pioneered in much ofthis research and in research on the ability of polycyclic hydrocarbons to cause malignant transformations in cultured cells(129). The importance of DMA binding for the carcinogenicityof polycyclic hydrocarbons on mouse skin was suggested in1964 by Brookes and Lawley (31, 32), who found a correlationbetween the carcinogenicity of several polycyclic hydrocarbons on mouse skin and the covalent binding of these hydrocarbons to mouse skin DNA. In 1957, the metabolism of BP toseveral hydroxylated products and to quiÃ±ones by a NADPH-dependent monooxygenase in liver microsomes was described(79), and several years later the liver microsomal metabolismof BP to DNA-bound metabolites was demonstrated (110,112).

Although the possibility that epoxides (arene oxides) werereactive metabolites of polycyclic aromatic hydrocarbons wassuggested by Boyland in 1950 (26), it was 1968 before themetabolism of a polycyclic hydrocarbon to an arene oxide wasfirst reported (147, 148). In this study, Jerina and his associates demonstrated the metabolism of naphthalene to naphthalene 1,2-oxide by liver microsomes. Arene oxides are nowrecognized as the immediate metabolic precursors of trans-dihydrodiols (by hydration with epoxide hydrolase) and asintermediates in the formation of phenols by isomerization (95,145).

During a remarkable 5-year interval from 1973 to 1978,research in the laboratories of Borgen, Brookes, Gelboin,Grover and Sims, Harvey, Slaga, and Weinstein and by ourlaboratories in Nutley and Bethesda led to the demonstrationthat one of the diastereomeric BP 7,8-diol-9,10-epoxides (seeChart 17 for structures) is an ultimate carcinogenic metaboliteof BP. The metabolism of BP to BP 7,8-oxide, to BP 7,8-dihydrodiol, and to the diastereomeric BP 7,8-diol-9,10-epox-

DECEMBER 1982 4887



A. H. Conney

OHBP 4,5-DIHYDRODIOL BP 9.10-DIHYDRODIOL

BP 7,8-OIHYDRODIOL

P-450

BP 7,8-DIOL-9,10-EPOXIDE-t BP 7,8-DIOL-S, 10-EPOXIDE-2

Chart 17. Metabolism of BP to arene oxides, dihydrodiols. and the diaster-eomeric BP 7,8-diol-9,10-epoxides. P-450, cytochrome P-450; EH, epoxide

hydrolase.

ides by cytochrome P-450 and epoxide hydrolase is describedin Chart 17. In BP 7,8-diol-9,10-epoxide 1, the benzylic hy-droxyl group is cis to the oxygen of the oxirane ring. In BP 7,8-diol-9,10-epoxide 2, the benzylic hydroxyl group is trans to theepoxide oxygen." The approach that Dr. Jerina and I took for

the identification of proximate and ultimate carcinogenic metabolites of BP and other polycyclic aromatic hydrocarbonsincluded (a) the synthesis of known and potential metabolites,(b) the determination of the mutagenic activity of known andpotential metabolites, (c) the identification of metabolites, and(d) the determination of the carcinogenic activity of known andpotential metabolites. This collaborative research program andresearch by other laboratories have resulted in the identification of proximate and ultimate carcinogenic metabolites of BPand of several additional polycyclic aromatic hydrocarbons.This research has also led to a generalized theory (the bayregion theory) of carcinogenesis by the polycyclic aromatichydrocarbons. A description of our research on hydrocarboncarcinogenesis is given below.

Metabolism of BP to Arene Oxides and Dihydrodiols by PurifiedCytochrome P-450 and Epoxide Hydrolase

In 1974, we reported on the use of high-pressure liquidchromatography for the separation of BP metabolites (131),and similar studies were reported independently by Selkirk efal. (271). We also reported on the use of purified hepaticcytochrome P-450 and purified hepatic epoxide hydrolase for

the elucidation of pathways of BP metabolism (131). In thisstudy, a reconstituted purified cytochrome P-450c-dependent

monooxygenase system from MC-treated rats metabolized BP

to phenols and quiÃ±ones, but little or no metabolism to dihydrodiols occurred in the absence of epoxide hydrolase (Chart18). The addition of purified epoxide hydrolase to the monooxygenase system resulted in the appearance of Chromatographie peaks corresponding to the 4,5-, 7,8-, and 9,10-frans-

dihydrodiols of BP (see Chart 17 for structures), and there wasa concomitant decrease in phenolic metabolites (Chart 18). BP4,5-oxide that was formed during the incubation of BP with thepurified cytochrome P-450-dependent monooxygenase systemwas sufficiently stable to appear as a peak on the chromato-gram, and the accumulation of BP 4,5-oxide during the incubation was completely prevented by the addition of epoxidehydrolase to the monooxygenase system (Chart 18). Thechange in profile of metabolites of BP that was observed whenthe ratio of epoxide hydrolase to cytochrome P-450 was variedprovided strong evidence for the cytochrome P-450-dependentmetabolism of BP to its 4,5-, 7,8-, and 9,10-oxides which couldeither be metabolized to frans-dihydrodiols by epoxide hydro

lase or undergo isomerization to phenols. The use of purifiedcytochrome P-450 and epoxide hydrolase as tools for the

elucidation of metabolic pathways and as an approach fordemonstrating the metabolic activation and detoxification ofpolycyclic hydrocarbons is described in more detail elsewhere(131, 190, 236, 237, 290, 291, 293, 352, 354).

Mutagenic Activity of BP Metabolites

We chose to study the mutagenic activity of known andpotential BP metabolites as an initial test of their biologicalactivity because the mutagenic activity of chemicals providesa simple method for evaluating their ability to interact withgenetic material in a way that results in an altered and heritablephenotype, and we thought that studies on the mutagenicity ofBP derivatives would help in the determination of how BP isactivated to its ultimately reactive forms. The inherent mutagenic activities of potential BP metabolites and other BP derivatives were examined in Salmonella typhimurium strains TA98, TA 100, and TA 1538 and in Chinese hamster V-79 cells

(76, 296, 338, 349, 359). Both the bacterial and mammaliancells selected for testing the mutagenic activity of BP deriva-

TOTAL METABOLITES

ZOO 400 600 200 400 600 200 40O 600

UNITS OF EPOXIDE HYDROLASE ADDED

200 400 600 BOO

4 BP 7.8-diol-9,10-epoxide 1 has also been called Isomer II. the cis isomer,

the fast isomer, and the syn isomer. BP 7.8-diol-9,10-epoxide 2 has also beencalled Isomer I, the frans isomer, the slow isomer, and the anti isomer.

Chart 18. Profile of metabolites formed during the incubation of BP with apurified cytochrome P-450c-dependent monooxygenase system in the presenceand absence of purified epoxide hydrolase (13t). Diol Fractions 1. 2, and 3correspond to BP 9.10-dihydrodiol, BP 4,5-dihydrodiol, and BP 7,8-dihydrodiol,respectively. Phenol Fractions 1 and 2 each correspond to mixtures of phenolicmetabolites. Quinone Fraction 1 has the Chromatographie mobility of BP 1,6-,3,6-. and 4,5-quinones, whereas Quinone Fraction 2 has the Chromatographiemobility of BP 4,5-oxide and the 6,12- and 11,12-quinones of BP. QuinoneFraction 2 contains primarily BP 4,5-oxide.




tives lack detectable levels of polycyclic hydrocarbon-metabo

lizing enzymes and thus the parent hydrocarbon, BP, is inactivein these test systems.

In 1975, we reported that BP 4,5-oxide (K-region oxide) was

a potent mutagen in several strains of S. typhimuhum and inChinese hamster V-79 cells, and we indicated that BP 7,8-oxide and BP 9,10-oxide were weakly mutagenic (349). In thefollowing year, the diastereomeric BP 7,8-diol-9,10-epoxides

were reported to have high mutagenic activity (135, 235, 339,359). The results of our research with more than 30 derivativesof BP indicated that the diastereomeric BP 7,8-diol-9,10-epox-ides and BP m-9,10-epoxide (see Table 16 for structure) werethe most mutagenic compounds tested in S. typhimuriumstrains TA 98 and TA 100 and in Chinese hamster V-79 cells

(76, 359). It was of considerable interest that the relativemutagenic activities of the various compounds depended onthe test system used. In strain TA 1538 of S. typhimurium, BP4,5-oxide had about the same mutagenic activity as did BP7,8-diol-9,10-epoxide 1, but BP 4,5-oxide was a more potentmutagen in this strain of bacteria than was BP 7,8-diol-9,10-

epoxide 2 (76, 86, 338, 339, 359). In all 3 bacterial strains,BP 7,8-diol-9,10-epoxide 1 was a more potent mutagen thanwas BP 7,8-diol-9,10-epoxide 2, while thÃ¨reverse was true inChinese hamster V-79 cells (359). We found that BP 7,8-diol-9,10-epoxides 1 and 2 were, respectively, about 40- and 100-fold more mutagenic toward Chinese hamster V-79 cells thanwas BP 4,5-oxide (76, 339, 359). The high mutagenic activitiesof the BP 7,8-diol-9,10-epoxides and 9,10-epoxy-7,8,9,10-tetrahydro-BP are undoubtedly an underestimate of intrinsic

biological activity because these epoxides are unstable inaqueous solution and they undergo spontaneous solvolysisand hydronium ion-catalyzed solvolysis to biologically inactive

tetraols (327, 359). During the course of our studies, we foundthat BP 4,5-oxide and BP H4-9,10-epoxide were rapidly metab

olized to nonmutagenic products by purified epoxide hydrolase(354, 359), and we also found that the BP 7,8-diol-9,10-epoxides and other diol-epoxides were refractory to attack by

epoxide hydrolase (343, 354, 359).Since the chemically synthesized BP 4,5-oxide and the BP

7,8-diol-9,10-epoxides 1 and 2 used for the mutagenicity studies described above are racemates, the optically pure (+)- and(â€”)-enantiomers of each compound were prepared (54, 360),

and we determined their mutagenic activities (54, 348). Substantial differences were observed for the mutagenic activitiesof the (+)- and (â€”)-enantiomers of the BP 7,8-diol-9,10-epox

ides 1 and 2 in bacterial and mammalian cells (Table 8). Instrains TA 98 and TA 100 of S. typhimurium, the (â€”)-enantio-mer of BP 7,8-diol-9,10-epoxide 1 was 1.3 to 9.5 times more

mutagenic than were the other 3 optically active stereoisomers.In Chinese hamster V-79 cells, however, the (+)-enantiomer ofBP 7,8-diol-9,10-epoxide 2 was 6 to 18 times more mutagenic

than were the other 3 isomers. This study suggests the presence of highly stereoselective receptors, detoxification mechanisms and/or transport systems. The data also demonstratethat the relative mutagenic activities of the four BP 7,8-diol-9,10-epoxides depend on the test system used. It will be shownlater that the high mutagenic activity of the ( + )-BP 7,8-diol-9,10-epoxide 2 in Chinese hamster V-79 cells accurately pre

dicted the high tumorigenicity of this isomer in mice (Table 15;Chart 21).

Studies on the mutagenicity of the (+)-[4S,5fl]- and (-)-


Table 8Mutagenicity of optically pure enantiomers of the BP 7,8-diol-9,10-

epoxides (348)Mutagenic activity is expressed as revertants/nmol/plate for the S. typhimu

rium strains and as 8-azaguanine-resistant colonies/nmol/105 surviving cells forthe Chinese hamster V-79 cells.

Mutagenic activity

S. typhimurium Chinesehamster V-

Compound tested Strain TA 98 Strain TA 100 79 cells

<-)-BP 7,8-diol-9,10-epoxide 1

( + )-BP 7,8-diol-9,10-epoxide 1

5200 9500 60

3900 5200 34

1800 1000 22

(-)-BP 7,8-diol-9,10-epoxide 2

2500 6000 400

(+)-BP 7.8-diol-9,10-epoxide 2

[4R,5S]-enantiomers of BP 4,5-oxide revealed that the (â€”)-enantiomer was more mutagenic than the ( + )-enantiomer in S.

typhimurium strains TA 98 and TA 100 and in Chinese hamsterV-79 cells (54). When mixtures of the enantiomers were studiedin Chinese hamster V-79 cells, synergistic cytotoxic and mu

tagenic responses were observed. The greatest cytotoxic andmutagenic responses occurred with a 3:1 mixture of the (-)-and (-f-)-enantiomers of BP 4,5-oxide, respectively (Table 9).

Further research is needed to determine the mechanism of thissynergism.

Specificity in the Metabolism of BP to Optically Active Enantiomers

Since optically active enantiomers have different biologicalactivities, the stereochemical course of BP metabolism by thecytochrome P-450-dependent monooxygenase system and by

epoxide hydrolase may play a critical role in the expression ofthe biological activity of BP. Accordingly, we investigated thebiotransformation of BP to its optically active metabolites (151,185, 292, 295, 297, 298), and similar studies on the stereo-chemical course of BP metabolism were done independentlyby Gelboin and his associates (135, 362-365). Liver micro-somal enzymes from rats treated with MC metabolized BP tothe (â€”)-enantiomers of BP 4,5-dihydrodiol, BP 7,8-dihydrodiol,and BP 9,10-dihydrodiol to a >10-fold excess relative to the(+)-enantiomers (295, 297, 298, 362, 365). The metabolism

DECEMBER 1982 4889



A, H. Conney

of BP to the (+M7S.8S]- and (-)-[7fÃ®,8fÃ®]-enantiomers of BP7,8-dihydrodiol by liver microsomes from control rats and from

rats treated with phÃ©nobarbital or MC is shown in Table 10. Inall cases, the (â€”)-[fÃ,Ã±]-enantiomerwas the major metabolite

formed. Studies with other hydrocarbons revealed that BA,DBA, chrysene, and phenanthrene were also metabolized byrat liver microsomes predominantly to the [fÃ®,fl]-enantiomer ofseveral dihydrodiols (292). Interestingly, the degree of enan-tiomeric purity of the metabolites formed depended on thesource of microsomes (kinds of cytochrome P-450s), the hydrocarbon substrate studied, and the positional dihydrodiolisomer measured (292).

Marked stereoselectivity occurred for the metabolism of theoptically pure ( + )-[S,S]- and (-)-[fl,fi]-enantiomers of BP 7,8-dihydrodiol to the optical enantiomers of the BP 7,8-diol-9,10-

Table 9

Mutagenic and cytotoxic activity of mixtures of optically pure enantiomers ofBP 4,5-oxide in Chinese hamster V-79 cells (54)

Cells were treated for 4 hr with 30 nmol of BP 4.5-oxide in 5 ml of medium.

% oftotal(

+ )-BP[4S,5fl]-oxide0100go755025100dose(-)-BP[4R,5SJ-oxide001025507590100Celldeath(%)05152238461718Mutationfrequency(8-azaguanine-resistantcolonies/105surviving

cells)02.34.410.618.124.415.812.3

epoxides 1 and 2 by rat liver microsomes (295, 298). Incubation of the (-)-[R,fl]-enantiomer of BP 7,8-dihydrodiol (the

metabolically predominant enantiomer) with liver microsomesfrom rats pretreated with MC resulted in the stereoselectivemetabolism of this dihydrodiol to the (+)-BP 7,8-diol-9,10-epoxide 2 to a 6-fold greater extent than to the (-)-BP 7,8-diol-9,10-epoxide 1 (Table 11). When the (+)-[S,S]-enantiomerof BP 7,8-dihydrodiol was used as the substrate, highly stereoselective metabolism to ( + )-BP 7,8-diol-9,10-epoxide 1 wasobserved. Interestingly, liver microsomes from MC-treated ratsmetabolized both the ( + )- and (-)-enantiomers of BP 7,8-

dihydrodiol with greater stereoselectivity than did liver microsomes from control rats or from rats treated with phÃ©nobarbital(Table 11 ). These differences in stereoselectivity in the metabolism of the BP 7,8-dihydrodiols to specific diol-epoxide iso-

mers probably reflect differences in the proportion of the various cytochrome P-450s present in liver microsomes from

control rats and from rats treated with phÃ©nobarbital and MC.Gelboin and Coon have recently shown that several purifiedcytochrome P-450 isozymes from rabbit liver microsomes have

Table 10Optical purity of BP 7,8-dihydrodiol formed during the incubation of BP with

liver microsomes from control and induced rats (151, 295)

Rats were given i.p. injections of sodium phÃ©nobarbital (75 mg/kg) or MC (25mg/kg) once daily for 4 days, and the animals were killed the following day.Liver microsomes were prepared and incubated with BP and NADPH.

% of each enantiomer formed

Pretreatment (+H7S.8S] (-H7R.8R]

ControlPhÃ©nobarbitalMC

939296

Table 11Metabolism of optically pure enantiomers of BP 7.8-dihydrodiol to the BP 7,8-diol-9,10-epoxides by rat

liver microsomes (295)

Rats were given i.p. injections of sodium phÃ©nobarbital (75 mg/kg) or MC (25 mg/kg) once daily for 4days, and the animals were killed the following day. Liver microsomes were prepared and incubated withoptically pure BP 7,8-dihydrodiol and NADPH. Tetraols and triols derived from the BP 7.8-diol-9,1O-epoxides 1 and 2 were quantified.

Substrate Products formed

% of total metabolites formed

Control PhÃ©nobarbital MCmicrosomes microsomes microsomes

HO1

OH

(-)-Diol-epoxide 1

(-)-BP [7fl,8fl]-dihydrodiol

OH

h)-Diol-epoxide 2

24

53

20

51

13

83

)-BP [7S.8S]-dihydrodiol

OH

h)-Diol-epoxide

o.

OH

(-)-Diol-epoxide 2

53

10

54

12

90





markedly different stereoselectivities in their metabolism of the(-)-[R,R] enantiomer of BP 7,8-dihydrodiol to BP 7,8-diol-9,10-epoxides 1 and 2 (97, 98). Since only one of the 4possible optically active BP 7,8-diol-9,10-epoxides has high

tumorigenic activity (Table 15; Chart 21), our results and thoseof Gelboin and Coon indicate that factors influencing the relative amounts or activities of the multiple cytochrome P-450s in

an organism may influence the amount of BP that is metabolized to its ultimate carcinogenic metabolite.

Covalent Binding of BP Metabolites

In 1973, Borgen et al. (23) reported more extensive covalentbinding of hydrocarbon metabolites to DMA when BP 7,8-

dihydrodiol was metabolized by hamster liver microsomes thanwhen BP or several of its biotransformation products weremetabolized by this system. The following year, Sims et al.(276) reported the covalent binding of a BP 7,8-diol-9,10-

epoxide to DMA in cultured cells that were treated with BP.Subsequent studies by Weinstein and his associates and byother investigators demonstrated that the ( + )-enantiomer ofBP 7,8-diol-9,10-epoxide 2, covalently bound to the 2-amino

group of guanine in nucleic acids, was the major nucleic acidadduct observed when BP was metabolized by bovine bronchial expiants (142, 233, 319), by 10TV2 mouse embryo fibro-

blasts (34), and by expiants or cultured cells from humanbronchus, lung, and colon (11, 143, 272). In some of thesestudies, evidence was also presented for the formation of smallamounts of nucleic acid adducts derived from BP 7,8-diol-9,10-epoxide 1. Studies with cultured BHK 21 /C13 cells (167,

259), cultured hamster embryo cells (14, 140), secondarymouse embryo fibroblast cells (259), baby hamster kidney cells(273), and human alveolar tumor cells (47) provided additionalevidence for the binding of both BP 7,8-diol-9,10-epoxides 1

and 2 to DNA after exposure of the cells to BP. It is ofconsiderable interest that the profile of DNA adducts dependson the test system studied. In hamster embryo cell culturesexposed to BP, there is a higher amount of the guanine adductof BP 7,8-diol-9,10-epoxide 1 and an adenine adduct than in10T'/2 cells (14, 140).

In our studies with a target tissue susceptible to BP carci-nogenesis, both (+)-BP 7,8-diol-9,10-epoxide 2 and (+)-BP7,8-diol-9,10-epoxide 1 were found covalently bound to DNA,RNA, and protein of skin after topical application of BP to thebacks of mice (1 72, 230). Interestingly, the relative amounts ofthe 2 diol-epoxides bound depended on whether binding to

DNA, RNA, or protein was measured, but in all cases theamount of ( + )-BP 7,8-diol-9,10-epoxide 2 that was bound wasgreater than the amount of bound ( + )-BP 7,8-diol-9,10-epox

ide 1. Our studies and those using tissue culture systemsdemonstrated that most of the binding of the BP diol-epoxidesto DNA and RNA occurred at the exocyclic 2-amino group ofguanine. The metabolism of BP to diol-epoxides and the structures of the guanine adducts of the BP diol-epoxides that were

isolated from mouse skin DNA and RNA after application of BPare shown in Chart 19. More recently, evidence was presentedfor the covalent binding of 9-hydroxy-BP 4,5-oxide to DNA (as

a minor adduct) after application of BP to mouse skin (312). Itis unlikely that this adduct has an important role in the initiationof skin tumors since BP 4,5-oxide and 9-hydroxy-BP have little

or no tumorigenic activity on mouse skin (155, 202).

( > BP 7.B OIHYOBODIOl (tl-Â»P T,*-DIOL-9.IO IPOXIDf ?

(+ÃŒ-SPT,Â«-OIHYOROOIOL

Ã”M

I*;-BP 7.1-OIOL-9.IO-EPOXIOE-I GUANINE 4DDUCT

Chart 19. Formation of BP 7,8-diol-9,10-epoxide adducts of guanine in DNAand RNA of mouse skin (172, 230). Mice were treated topically with BP, andnucleic acid adducts of the BP 7.8-diol-9.10-epoxides that were formed from thecis and trans addition of the 2-amino group of guanine to the oxirane ring of thediol-epoxides were isolated. The major adduct observed was from the transaddition of the 2-amino group of guanine across the epoxide group of (+)-BP7,8-diol-9.10-epoxide 2.

When the (+)- and (-)-enantiomers of BP 7,8-diol-9,10-epoxide 2 were added to double-stranded DNA in vitro, Meehanand StrÃ¤ub(222) found that the ( + )-enantiomer was covalentlybound to the 2-amino group of deoxyguanosine to a 20-foldgreater extent than was the (-)-enantiomer and that small and

approximately equal amounts of both enantiomers were boundto the exocyclic amino group of deoxyadenosine. Interestingly,the large difference in covalent binding of the (+)- and ( â€”¿�)-enantiomers of BP 7,8-diol-9,10-epoxide 2 to the 2-aminogroup of deoxyguanosine did not occur when single-stranded

DNA was used. These results suggest a highly stereoselectiveorientation (noncovalent binding) of the ( + )-enantiomer of BP7,8-diol-9,10-epoxide 2 to chiral centers in double-strandedDNA prior to the covalent binding of this diol-epoxide to the 2-

amino group of deoxyguanosine. The highly stereoselectivecovalent binding of the ( + )-enantiomer of BP 7,8-diol-9,10-epoxide 2 to the 2-amino group of deoxyguanosine in double-

stranded DNA is in good agreement with the high mutagenicityof this enantiomer in Chinese hamster V-79 cells (Table 8) and

with the high tumorigenicity of this enantiomer in mice (Table15; Chart 21).

Tumorigenicity of BP Metabolites

Since not all mutagens are carcinogens and since covalentbinding of chemicals need not be accompanied by tumorformation, the identification of proximate and ultimate carcinogenic metabolites can only come from carcinogenicity studies. Because of these considerations, carcinogenicity studiesin experimental animals have been an essential part of ourprogram to identify proximate and ultimate carcinogenic metabolites of BP and other polycyclic aromatic hydrocarbons.

Tumorigenicity of Racemic BP 7,8-oxide, BP 7,8-dihydrodiol, and the BP 7,8-diol-9,10-epoxides on Mouse Skin. Inour initial carcinogenicity studies, we found that application of0.4 /tmol of BP or BP 7,8-oxide every 2 weeks to the skin ofmice for 60 weeks resulted in a 90 to 100% tumor incidence.At an equimolar dose, BP 9,10-oxide and BP 11,12-oxide (K-region oxide) were inactive as carcinogens, and BP 4,5-oxide(K-region oxide) produced only one skin tumor among the 30

DECEMBER 1982 4891



A. H. Conney

mice that were treated (202, 331). When a lower dose ofhydrocarbon was studied, BP 7,8-oxide was significantly less

carcinogenic than was BP (Chart 20). HistolÃ³gica! examinationrevealed that most of the tumors in the treated animals weresquamous cell carcinomas.

BP 7,8-dihydrodiol was considerably more tumorigenic thanwas BP 7,8-oxide when tested as a complete carcinogen onmouse skin. The application of 0.15 /tmol of BP or BP 7,8-

dihydrodiol to mouse skin once every 2 weeks for 60 weeksresulted in a tumor incidence of 100%, whereas only 20% ofthe mice treated with an equimolar amount of BP 7,8-oxide hadskin tumors (Chart 20) (203). BP 7,8-dihydrodiol was at least

equipotent to BP as a complete carcinogen at several doses(201, 203), and other studies have shown that BP 7,8-dihydro

diol is a potent tumor initiator on mouse skin (55, 285). Incontrast to these observations, BP 4,5-dihydrodiol and BP9,10-dihydrodiol have little or no tumorigenic activity (280,296). It was of considerable interest that BP H4-7,8-epoxideand BP H4-7,8-diol, compounds related to BP 7,8-oxide andBP 7,8-dihydrodiol but with the double bond saturated withhydrogen at the 9,10-position of the molecule, were inactive in

eliciting tumors on mouse skin (201, 203) (Chart 20). Thestrong carcinogenic activities of BP 7,8-oxide and BP 7,8-

dihydrodiol combined with the lack of carcinogenicity of thesecompounds when the 9,10-double bond was removed stronglysuggested that the carcinogenicity of BP 7,8-oxide and BP7,8-dihydrodiol on mouse skin was due to the metabolic conversion of these compounds to one or more of the BP 7,8-diol-9,10-epoxides.

Although BP 7,8-diol-9,10-epoxide 1 was inactive as a complete carcinogen on mouse skin, BP 7,8-diol-9,10-epoxide 2

did have very weak tumorigenic activity when compared to BP(201). In these studies, 100% of the mice had skin tumorswhen they were treated with 0.4 jumol of BP once every 2weeks for 60 weeks, and 13% of the animals had skin tumorswhen they were treated with an equimolar dose of BP 7,8-diol-9,10-epoxide 2. Additional studies by Slaga ef al. (281, 285)indicated that BP 7,8-diol-9,10-epoxide 2 had considerably

weaker activity than did BP as a tumor initiator on mouse skin.Although BP 7,8-diol-9,10-epoxide 2 was only weakly carcinogenic on mouse skin and BP 7,8-diol-9,10-epoxide 1 was

inactive, we found that both compounds were highly active incausing hypertrophy and hyperplasia when they were appliedtopically (30).

High Tumorigenicity of Racemic BP 7,8-dihydrodiol andBP 7.8-diol-9,10-epoxide 2 in Newborn Mice. Since the newborn mouse is highly susceptible to the effects of many chemical carcinogens (96, 304, 305), we tested potential proximateand ultimate carcinogenic metabolites of BP and other poly-

cyclic aromatic hydrocarbons in a newborn mouse tumormodel. In these studies, the hydrocarbons were injected i.p.into Swiss-Webster mice on the first, 8th, and 15th days of life,

and the animals were killed several months later. The predominant tumors observed were pulmonary tumors (mostly adenomas with some adenocarcinomas), malignant lymphomas, andliver tumors. The kinds of tumors observed depended on thehydrocarbon studied, the dose administered, and the durationof the study. The newborn mouse tumor model has been aparticularly useful test system for identifying proximate andultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. We found that BP 7,8-oxide was moderately active in

CO

OC.O

13

O

S

Otr

1001-

90

80

70

60

50

40

3O

IO

BP 7,8-DIHYDRODIOL

J I I I i A

BP 7,8-OXIDE

I />-c-o

H4-7,8-EPOXIDEand

H4-7,8-DIOL

l

5 IO 15 20 25 30 35 40 45 50 55 60

WEEKS ON TESTChart 20. Tumorigenicity of BP, BP 7,8-oxide, BP 7,8-dihydrodiol, 7,8-epoxy-

7,8.9,10-tetrahydro-BP(H4-7,8-epoxide), and (rans-7.8-dihydroxy-7,8,9,10-tet-rahydro-BP (H4-7,8-dioD in mice treated topically with 0.15 /xmol of compoundonce every 2 weeks for 60 weeks (201, 203).

causing pulmonary tumors when injected into newborn micewhereas BP 4,5-oxide, BP 9,10-oxide, and BP 11,12-oxide

had little or no tumorigenic activity in this animal model (336).Additional studies revealed that BP 7,8-dihydrodiol (1400nmol) administered i.p. to newborn mice during a 15-day interval was at least 10 times more active than an equimolar doseof BP in causing malignant lymphomas and pulmonary tumors(Table 12, Experiment 1) (153). BP 7,8-diol-9,10-epoxides 1

and 2 were highly toxic, and survival of the animals could beachieved only when 1/50 of the above dose of the diol-epox-

ides was administered. A comparison of the number of pulmonary tumors after a 1400-nmol dose of BP, a 1400-nmoldose of BP 7,8-dihydrodiol, and a 28-nmol dose of BP 7,8-diol-9,10-epoxide 2 suggested that BP 7,8-dihydrodiol and BP 7,8-diol-9,10-epoxide 2 were, respectively, approximately 12- and40-fold more tumorigenic than was BP (Table 12, Experiment1). In an additional experiment, we compared the tumorigenicityof low equimolar doses of BP, BP 7,8-dihydrodiol, BP 7,8-diol-9,10-epoxide 1, BP 7,8-diol-9,10-epoxide 2, and the tetraolswhich result from the hydrolysis of BP 7,8-diol-9,10-epoxide 2in aqueous medium (Table 12, Experiment 2). Animals treatedwith 28 nmol of BP during the first 15 days of life had abouttwice as many pulmonary tumors per mouse as did controlanimals. In this study, BP 7,8-diol-9,10-epoxide 1 and the BP





Table 12

Tumorigenicity of BP, BP 7,8-dihydrodiol, and the BP 7,8-diol-9,10-epoxides in newborn mice (153, 157)

Mice were given i.p. injections of 1/7. 2/7, and 4/7 of the total dose of compound on the first, 8th, and15th days of life, respectively. The studies were terminated when the animals were 21 to 24 weeks old inExperiment 1 and 28 weeks old in Experiment 2.

Experiment12CompoundadministeredControlBPBP

7.8-dihydrodiolBP7,8-diol-9,10-epoxide2ControlBPBP

7.8-dihydrodiolBP7.8-diol-9,10-epoxide1BP7,8-diol-9.10-epoxide2Tetraols

(from BP7,8-diol-9,10-epoxide2)Total

dose(nmol)0140014002802828282828%

ofmicewithmalig

nantlym-phoma00750000022%ofmicewithlung tu

mors484928112196610865Lungtumors/mouse<0.16.375.04.90.130.241.770.144.420.05

tetraols derived from BP 7,8-diol-9,10-epoxide 2 had little orno tumorigenic activity, whereas BP 7,8-diol-9,10-epoxide 2and BP 7,8-dihydrodiol were highly tumorigenic at the 28-nmol

dose tested. After correcting for the small number of lungtumors in control mice, the data in Experiment 2 indicate thatBP 7,8-dihydrodiol and BP 7,8-diol-9,10-epoxide 2 were, respectively, about 15- and 40-fold more active than BP incausing pulmonary tumors (157). The studies described inTable 12 indicate that BP 7,8-dihydrodiol is a proximate carcinogenic metabolite and that BP 7,8-diol-9,10-epoxide 2 is an

ultimate carcinogenic metabolite of BP in the newborn mouse.These data provided the first demonstration of proximate andultimate carcinogenic metabolites for the polycyclic aromatichydrocarbon class of carcinogens. Although the reason(s) forthe high tumorigenic activity of racemic BP 7,8-diol-9,10-epox-

ide 2 in newborn mice and the much weaker activity of thiscompound on mouse skin is not known, our results illustratethe importance of studies with several animal models duringthe evaluation of the carcinogenicity of chemicals.

High Tumorigenic Activity of 2-Hydroxy-BP. A comparisonof the carcinogenic activity of BP with that of its 12 possibleisomerie phenols revealed that only 2- and 11-hydroxy-BPwere complete carcinogens on mouse skin (155, 331). 2-Hydroxy-BP was almost equipotent to BP in causing skintumors; 11-hydroxy-BP was weakly active; and 1-, 3-, 4-, 5-,6-, 7-, 8-, 9-, 10-, and 12-hydroxy-BP were inactive. 2-Hydroxy-BP was about 4-fold more active than an equimolar dose

of BP in causing pulmonary tumors in the newborn mousetumor model, and 6-hydroxy-BP was inactive (53). Additionalstudies with the 12 isomerie BP phenols indicated that only 2-hydroxy-BP had strong tumor-initiating activity in an initiation-promotion model on mouse skin (278). 2-Hydroxy-BP is the

only known example of a phenolic polycyclic aromatic hydrocarbon with strong carcinogenic activity, and additional studiesare needed to determine how 2-hydroxy-BP exerts its carcinogenic effect and to determine whether or not 2-hydroxy-BP

is a metabolite of BP.Tumorigenicity of Optically Active Metabolites of BP. An

examination of the tumorigenic activity of the (+)-[7f?,8S]- and(-H7S,8ft]-enantiomers of BP 7,8-oxide revealed that the(+)-enantiomer, which is the major enantiomer formed meta-

Table 13Tumorigenicity of optically pure enantiomers of BP 7,8-oxide on mouse skin

and in the newborn mouse (185)Skin study* Newborn study

% of mice % of miceCompound adminis- with skin Skin tumors/ with lung Lung tumors/

tered tumors mouse tumors mouse

Control(-)-BP[7S.8R}-oxide(+>-BP[7fl.8S]-oxide(Â±)-BP

7,8-oxide71138670.070.110.761.48132084890.140.252.283.60

Mice received a single topical application of 100 nmol of compound followed7 days later by twice-weekly applications of 16 nmol of 12-O-tetradecanoylphor-bol-13-acetate for 25 weeks.

" Mice were given i.p. injections of 100, 200, and 400 nmol of BP 7,8-oxide

on the first, 8th, and 15th days of life, respectively. The animals were killed at 31to 35 weeks of age.

bolically from BP, is severalfold more active than the (-)-enantiomer as an initiator of tumors on mouse skin and incausing pulmonary tumors in newborn mice (185) (Table 13).Interestingly, in both tumor models, we found that racemic BP7,8-oxide produced more tumors than either optical enantiomer

alone (Table 13). Without any enantiomeric interaction, thetumorigenic activity of racemic BP 7,8-oxide should have heenintermediate to the activity of the (+)- and (-)-enantiomers.

This is not the case, and our data demonstrate a synergisticinteraction for the carcinogenicity of the optical enantiomers.Further studies are needed to determine the mechanism of thissynergistic interaction.

An examination of the tumorigenic activities of the (+)-[7S.8S]- and (-)-[7fl,8fl]-enantiomers of BP 7,8-dihydrodiol(158, 199) revealed that (-)-BP 7,8-dihydrodiol, which is themajor enantiomer formed metabolically from BP, was several-fold more active than (+)-BP 7,8-dihydrodiol as a tumor initiator

on mouse skin (Chart 21) and in causing pulmonary tumorsand malignant lymphomas in newborn mice (Table 14).

When the 4 optically active BP 7,8-diol-9,10-epoxides were

tested for tumorigenicity on mouse skin and in newborn mice(41, 279), significant tumorigenicity in both tumor models wasfound only for (+)-BP 7,8-diol-9,10-epoxide 2 (Table 15; Chart21), which is the major diol-epoxide isomer formed metabolically from BP. Although (+)-BP 7,8-diol-9,10-epoxide 2 was

DECEMBER 1982 4893



A. H. Conney

Chart 21. Tumor-initiating activity of optically pure enantiomers of BP 7,8-dihydrodioland the diastereomeric BP 7,8-diol-9,10-epoxides on mouse skin (199, 279). In Experiment A, mice were treated topically with 100nmol of BP or the ( + )- and (â€”)-enantiomers ofBP 7,8-dihydrodiol. Eleven days later, themice were treated with 16 nmol of 12-O-tetra-decanoylphorbol-13-acetate twice weekly for21 weeks.ln Experiment B, mice were treatedtopically with 200 nmol of BP or the (+>- and<â€”)-enantiomers of BP 7,8-diol-9,10-epoxides1 and 2. Seven days later, the mice receivedtwice weekly applications of 10 fig of 12-O-tetradecanoylphorbol-13-acetate

f. 3

O

(-I-BP 7.8-OIHYOROOIOL

7 9 II I3 IS I7 I9 2l 7 9

WEEKS OF PROMOTION

[Â«-BP 7.8-DIOL-9,IO-EPOXIDE-2

13 A 17 19 ZI 23 25

less active than BP as a tumor initiator on mouse skin (Chart21), this diol-epoxide isomer was 50 to 100 times more active

than BP in causing pulmonary tumors when injected into newborn mice (Tables 12 and 15). The injection of only 7 or 14nmol of (+)-BP 7,8-diol-9,10-epoxide 2 into newborn mice

during the first 15 days of life caused a high incidence ofpulmonary tumors, but little or no tumorigenicity was observedafter an equimolar dose of BP or the other 3 diol-epoxideisomers (Table 15). The data in Table 14 indicating that a highdose of (+)-BP [7S,8S]dihydrodiol has tumorigenic activitysuggest that ( + )-BP-7,8-diol-9,10-epoxide 1, the major diol-epoxide metabolite of ( + )-BP [7S,8S]dihydrodiol (Table 11),may have tumorigenic activity if a sufficiently high dose couldbe given. It is of interest that the highly tumorigenic ( + )-BP7,8-diol-9,10-epoxide 2 was the most mutagenic of the 4 diol-epoxides of BP that were tested in Chinese hamster V-79 cellswhereas the relative mutagenic activities of the 4 diol-epoxides

in S. typhimurium strain TA 98 or TA 100 did not predict thetumorigenic activities of these compounds (Table 8).

The stereochemical course for the metabolism of BP to itsproximate and ultimate carcinogenic metabolites and the absolute stereochemistry of each metabolite are summarized inChart 22. Major pathways for the metabolism of BP to its diol-epoxides by liver microsomal enzymes are given by the heavyarrows, and minor pathways are given by the thin arrows. Themetabolism and carcinogenicity studies described here havedemonstrated that BP is metabolized by cytochrome P-450 tothe proximate carcinogen (+)-BP 7,8-oxide [7fl,8S] to a muchgreater extent than to the ( â€”¿�)-enantiomerof this arene oxide.(+)-BP 7,8-oxide is then stereospecifically metabolized byepoxide hydrolase to the proximate carcinogen (-)-BP 7,8-dihydrodiol [7R,8R] which in turn is stereoselectively metabolized by cytochrome P-450 to the ultimate carcinogen ( + )-BP7,8-diol-9,10-epoxide 2 [7fl,8S,9S,10fl]. It is of interest to

note that liver enzymes metabolize BP predominantly to theonly BP 7,8-diol-9,10-epoxide isomer with high tumorigenic

activity.

Bay Region Theory of Polycyclic Aromatic Hydrocarbon Car-

cinogenesis

Several observations made during the course of our researchwith Dr. Jerina led to a new theory (the bay region theory) thathas successfully predicted proximate and ultimate carcinogenic metabolites of more than a dozen polycyclic aromatichydrocarbons. The prototype of a bay region in a polycyclichydrocarbon is the sterically hindered region between C-4 and

Table 14Tumorigenicity of optically pure enantiomers of BP 7,8-dihydrodiol in newborn

mice (158)

Mice were given i.p. injections of 1/7, 2/7, and 4/7 of the total dose ofcompound on the first, 8th, and 15th days of life, respectively. The animals werekilled at 17 weeks of age.

CompoundadministeredControlBP(-)-BP

[7R,8fi)-dihydrodiol(+)-BP

[7S,8Sl-dihydrodiolTotal

dose(nmol)01400140700140700%

ofmicewithma

lignantlym-phoma0047006%ofmicewithlungtumors1074981001654Lung

tumors/mouse0.104.19.332.20.22.3

Table 15

Pulmonary tumors in newborn mice treated with optically pure enantiomers ofthe diastereomeric BP 7,3-diol-9.10-epoxides (41)

Mice were given i.p. injections of 1/7, 2/7, and 4/7 of the total dose ofcompound on the first, 8th and 15th days of life, respectively. The animals werekilled at 34 to 37 weeks of age.

Total doseCompound administered(nmol)ControlBP(-)-BP

7,8-diol-9.10-epoxide1(+ )-BP 7,8-diol-9,10-epoxide1(-)-BP

7.8-diol-9,10-epoxide2(+)-BP7,8-diol-9,10-epoxide2BP(-)-BP

7,8-diol-9,10-epoxide1(+ )-BP 7,8-diol-9,10-epoxide1(-)-BP

7.8-diol-9,10-epoxide2(+)-BP7,8-diol-9,10-epoxide 20777771414141414%

of micewith lung tu

mors1118131497114221512100Lungtumors/

mouse0.120.220.150.150.091.720.150.250.340.137.67

C-5 of phenanthrene. Examples of bay regions and bay-regiondiol-epoxides of polycyclic aromatic hydrocarbons are given inChart 23. The bay region theory suggested that bay-regiondiol-epoxides of polycyclic aromatic hydrocarbons are primecandidates for ultimate carcinogens and that the amount ofmetabolism of the parent hydrocarbon to bay-region diol-epoxides should be important for the expression of the carcinogenicity of the parent hydrocarbon. The findings that led to thebay region theory, which was first proposed by Jerina and Daly(146), included (a) high chemical reactivity and mutagenic





soÂ«?

<Â»>-BP7.8-DIOL-9.10-EPOXIDE-2

(-)-BP 7.8-OIOL-9.10-EPOXIDE-1

P..

OHM-BP 7,8-OIOL-9,tO-EPOXIDE-1

(-)-BP 7,8-OXIDEOH

(Â»)-BP 7,8-OIHYDRODIOL

OH(-)-BP 7,8-OIOL-9,10-EPOXIDE-2

Chart 22. Formation and absolute stereochemistry of BP metabolites responsible for the carcinogenicity of the parent hydrocarbon. Heavy arrows, indicate themajor metabolic pathways.

Boy region Bay region Bay region

0-,,,.-OH

0,,

Chart 23. Structures of bay-region diol-epoxides ofseveral polycyclic aromatic hydrocarbons.

8 0

PHENANTHRENE

DIOL-EPOXIDE

Bay region

Ã›Ã›OTBENZ[O]ANTHRACENE

DIOL-EPOXIDE

Bay region

7 6 Bay region

CHRYSENE

DIOL-EPOXIDE

Bay region

lo^^/XÂ»Bay region

DIBENZ[a,h]ANTHRACENE BENZO[a]PYRENE

DIOL-EPOXIDE DIOL-EPOXIDE

7.12-DIMETHYL-

BENZ [o] ANTHRACENE

DIOL-EPOXIDE

activity of bay-region epoxides of BP relative to non-bay-region

epoxides (149,359), (b) observations from a survey of theliterature indicating that substituents which could block theformation of bay-region diol-epoxides on the angular benzoring of BA derivatives inhibited the carcinogenicity of thesecompounds (146), and (c) the results of Dewar perturbationalmolecular orbital calculations predicting a greater ease ofcarbocation formation at the benzylic carbon atom for bay-region epoxides and diol-epoxides than for their correspondingnon-bay-region epoxides and diol-epoxides (149).

Examination of the properties of the BP 7,8-diol-9,10-epox-ides that account for their high biological activity revealed that9,10-epoxy-7,8,9,10-tetrahydro-BP (BP H4-9,10-epoxide;bay-region epoxide) is an extremely potent mutagen whereas7,8-epoxy-7,8,9,10-tetrahydro-BP (BP H4-7,8-epoxide; non-

bay-region epoxide) is a considerably less potent mutagen(359) (Table 16). In addition, the bay-region BP 7,8-diol-9,10-epoxides 1 and 2 are much more mutagenic than are BP 7,10-diol-8,9-epoxide, the BP 9,10-diol-7,8-epoxides 1 and 2, orthe BP 7,8- and 9,10-oxides (75, 296, 349, 359) (Table 16).These results indicate that a saturated benzo ring and a bay-region 9,10-epoxide group in BP are required for high muta

genic activity, but hydroxyl groups on the benzo ring are notrequired.

The mutagenicities of bay-region and non-bay-region tetra-

hydroepoxides of BP, BA, chrysene, and phenanthrene werecompared to the AE Â¡,.1,,,//â€¢;values which are calculated indices

for the ease of carbocation formation at the benzylic carbonatom. It can be seen in Table 17 that, for each of the 4 pairs oftetrahydroepoxides that we tested, the bay-region tetrahydro-

DECEMBER 1982 4895



A. H. Conney

Table 16Mutagenicity of benzo-ring epoxides and arene oxides of BP (75, 296, 349, 359)

Relative mutagenic activity

Compound

S. typhimurium

Chinese hamsterStrain TA 98 Strain TA 100 V-79 cells

BP 7.8-diol-9.10-epoxide 1 100 100 40

BP 7,8-diol-9,10-epoxide 2 35 65 100

BP 7,10-diol-8,9-epoxide 0.4

BP 9,10-diol-7,8-epoxide 1 0.4

tBP 9,10-diol-7,8-epoxide 2 0.2

BPHÂ«-9,10-epoxide 95 90 40

BP Hâ€ž-7.8-epoxide 10 0.2

BP9,10-oxide 0.6

BP 7,8-oxide 0.6

epoxide has higher mutagenicity and a higher AEdeioc//Jvaluethan does the corresponding non-bay-region tetrahydroepox-

ide (343, 346, 352, 359). Recent studies have also indicatedsimilar results for the heterocyclic hydrocarbon benz(c)acridinewhere the bay-region tetrahydro-1,2-epoxide is more mutagenic than the non-bay-region tetrahydro-3,4-epoxide (342).

Proximate and Ultimate Carcinogenic Metabolites of BA

Studies with BA were initiated as an early test of the bayregion theory. Even though BA is only a weak carcinogen,perturbational molecular orbital calculations indicated an unusual ease of carbocation formation at the bay-region 1-position for the BA 3,4-diol-1,2-epoxides when compared withother BA diol-epoxides (149). Because of these considerations,Dr. Jerina and our laboratory predicted that BA 3,4-dihydrodiol

would be a prime candidate for a proximate carcinogenicmetabolite of BA and that the diastereomeric BA 3,4-diol-1,2-

epoxides 1 and 2 (epoxide oxygen cis and frans to the benzylic

hydroxyl group, respectively) would be prime candidates forultimate carcinogenic metabolites of BA. The expected pathway for the metabolism of BA to its bay-region diol-epoxides is

shown in Chart 24. An examination of the metabolism of BA byrat liver microsomes or by a combination of a purified cyto-chrome P-450-dependent reconstituted monooxygenase sys

tem and purified epoxide hydrolase revealed that BA wasindeed metabolized to BA 3,4-dihydrodiol, but the amount ofthis dihydrodiol formed relative to the 5,6- and 8,9-dihydrodiols

was very low (293, 302). The predominant enantiomer of BA3,4-dihydrodiol that was formed metabolically had (-H3fl,4fi]

absolute stereochemistry (294), and studies on the furthermetabolism of the (-)-[3f?,4R]-enantiomer of BA 3,4-dihydro

diol by rat liver microsomes revealed the stereoselective formation of BA 3,4-diol-1,2-epoxide 2 (292).

We evaluated the metabolic activation to mutagens of all 5metabolically possible frans-dihydrodiols of BA, and the prediction that BA 3,4-dihydrodiol would be metabolized to muta-





Table 17Mutagenic activity and Ã E.,.., ,'/i values for bay-region and non-bay-region tetrahydroepoxides (343, 346, 352, 359)

Relative mutagenic activity

Compound

S. typhimurium

Strain Strain Chinese ham-TA98 TA100 ster V-79 cells

3 BP HÂ«-7,8-epoxide 0.488 0.4

876

BPH.-9,10-epoxide

4 BA H4-3,4-epoxide

BA H.-1,2-epoxide

0.794 100 100 100

0.628 25 20

0.766 100 100 100

7 6

s o

* Chrysene H4-1,2-epoxide 0.526 6 19

Chrysene H4-3,4-epoxide 0.640 100 100

Phenanthrene H4-1,2-epoxide 0.540 8 12

100

Phenanthrene H4-3,4-epoxide 0.658 100 100 100

gens more extensively than BA or the 1,2-, 5,6-, 8,9-, and10,11-dihydrodiols of BA was confirmed (353). BA 3,4-dihy-drodiol was metabolized by a highly purified cytochrome P-450-dependent monooxygenase system to products which

were at least 10 times more mutagenic to S. typhimurium strainTA 100 than were the metabolites formed from BA or the other4 metabolically possible frans-dihydrodiols of BA (Chart 25).

An examination of the mutagenic and cytotoxic activities ofseveral diol-epoxides of BA (see Table 18 for structures) revealed that the bay-region BA 3,4-diol-l ,2-epoxides 1 and 2were many times more mutagenic than were the BA 8,9-diol-10,11-epoxides 1 and 2 or the BA 10,11-diol-8,9-epoxides 1and 2 (343) (Table 18). It was of interest that 1,2-epoxy-1,2,3,4-tetrahydro-BA (BA H4-1,2-epoxide; see Table 17 for

structure) had somewhat higher mutagenic activity than did theBA 3,4-diol-1,2-epoxides, indicating that hydroxyl groups in

the benzo ring are not required for high biological activity(343).

In accord with observations on the mutagenic activity of theBA derivatives, we found that BA 3,4-dihydrodiol was at least10 times more tumorigenic than BA or the 1,2-, 5,6-, 8,9- or10,11-dihydrodiols of BA when tested as a tumor initiator on

mouse skin (Table 19) (351). In addition, the injection of 2800nmol of BA 3,4-dihydrodiol into newborn mice during the first

15 days of life caused at least 30 times more pulmonary tumorsthan did an equimolar dose of BA or the other metabolicallypossible BA dihydrodiols, and BA 3,4-dihydrodiol also hadexceptionally high activity in causing malignant lymphomas(Table 19) (335). Examination of the tumorigenicity of the bay-region BA 3,4-diol-1,2-epoxides as initiators of skin tumors inmice (191) revealed that BA 3,4-diol-1,2-epoxide 1 and BA3,4-diol-1,2-epoxide 2 were many times more tumorigenic thanBA and that BA 3,4-diol-1,2-epoxide 2 was somewhat moretumorigenic than BA 3,4-dihydrodiol (Table 20). Injection of280 nmol of these compounds into newborn mice during theirfirst 15 days of life indicated that BA 3,4-diol-1,2-epoxide 2

DECEMBER 1982 4897



A. H. Conney

OÃ›OBA 3,4-DIOL-1,2-EPOXIDE-l

Bay region : P-4SO

Chart 24. Metabolism of BA to a BA 3,4-dioH,2-epoxide. Although BA 3,4-diol-1,2-epoxide 2 has been identified as a metaboliteof BA 3,4-dihydrodiol. the metabolic formation 10of BA 3.4-diol-1,2-epoxide 1 has not yet beendemonstrated.

000BA 3.4-OXIDE

E POXlDE

8A 3,4-DIHYDRODIOL

P-450

BA 3.4-DIHYDROOIOL

â€”¿� U Ã–MBA 10, M-OIHYDRODIOL

Â«BA5.6-DIHYORODIOL* BA 1,2-DIHYDRODIOL

-o BA 8,9-DIHYDROOIOL-OBA

20 40

CYTOCHROME

60 80P-450 (PMOL)

Chart 25. Metabolism of BA and its dihydrodiols to mutagens by a purifiedcytochrome P-450c-dependent monooxygenase system from rat liver micro-somes (353).

was at least 30-fold more active than BA 3,4-dihydrodiol andat least 60-fold more active than BA in causing lung tumorswhereas BA 3,4-diol-1,2-epoxide 1 was less than one-tenth asactive as its diastereomer (Table 20) (330). High tumor-initiating activity of BA 3,4-dihydrodiol and BA 3,4-diol-1,2-epoxide

2 on mouse skin was also reported by Slaga and his associates,and these investigators found that BA 1,2-diol-3,4-epoxide 2,BA 10,11-diol-8,9-epoxide 2, and the BA 8,9-diol-10,11-epox-

ides 1 and 2 had very low or no tumorigenic activity (284). It isof interest that, although evidence has been presented that BA8,9-diol-10,11-epoxide 2 and BA 3,4-diol-1,2-epoxide 2 are

both covalently bound to DMA after application of BA to mouseskin (90), only the latter diol-epoxide is highly tumorigenic. The

results of the tumorigenicity studies described here, coupledwith the observation that small amounts of BA 3,4-dihydrodiol

OUOÂ»OH

BA 3.4-DIOL-1.2-EPOXIDE-2

Table 18Mutagenicity of BA diol-epoxides (76, 343)

Mutation frequency

Compound

Chinese hamsterV-79 cells (8-

S. typhimurium azaguanine-re-strain TA 100 sistant colonies/

(histidine revert- nmol/105 surviv-

ants/nmol/plate) ing cells)

BA 3,4-diol-1,2-epoxide 2


1650

850

16

50

BA 8,9-diol-10.1 1-epoxide 2


OH

50

8.5

10.9

0.06

0.10

0.03

0.21


and BA 3,4-diol-1,2-epoxide 2 are formed metabolically fromBA by liver microsomes (292-294), indicate that BA 3,4-dihy

drodiol is a proximate carcinogenic metabolite of BA and that





Table 19

Tumorigenicity of BA dihydrodiols on mouse skin and in newborn mice (335, 351)

Skin study Newborn study

% of mice% of mice with malig- % of mice

with skin tu- Skin tumors/ nant lym- with lung tu- Lung tumors/

Compound administered mors mouse phoma mors mouse

Control

23

0.03

0.30 37

0.05

1.59

Â°-07 17 0.17

BA 1,2-dihydrodiol

80 4.80 96 54.3

20 0.20

OH

BA 5.6-dihydrodiol

O 17 0-21 11 0.11

14 0.14 0.03

BA 10.11-dihydrodiol

* Mice received a single topical application of 2 /imol of compound followed 18 days later by twice-

weekly applications of 16 nmol of 12-O-tetradecanoylphorbol-l 3-acetate for 20 weeks.b Mice were given i.p. injections of 0.4, 0.8, and 1.6 /Â»molof compound on the first, 8th, and 15th days

of life, respectively. The animals were killed at 22 weeks of age.

BA 3,4-diol-1,2-epoxide 2 is an ultimate carcinogenic metabolite of BA. It is of interest that BA 3,4-diol-1,2-epoxide 1 isalso more tumorigenic than BA, but this diol-epoxide has not

yet been identified as a metabolite of BA. Metabolism studiessuggest that the low carcinogenic activity of BA is caused atleast in part from a low rate of conversion of BA to BA 3,4-dihydrodiol relative to the formation of other dihydrodiols of

BA. Only 1 to 4% of the total metabolites of BA formed by ratliver microsomes can be accounted for as its 3,4-dihydrodiolwith a bay-region double bond (293). Studies on the tumori-genicity of the (+)- and (-)-enantiomers of BA 3,4-dihydrodiolrevealed that the (-)-{3fl,4fl]-enantiomer, which is the meta-

bolically predominant enantiomer (292), is much more tumorigenic than the (+H3S,4S]-enantiomer on mouse skin and in

DECEMBER 1982 4899



A. H. Conney

Table 20Tumorigenicity of bay-region diol-epoxides of BA on mouse skin and in the newborn mouse (191, 330)

Skin study* Newborn study0

Compound administered

% of mice % of micewith skin tu- Skin tumors/ with lung tu- Lung tumors/

mors mouse mors mouse

Control 0.03

0.07

13

16

0.15

0.22


OH

BA 3,4-diol-l ,2-epoxide 2

45

43

70

1.30

0.60

1.90

35

42

100

0.37

0.56

13.3

' Mice received a single topical application of 0.4 Â«molof compound followed 7 days later by twice-

weekly applications of 16 nmol of 12-O-tetradecanoylphorbol-13-acetate for 20 weeks." Mice were given i.p. injections of 40. 80, and 160 nmol of compound on the first, 8th, and 15th days

of life, respectively. The animals were killed at 26 weeks of age.

newborn mice (191, 330). Studies are now in progress todetermine the tumorigenicity of the 4 optically active isomersderived from the diastereomeric BA 3,4-diol-1,2-epoxides.

Proximate and Ultimate Carcinogenic Metabolites of Chrysene

Chrysene is a weak carcinogen with 2 equivalent bay regions(Table 21 ). The bay region theory predicts that a chrysene 1,2-diol-3,4-epoxide is an ultimate carcinogenic metabolite of chrysene and that chrysene 1,2-dihydrodiol is a proximate carci

nogenic metabolite. Chrysene is metabolized predominantly toits 1,2- and 3,4-dihydrodiols (213, 237). Liver microsomesfrom MC-treated rats produced the (-H1 fÃ®,2fÃ®]-enantiomerofchrysene 1,2-dihydrodiol (bay-region diol-epoxide precursor)

with 80% enantiomeric purity (237). However, when liver microsomes from phenobarbital-treated rats were used, the chrysene 1,2-dihydrodiol that was formed was only 10% enantio-merically pure (237). Metabolism of the metabolically formed(â€”H1".2ft}-enantiomer of chrysene 1,2-dihydrodiol by livermicrosomes from MC-treated rats resulted in the predominantformation of a bay-region 1,2-diol-3,4-epoxide in which theoxirane ring and the hydroxyl group are frans (Isomer 2) (237).

Metabolic activation of chrysene 1,2-dihydrodiol by livermicrosomes or a purified cytochrome P-450-dependent mono-oxygenase system resulted in the formation of metabolites thatwere at least 20 times more mutagenic to strain TA 100 of S.typhimurium than were the metabolites formed from chryseneor chrysene 3,4- or 5,6-dihydrodiol (355). When the doublebond at the bay-region 3,4-position of chrysene 1,2-dihydrodiol

was saturated, the resulting tetrahydrodiol (chrysene H4-1,2-

diol) could not be metabolized to mutagens, suggesting that abay-region chrysene 1,2-diol-3,4-epoxide was the active mutagenic metabolite formed from chrysene 1,2-dihydrodiol(355). Additional studies revealed that the 1,2-diol-3,4-epox-ides of chrysene which have a bay-region epoxide either cis

(Isomer 1) or frans (Isomer 2) to the benzylic hydroxyl grouphave high mutagenic activity in bacterial and mammalian cells(346).

Studies on the tumorigenicity of chrysene and its metabolically possible frans-dihydrodiols on mouse skin revealed thatchrysene 1,2-dihydrodiol has higher tumor-initiating activitythan chrysene (200, 283) whereas the 3,4- and 5,6-dihydro-

diols of chrysene have no appreciable tumorigenic activity(Table 21) (200). frans-1,2-DihydroxyÂ«.t.2,3,4-tetrahydrochry-sene, which has a saturated bay-region 3,4-bond, has lessthan 25% of the tumorigenic activity of chrysene 1,2-dihydro

diol (200). Tumorigenicity studies in the newborn mouse indicated that chrysene 1,2-dihydrodiol and chrysene 1,2-diol-3,4-epoxide 2 are at least 10- and 50-fold more active, respectively,than chrysene in causing pulmonary tumors (Table 21), andchrysene 1,2-diol-3,4-epoxide 1 has little or no tumorigenicactivity in this test system (38). Recent studies on the tumorigenic activities of optically active metabolites of chrysene haveshown that the metabolically formed (-)-chrysene 1,2-dihydrodiol and (+)-chrysene 1,2-diol-3,4-epoxide 2 are more tumor

igenic on mouse skin and in the newborn mouse than theirmirror image enantiomers (52). The results of these studies





Table 21

Tumorigenicity of chrysene and its derivatives on mouse skin and in newborn mice (38, 200, 346)Skin study* Newborn study*


%of% of % of male

mice with Skin tu- mice with mice with Liver tu-skin tumors/ lung tu- Lung tu- liver tumors/male

mors mouse mors mors/mouse mors mouse

Control

Chrysene

52

79

0.07

1.45

3.38

15

17

73

Chrysene 1,2-dihydrodiol


10 0.10 13

0.07 23


Chrysene H4-3,4-epoxide

77

Chrysene 1,2-diol-3,4-epoxide 1OH

1.50 71

29

98

0.17

0.34 25 0.42

2.07 46 0.67

0.18

0.27 0 0

1.24 38 0.58

0.34 8 0.08

15.91 39 0.87Chrysene 1,2-diol-3,4-epox>de 2

Mice received a single topical application of 4 //mol of compound except that animals treated with chrysene H4-3,4-epoxide received 2 pmol of compound in a separate experiment. Seven days later, the mice were treated with 16nmol of 12-O-tetradecanoylphorbol-13-acetate twice weekly for 25 weeks.

" Mice were given i.p. injections of 0.2, 0.4, and 0.8 /imol of compound on the first, 8th, and 15th days of life,

respectively. The animals were killed at 38 to 42 weeks of age.

indicate that the (-H1fl-2fl]-enantiomer of chrysene 1,2-di-hydrodiol and the (+)-enantiomer of chrysene 1,2-diol-3,4-epoxide 2 are proximate and ultimate carcinogenic metabolitesof chrysene.

Proximate and Ultimate Carcinogenic Metabolites of DBA

DBA is a strong carcinogen with 2 equivalent bay regions(Table 22). In contrast to the weak carcinogen BA which ismetabolized only slightly to its 3,4-dihydrodiol, DBA is extensively metabolized to its DBA 3,4-dihydrodiol with a doublebond in the bay-region 1,2-position, and significant amounts ofthe 1,2- and 5,6-dihydrodiols are also formed (236, 275). Thebay region theory predicts that DBA 3,4-dihydrodiol is a proximate carcinogenic metabolite of DBA and that one or more ofthe DBA 3,4-diol-1,2-epoxides are ultimate carcinogenic metabolites of DBA. Metabolic activation of DBA and the 1,2-,

3,4- and 5,6-dihydrodiols of DBA by liver microsomes or by ahighly purified cytochrome P-450-dependent monooxygenasesystem revealed that DBA 3,4-dihydrodiol was activated toproducts that were more mutagenic to strain TA 100 of S.typhimurium than were the metabolites of DBA or the metabolites of the 1,2- or 5,6-dihydrodiols of DBA (357). Saturation ofthe bay-region double bond at the 1,2-position of DBA 3,4-dihydrodiol produced a tetrahydrodiol (DBA Hâ€ž-3,4-diol)whichwas poorly activated by microsomes or the purified monooxygenase system (357). The high mutagenic activity of DBA 3,4-dihydrodiol after metabolism and the importance of the doublebond at the bay-region 1,2-position of the dihydrodiol stronglysuggest that a bay-region 3,4-diol-1,2-epoxide is an ultimate

mutagenic metabolite of DBA.Examination of the tumorigenicity of the 1,2-, 3,4-, and 5,6-

dihydrodiols of DBA revealed that only the 3,4-dihydrodiol of

DBA had significant tumorigenic activity on mouse skin and in

DECEMBER 1982 4901



A. H. Conney

Table 22

Tumorigenicity of DBA dihydrodiols on mouse skin and in newborn mice (40)Skin study" Newborn study*

Compound administeredDose

(nmol)

%ofmice withskin tu

mors

Skin tumors/mouse

Dose(nmol)

% of micewith lungtumors

%ofmale

Lung tu- mice withmors/ liver tu-mouse mors

Control

10160

10160

DBA 1,2-dihydrodiol

OH

DBA 3,4-dihydrodiol

DBA 5,6-dihydrodiol

â€ž¿�â€¢OH

10160

10160

160

10

2169

1418

3157

143

29

0.10

0.362.14

70420

0.140.18

0.691.52

0.170.03

0.68

70420

70420

70420

12

88100

1118

8399

0.14

3.6146.90

0.110.21

1.9928.06

0.080.07

DBA H4-3,4-diol

" Mice received a single topical application of compound followed 7 days later by twice-weekly applications of 16nmol of l2-O-tetradecanoylphorbol-13-acetate for 25 weeks.

6 Mice were given i.p. injections of 1/7, 2/7, and 4/7 of the total dose of compound on the first, 8th, and 15th days

of life, respectively. The animals were killed at 25 to 29 weeks of age.

the newborn mouse (40) (Table 22). Although DBA 3,4-dihy

drodiol was somewhat less active than DBA in causing skinand pulmonary tumors, this dihydrodiol was uniquely active incausing liver tumors (Table 22). DBA H4-3,4-diol, with thesaturated bay-region double bond, was considerably less tu-morigenic than DBA 3,4-dihydrodiol on mouse skin. The hightumor-initiating activity of DBA 3,4-dihydrodiol on mouse skin

was also observed by Slaga and his associates, but theseinvestigators indicated that DBA 3,4-diol-1,2-epoxide 2 had

little or no tumorigenicity in this animal model (283). AlthoughDBA 3,4-diol-1,2-epoxide 2 was reported to be inactive as a

tumor initiator on mouse skin, the overall results of studies withDBA and its derivatives suggest that DBA 3,4-dihydrodiol andone or more of the bay-region DBA 3,4-diol-1,2-epoxides are

proximate and ultimate mutagenic and carcinogenic metabolites of DBA.

Proximate and Ultimate Carcinogenic Metabolites of B(c)Ph

B(c)Ph is a weak carcinogen with a bay region between 2angular benzo rings (Table 23). The bay region theory predicts

that a B(c)Ph 3,4-diol-1,2-epoxide, if formed metabolically, is

a prime candidate as an ultimate carcinogenic metabolite ofthe parent hydrocarbon. We found that B(c)Ph 3,4-dihydrodiol,the precursor of the bay-region diol-epoxides of B(c)Ph, was

metabolized to mutagens by liver microsomes or a purifiedcytochrome P-450-dependent monooxygenase system sever-alfold more effectively than was B(c)Ph or its 1,2- and 5,6-dihydrodiols (344). When the bay-region double bond at the1,2-position of B(c)Ph 3,4-dihydrodiol was saturated with hy

drogen, the resulting tetrahydrodiol could not be metabolicallyactivated to mutagens. These data suggest that one or more ofthe bay-region B(c)Ph 3,4-diol-1,2-epoxides is an ultimate mu

tagenic metabolite of B(c)Ph. In accord with these observations, we found that B(c)Ph 3,4-diol-1,2-epoxide 1, in whichthe bay-region epoxide oxygen and the benzylic hydroxylgroup are c/s and B(c)Ph 3,4-diol-1,2-epoxide 2, in which thebay-region epoxide oxygen and the benzylic hydroxyl group

are frans, have strong mutagenic activity in bacterial andmammalian cells (344).

Studies on the tumor-initiating activity of B(c)Ph and its





metabolically possible dihydrodiols and bay-region diol-epox-ides on mouse skin revealed that B(c)Ph 3,4-dihydrodiol wasmore tumorigenic than B(c)Ph whereas the 1,2- and the 5,6-

dihydrodiols of B(c)Ph were inactive (197) (Table 23). Whenthe analog of B(c)Ph 3,4-dihydrodiol with a saturated 1,2-bondin the bay region [B(c)Ph H4-3,4-diol] was tested, it was inactive(Table 23), suggesting that B(c)Ph 3,4-dihydrodiol requiredmetabolism at the bay-region 1,2-position for tumorigenicity(197). The data in Table 23 also demonstrate that the diaster-eomeric bay-region 3,4-diol-1,2-epoxides of B(c)Ph are at least50- to 100-fold more active than is B(c)Ph as tumor initiatorson mouse skin (150, 197). An interesting aspect of this studyis the extraordinarily high tumorigenic activity on mouse skindisplayed by both diastereomers. The B(c)Ph 3,4-dioM ,2-epoxides are much more tumorigenic than BA 3,4-diol-1,2-

epoxide 2 (Table 20), which had previously been the mostactive diol-epoxide tested on mouse skin (191, 284). It is ofconsiderable interest that the highly tumorigenic B(c)Ph bay-region diol-epoxides have relatively low chemical reactivity asmeasured by either their spontaneous or their acid-catalyzed

solvolysis in water (150). Further studies are needed to providean explanation for the greater than expected tumorigenicity ofthe bay-region diol-epoxides of B(c)Ph.

Proximate and Ultimate Carcinogenic Metabolites of MC

MC is a strong carcinogen, and the structure of this compound is shown in Table 24. Studies on the metabolism of MCby purified cytochrome P-450 in the presence and absence ofpurified epoxide hydrolase failed to provide evidence for theformation of significant amounts of the 9,10-dihydrodiol with abay-region 7,8-double bond (290, 291). Since 2 major metabolites of MC are 1- and 2-hydroxy-MC, we studied the metab

olism of these alcohols to determine if they were converted tothe 9,10-dihydrodiols (290, 291). (Â±M-Hydroxy-MC was me

tabolized by rat liver microsomes and by a purified cytochromeP-450-dependent monooxygenase system in the presence ofepoxide hydrolase to a pair of diastereomeric irans-9,10-di-hydrodiols in which the 10-hydroxyl group is either c/s or fransto the 1-hydroxyl group (290, 291). Upon metabolic activationby monooxygenases, we found that one of these 9,10-dihydro-

diols was among the most mutagenic MC derivatives testedtoward both bacterial and mammalian cells (347).

Studies on the tumorigenic activity of the two 9,10-dihydrodiols of 1-hydroxy-MC revealed that one of these compoundshad about the same activity as did MC and was considerablymore active than was 1-hydroxy-MC in initiating tumors onmouse skin (186). The metabolically predominant isomer of 1-hydroxy-MC 9,10-dihydrodiol was more active than MC or 1-hydroxy-MC in causing pulmonary and liver tumors in thenewborn mouse (186) (Table 24). In these studies, 1-hydroxy-MC 9,10-dihydrodiol was about 10-fold more active than MCin causing liver tumors in male mice, and 1-hydroxy-MC wasinactive (Table 24). These results indicate that 1-hydroxy-MC9,10-dihydrodiol with an adjacent bay-region double bond is a

proximate carcinogenic metabolite of MC. In our studies (186),as well as those by others (46, 274), 2-hydroxy-MC and 2-keto-MC had strong tumorigenic activity. It would be of interestto determine the tumorigenic activity of the 9,10-dihydrodiolsof 2-hydroxy-MC and 2-keto-MC.

Studies by Malaveille ef al. (214) have shown that MC 9,10-

Table 23Tumor-initiating activity of B(c)Ph and its derivatives on mouse skin (150, 197)

Mice received a single topical application of compound followed 7 days laterby twice weekly applications of 16 nmol of 12-O-tetradecanoylphorbol-13-ace-tate for 20 weeks.


% of micewith tu- Tumors/

Dose (/imol) mors mouse

Control

B(c)Ph 1,2-dihydrodiol

...OH

B(c)Ph 3,4-dihydrodiol

B(c)Ph 5.6-dihydrodiol

.Â»OH

B(c)Ph 3,4-diol-1,2-epoxide 1

0.42.0

0.42.0

0.42.0

0.42.0

0.42.0

0.0250.4

0.0250.4

1738

2847

310

5387

5780

0.170.59

0.030.03

0.411.07

0.070.03

0.030.13

1.636.47

1.537.13

B(c)Ph 3,4-diol-1,2-epoxide 2

dihydrodiol is metabolically activated to a potent mutagen, andstudies by Chouroulinkov et al. (57) have shown that the 9,10-

dihydrodiol of MC was more active as a tumor initiator thanwere the 4,5-, 7,8-, or 11,12-dihydrodiols of MC. In this laterinvestigation, MC 9,10-dihydrodiol was somewhat less tumor

igenic than MC. Additional evidence that MC is activated viathe formation of bay-region diol-epoxides was obtained fromstudies on the nature of the DNA-bound adducts of MC (91,

168, 169, 311). These studies indicated that several of thecovalently bound adducts were saturated in positions 7, 8, 9,and 10 of the hydrocarbon which is consistent with the formation and covalent binding of 9,10-diol-7,8-epoxides of MC.In further studies, it was concluded that one of the DNA-boundadducts is a 1- or 2-hydroxy derivative of MC saturated in

positions 7, 8, 9, and 10 of the molecule (169). The availabledata indicate that one or more of the MC 9,10-diol-7,8-epox-ides and/or their 1- and 2-hydroxy derivatives are ultimate

carcinogenic metabolites of MC.

DECEMBER 1982 4903



A. H. Conney

Table 24

Tumorigenicity of MC and its derivatives in newborn mice (186)

Mice were given i.p. injections of 7, 14, and 28 nmol of compound on the first, 8th, and 15th days of life,respectively. The animals were killed at 36 to 39 weeks of age.

Pulmonary tumors Hepatic tumors


% of male% of mice Tumors/ mice with Tumors/male

with tumors mouse tumors mouse

11

82

0.11

1.77

6

19

0.06

0.43

0.08

26 0.29

OH1-Hydroxy-MC

1-Hydroxy-MC 9,10-dihydrodiol

94 4.47 81 4.33

86 2.08 14 0.14

67 1.03 35 0.35

HO

2-Hydroxy-MC

MC 11.1 2-0 xido

14

13

0.20

0.13 0.07

MC 11,12-dihydrodiol

Proximate and Ultimate Carcinogenic Metabolites of DB(a,h)Pand DB(a,i)P

DB(a,h)P and DB(a,i)P are hexacyclic aromatic hydrocarbonsthat have high tumorigenic activity in mice, and both of thesehydrocarbons have 2 bay regions. The structures of thesecompounds and some of their derivatives are given in Table25. Quantum mechanical calculations designed to predict the

ease of triol carbocation formation from diol-epoxides indicatedthat the bay-region diol-epoxides of DB(a,h)P and DB(a,i)P

should have very high chemical reactivity (149), and studies oftheir solvolysis have confirmed these predictions (345). Recentstudies by Hecht ef al. (125) have resulted in the identificationof DB(a,i)P 3,4-dihydrodiol as a metabolite of DB(a,i)P that isformed by rat liver.

Mutagenicity studies with several benzo-ring derivatives of





Table 25Tumorigenicity of DB(a,h)P, DB(a,i)P, and their derivatives on mouse skin and in the newborn mouse (51)

Skin study Newborn study



% of mice % of mice % of malewith tu- Tumors/ with tu- Tumors/ mice with Tumors/

mors mouse mors mouse tumors male mouse

DB(a,ri)P

DB(a.h)P 1,2-dihydrodiol

DB(a,r))PH4-1,2-diol

DB<a,r>)P1,2,-dtol-3,4-epoxide 2

DB(a,/)P

DB(a,/)P3,4-dihydrodiol

DB(a,/)PH4-3,4-diol

DB(a,/)P3,4-diol-1.2-epoxide2

2,10-Difluoro-DB(a,/)P

10

61

0.10 27

72 5.52

80 4.40

67 3.60

50 1.83

79 5.25

97

0.61

5.07 44

98 17.0

82

95

97 4.40

81 5.00 100 33.3

1.64

67 2.03

95

86

25

4.13

0.88

41 3.76

1.74 28

5.54 26

0.80

1.37

54 0.82

67 4.48

88 3.30

2.57 25

0.43

0.25

0.03

' Mice received a single topical application of 600 nmol of compound followed 7 days later by twice-weeklyapplications of 16 nmol of 12-O-tetradecanoylphorbol-13-acetate for 16 weeks.

" Mice were given i.p. injections of 12.5, 25, and 50 nmol of compound on the first, 8th, and 15th days of life,

respectively. The animals were killed at 49 to 54 weeks of age.

DB(a,h)P and DB(a,i)P showed that DB(a,h)P 1,2-dihydrodioland DB(a,i)P 3,4-dihydrodiol, the expected dihydrodiol precursors of the bay-region diol-epoxides, were metabolized tomutagenic products by a cytochrome P-450-dependent mono-

oxygenase system to a greater extent than their respectiveparent hydrocarbons, and the tetrahydrodiol derivatives with

no adjacent bay-region double bond were only poorly activated

to mutagenic metabolites (345). Additional studies have shownthat synthetic bay-region diol-epoxides of DB(a,h)P andDB(a,i)P, in which the benzylic hydroxyl group and the oxiranering are frans (Isomer 2), have high inherent mutagenic activityin bacterial and mammalian cells (345).

DECEMBER 1982 4905



|;!Â»'-

.

A. H. Conney

The results of tumorigenicity studies with DB(a,h)P, DB(a,i)P,and several of their derivatives are summarized in Table 25(51). DB(a,h)P 1,2-dihydrodiol and DB(a,i)P 3,4-dihydrodiol,the immediate metabolic precursors of the bay-region diol-epoxides, had tumor-initiating activity on mouse skin about

equal to their respective parent hydrocarbons (Table 25). Innewborn mice, these dihydrodiols were the most tumorigenicdibenzopyrene derivatives tested, and they had 3- to 8-fold

greater activity than their parent hydrocarbons in producingpulmonary tumors. The bay-region diol-epoxides DB(a,h)P 1,2-diol-3,4-epoxide 2 and DB(a,i)P 3,4-diol-1,2 epoxide 2 had

less tumorigenic activity than did their dihydrodiol precursors,both on mouse skin and in the newborn mouse. These bay-region diol-epoxides were also less active than their parent

hydrocarbons on mouse skin, and they had tumorigenic activityequal to or somewhat less than their parent hydrocarbons inthe newborn mouse. DB(a,h)P H4-1,2-diol and DB(a,i)P H4-3,4-

diol differ from their corresponding dihydrodiols in that theadjacent bay-region double bonds are saturated with hydrogen. As expected, these tetrahydrodiols had only 10 to 12% ofthe tumorigenic activity of the dihydrodiols for the lung ofnewborn mice, but both tetrahydrodiols had appreciable tumor-initiating activity on mouse skin, and DB(a,i)P H4-3,4-diol had

significant tumorigenic activity for the liver in newborn mice.Although these results suggest that non-bay-region metabolites

may be responsible for some of the tumorigenic effects ofDB(a,h)P and DB(a,i)P, these hydrocarbons are symmetricalmolecules with 2 equivalent bay regions, and the unexpectedlyhigh tumorigenic activity of the tetrahydrodiols on mouse skinand for the liver may be due to the formation of a diol-epoxide

in the other bay region of the molecule. Evidence in support ofthis concept comes from the complete lack of activity of 2,10-difluoro-DB(a,i)P as a tumor initiator on mouse skin and incausing pulmonary and liver tumors in the newborn mouse(Table 25). Low tumorigenicity of 2,10-difluoro-DB(a,i)P was

also observed by Hecht ef a/. (125) and by Boger ef al. (21 ).The difluoro derivative, which has fluorine in both benzo rings,undergoes little or no metabolism to dihydrodiols that areprecursors of bay-region diol-epoxides (125). The availabledata suggest that bay-region diol-epoxides are ultimate muta-

genie and carcinogenic metabolites of DB(a,h)P and DB(a,i)P.

Mutagenic and Carcinogenic Activities of Bay-Region Epoxidesand Diol-Epoxides of Phenanthrene and B(e)P

Phenanthrene, the simplest polycyclic aromatic hydrocarbonwith a bay region, is generally considered to have little or nocarcinogenic activity. The bay-region 3,4-epoxy-1,2,3,4-te-trahydrophenanthrene (phenanthrene H4-3,4-epoxide) is a

moderately potent mutagen in strains TA 98 and TA 100 of S.typhimurium and in Chinese hamster V-79 cells, but the dias-tereomeric bay-region 1,2-diol-3,4-epoxides 1 and 2 of phen

anthrene are considerably less mutagenic (346). The structures of these compounds are shown in Table 26.

Examination of the tumorigenic activity of several phenanthrene derivatives revealed that the metabolically possible1,2-, 3,4-, and 9,10-dihydrodiols of phenanthrene have little or

no activity as tumor initiators on mouse skin (346). Interestingly, phenanthrene H4-3,4-epoxide (bay-region tetrahydro-

epoxide) is weakly active as a tumor initiator on mouse skin(346) and in causing pulmonary and hepatic tumors wheninjected into newborn mice (Table 26) (38). Studies on the

Table 26

Tumorigenicity of phenanthrene and its derivatives in newborn mice (38)

Mice were given Â¡.p.injections of 0.2, 0.4. and 0.8 fimol of compound on thefirst, 8th, and 15th days of life, respectively. The animals were killed at 38 to 42weeks of age.


% of micewith tu- Tumors/

Compound administered mors mouse

% of malemice with Tumors/

tumors male mouse

Control 15

17

0.17

0.20

Boy region

Phenanthrene

45 0.74 15 0.19

PhenanthreneH<-3,4-epoxide

14 0.18

Phenanthrene1.2-diol-3.4-epoxide 1

16 0.16 0.09

Phenanthrene1,2,-diol-3,4-epoxide 2

tumorigenicity of the diastereomeric phenanthrene-1,2-diol-3,4-epoxides in the newborn mouse tumor model revealed thatboth of these bay-region diol-epoxides are inactive in causing

pulmonary tumors (38). The greater tumorigenic activity ofphenanthrene H4-3,4-epoxide relative to the bay-region diol-

epoxides is in accord with the relative mutagenic activities ofthese compounds in bacteria and in mammalian cells (346).

B(e)P, a symmetrical hydrocarbon with 2 identical bay regions, has little or no tumorigenic activity in the mouse. Thestructures of B(e)P and some of its possible proximate andultimate carcinogenic metabolites are shown in Table 27. Recent studies have shown that very little B(e)P is metabolized toB(e)P 9,10-dihydrodiol (expected precursor of the bay-regiondiol-epoxides) by cultured hamster embryo cells (211) and ratliver microsomes (212). In addition, little or no B(e)P 9,10-dihydrodiol is converted to the bay-region diol-epoxides by rat

liver microsomes (356). Studies on the tumorigenicity of B(e)Pand B(e)P 9,10-dihydrodiol revealed that these compoundshave no significant tumor-initiating activity on mouse skin and

fail to induce lung tumors in newborn mice (39). However,B(e)P 9,10-dihydrodiol did produce a significant incidence ofhepatic tumors when injected into newborn male mice, suggesting that some bay-region diol-epoxide may have been

formed (Table 27). Studies on the intrinsic mutagenicity of thebay-region diol-epoxides of B(e)P indicate that these com-





Table 27

Tumorigenicity of B(e)P and its derivatives in newborn mice (39, 50)

In Experiment 1, the mice were given i.p. injections of 0.4. 0.8, and 1.6 (imol of compound on the first, 8th, and 15thdays of life, respectively. The animals were killed at 62 to 66 weeks of age. In Experiment 2, the mice were given i.p.injections of 0.1, 0.2, and 0.4 /imo! of compound on the first, 8th, and 15th days of life, respectively. The animals werekilled at 39 to 43 weeks of age.


Experiment Compound administered

% of male% of mice Tumors/ mice with tu- Tumors/male

with tumors mouse mors mouse

Control

Boy rtglon

B(e)P

B(e)P 4,5-dihydrodiol

HO'

B(e)P9,10-dihydrodiol

Control

B(e)P

B(e)P 9,10-diol-11,12-epoxide 1

B(e)P 9.10-diol-11,12-epoxide 2

48

41

0.68

0.56

11

21

0.11

0.21

51 0.88 17 0.22

35

18

18

0.63

0.28

0.21

61 0.82

0.06

35 0.44 0.05

25 0.39 21 0.21

pounds do have significant mutagenic activity, but they areonly 1 to 10% as active as the corresponding bay-region 7,8-diol-9,10-epoxides of BP in bacterial and mammalian cells(341). When the diastereomeric bay-region diol-epoxides of

B(e)P were injected into newborn mice, it was found that B(e)P9,10-diol-11,12-epoxide 1 produced a small increase in the

incidence of pulmonary tumors when compared to controls orto B(e)P and that B(e)P 9,10-diol-11,12-epoxide 2 produced

an increase in the number of hepatic tumors in male mice (50)(Table 27). B(e)P and B(e)P 4,5-oxide (K-region oxide; data

not shown) were nontumorigenic in the newborn mouse tumormodel. The data described in Table 27 suggest that the bay-region 9,10-diol-11,12-epoxides of B(e)P are weakly active

ultimate carcinogens.

Other Hydrocarbons That Fit the Predictions of the Bay-Region

Theory

Our collaborative research with Dr. Jerina's laboratory that

is described above has demonstrated proximate and ultimatecarcinogens and mutagens for 10 different polycyclic aromatichydrocarbons. For each hydrocarbon, the proximate and ultimate carcinogens and mutagens that we identified were inagreement with the bay region theory. Further support for thebay region theory has come from studies with additional hydrocarbons in several other laboratories.

Examination of the fluorescent properties of DNA-boundadducts derived from 7-methylbenz(a)anthracene (303, 310)and 7,12-dimethylbenz(a)anthracene (20,100,139, 231.311 )

DECEMBER 1982 4907



A. H. Conney

provided evidence for the covalent binding of the angular benzoring of these hydrocarbons to DMA. Studies with the 3,4-dihydrodiols of 7-methylbenz(a)anthracene and 7,12-dimeth-ylbenz(a)anthracene (expected precursors of the bay-regiondiol-epoxides) indicated that both of these dihydrodiols undergo metabolic activation to mutagens and cause malignanttransformations to a greater extent than do their parent hydrocarbons (215-218). Tumorigenicity studies on mouseskin revealed that the 3,4-dihydrodiols of 7-methylbenz(a)-anthracene, 8-methylbenz(a)anthracene, and 7,12-dimethyl-

benz(a)anthracene were more active tumor initiators thantheir parent hydrocarbons (56, 282, 332, 333). In addition, the3,4-dihydrodiols of 7,12-dimethylbenz(a)anthracene and 7-hy-droxymethyl-12-methylbenz(a)anthracene caused more lungand liver tumors than did 7,12-dimethylbenz(a)anthracene

when injected into newborn mice (334). HydrogÃ©nation of thebay-region 1,2-double bond of 7,12-dimethylbenz(a)anthra-cene resulted in 1,2,3,4-tetrahydro-7,12-dimethylbenz(a)an-thracene which had only one-tenth of the tumor-initiating activ

ity of the parent hydrocarbon on mouse skin (99). Although1,2,3,4-tetrahydro-7,12-dimethylbenz(a)anthracene was amuch less effective tumor initiator than the parent hydrocarbon,the tetrahydro derivative still possessed strong tumor-initiatingactivity, possibly by metabolic activation at non-bay-region

positions.Hecht and his associates have provided evidence for the

metabolic activation of 5-methylchrysene to bay-region 1,2-diol-3,4-epoxides (124, 126-128, 223). These studies indi

cated that the major DNA adduct observed after application of5-methylchrysene to mouse skin was derived from a bay-region1,2-diol-3,4-epoxide. In addition, 5-methylchrysene-1,2-dihy-drodiol (precursor of bay-region diol-epoxides) was metabol-ically activated to mutagens to a greater extent than was 5-methylchrysene, and 5-methylchrysene-1,2-dihydrodiol wasmore tumorigenic on mouse skin than was 5-methylchrysene

or its other metabolically possible dihydrodiols. Substitutionwith fluorine at positions 1, 3, or 12 of 5-methylchryseneinhibited the formation of the 1,2-dihydrodiol and inhibited

carcinogenicity.Evidence has been obtained for the metabolism of the strong

carcinogen 15,16-dihydro-11-methylcyclopenta(a)phenan-thren-17-one to a frans-3,4-diol-1,2-epoxide (bay-region diol-

epoxide) that is bound covalently to DNA (88). In addition, thefrans-3,4-dihydrodiol of 15,16-dihydro-11-methylcyclopen-ta(a)phenanthren-17-one (bay-region diol-epoxide precursor)

is metabolized to mutagens to a greater extent than is theparent compound (88), and this dihydrodiol is also more activethan the parent compound as a tumor initiator on mouse skin(87).

Studies with the carcinogenic 1,4-dimethylphenanthrenehave provided evidence that its 7,8-dihydrodiol (precursor ofthe bay-region diol-epoxide) is a major metabolite and that thisdihydrodiol is a proximate mutagenic metabolite of 1,4-dimeth

ylphenanthrene (183).Recent studies with several benzofluoranthene derivatives

are in accord with the bay region theory (9, 182).Benzo(o)fluoranthene 9,10-dihydrodiol, which is the immediateprecursor of a bay-region diol-epoxide, was as active as

benzo(b)fluoranthene in initiating tumors on mouse skin.Benzo(y')fluoranthene 9,10-dihydrodiol, which is the expected

metabolic precursor of a diol-epoxide in a 4-sided pseudo-bay

region, had appreciable tumor-initiating activity but was lessactive than benzoO)fluoranthene. Benzo(/0fluoranthene 8,9-dihydrodiol cannot form a bay-region diol-epoxide, and this

compound was inactive as a tumor initiator on mouse skin.

Summary and Concluding Remarks

Multiple cytochrome P-450s with characteristic but overlap

ping substrate specificities metabolize polycyclic aromatic hydrocarbons and many other classes of precarcinogens to theirproximate and ultimate carcinogenic metabolites, and the sameenzymes also participate in the detoxification of these compounds. The selective regulation of the cytochrome P-450enzymes by genetic and environmental factors is an importantarea of cancer research since changes in the activity or amountof specific forms of cytochrome P-450 will alter both the extentof metabolism of a substrate and the relative yields of itsvarious metabolites. I have given several examples of specificityin the induction and activation of cytochrome P-450 isozymes,

and I have indicated how this can alter the action of drugs,chemical carcinogens, and other environmental pollutants aswell as the metabolism and action of endogenous substratessuch as steroid hormones, lipid-soluble vitamins, and prosta-

glandins. The ability of microsomal enzyme inducers to inhibitthe action of some chemical carcinogens by enhancing theirdetoxification was pointed out as well as certain problems withthis approach to cancer prevention. The toxicological significance of the induction of the cytochrome P-450 system inmammals and in other living organisms depends on the cytochrome P-450 isozymes that are induced and on the identity ofthe chemicals in the environment of the organism. Markedinterindividual differences occur for the metabolism of chemicals in human beings, and these differences are caused bycomplex interactions between genes and environment. Theingestion of drugs, the smoking of cigarettes, exposure tohalogenated hydrocarbon insecticides or other environmentalpollutants, and changes in the diet can all influence the metabolism of chemicals in humans. It is likely that these same factorsinfluence the metabolism and action of environmental carcinogens in humans.

Bay-region diol-epoxides have been identified as importantultimate carcinogenic metabolites for the polycyclic aromatichydrocarbon class of carcinogens. The marked selectivity ofcytochrome P-450 and epoxide hydrolase for the metabolismof polycyclic aromatic hydrocarbons to specific isomers of diol-

epoxides and the critical importance of stereochemical factorsfor the expression of biological activity have been particularlyinteresting aspects of our research. Studies on the biotransformation of BP and chrysene by liver microsomes from MC-treated rats or by purified cytochrome P-450c and purified

epoxide hydrolase have demonstrated the stereoselective metabolism of these hydrocarbons to [R,R]-enantiomers of trans-dihydrodiols that are precursors of bay-region diol-epoxides.In each case, the predominant [f?,R]-dihydrodiol that is formedmetabolically is considerably more carcinogenic than its mirrorimage enantiomer. These [fl,fl]-dihydrodiols undergo stereo-selective metabolism predominantly to highly tumorigenic bay-region diol-epoxides with the epoxide oxygen frans to thebenzylic hydroxyl group. Examination of the carcinogenicity ofthe 4 optically active bay-region diol-epoxides of BP and the 4optically active bay-region diol-epoxides of chrysene demon-




strated that for each series of compounds only the metabol-ically predominant (+)-diol-epoxide 2 isomer with the epoxideoxygen trans to the benzylic hydroxyl group has high tumori-

genie activity.Although the bay-region theory has been remarkably suc

cessful in predicting proximate and ultimate carcinogenic metabolites of more than a dozen polycyclic aromatic hydrocarbons, it is possible that metabolites at non-bay-region positions

will be found that can function as ultimate carcinogens. Itshould be noted that some carcinogenic hydrocarbons do notpossess a bay region (see Ref. 122), and further research isneeded to identify proximate and ultimate carcinogenic metabolites of these compounde. It is of interest that some polycyclicaromatic hydrocarbons with a methyl group in an angular benzoring have carcinogenic activity (see Ref. 122). Although thesecompounds may undergo metabolic activation at non-bay-re

gion positions, at least some of these compounds have thepotential for bay-region diol-epoxide formation because they

are metabolized to dihydrodiols that involve the substitutedcarbon atom (340).

Since each of the polycyclic aromatic hydrocarbons that Ihave discussed is metabolized to multiple products, there is aneed for studies to determine the carcinogenic activity ofmixtures of metabolites of the polycyclic hydrocarbons. It islikely that some of these metabolites interact with each otherto alter the carcinogenic response. It was pointed out in Table13 that the simultaneous administration of the (+)- and (â€”)-enantiomers of BP 7,8-oxide causes a synergistic tumorigenicresponse in mice. In addition, combinations of the (+)- and(-)-enantiomers of BP 4,5-oxide have a synergistic mutageniceffect in Chinese hamster V-79 cells (Table 9).

The identification of bay-region diol-epoxides as ultimate

carcinogenic metabolites of many polycyclic aromatic hydrocarbons should help in the search for naturally occurring andsynthetic chemicals that inactivate these ultimate carcinogens.Recent studies demonstrated that riboflavin 5'-phosphate,

flavin adenine dinucleotide, and several plant phenols (ellagicacid, chlorogenic acid, caffeic acid, and ferulic acid) are antagonists of the mutagenic activity of bay-region diol-epoxides

(350, 358). Studies on the mechanisms of these effects indicate that riboflavin 5'-phosphate functions as an acid catalyst

and enhances the conversion of BP 7,8-diol-9,10-epoxide 2 to

biologically inactive tetraols (358) whereas ellagic acid formscovalent adducts with the diol-epoxide (268). The identificationand use of naturally occurring and synthetic chemicals thatantagonize ultimate carcinogens are potentially important approaches to cancer prevention.

Acknowledgments

I dedicate this manuscript to my wife, Diana, and to my past and presentcolleagues. I am very fortunate in having a wonderful wife and a wonderful groupof associates with a deep commitment to biomedicai research.

I thank Maria Devenney and Linda Gregg for their excellent assistance withthis manuscript.

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45. Campbell, T. C. Nutrition and drug-metabolizing enzymes. Clin. Pharmacol.Ther., 22. 699-706, 1977.

46. Cavalieri, E.. Roth, R., Althoff, J. Grandjean. C., Patii. K., Marsh, S., andMcLaughlin, D. Carcinogenicity and metabolic profiles of 3-methylcholanthrene oxygenated derivatives at the 1 and 2 positions. Chem.-Biol. Interact., 22. 69-81. 1978.

47. Cerutti. P. A., Sessions. F., Marinaran, P. V.. and Lusby, A. Repair of DNAdamage induced by benzo(a)pyrene diol-epoxides I and II in human alveolartumor cells. Cancer Res., 38. 2118-2124. 1978.

48. Cha. Y-N., and Bueding. E. Effect of 2(3Mert-butyl-4-hydroxyanisole administration on the activities of several hepatic microsomal and cytoplasmicenzymes in mice. Biochem. Pharmacol.. 28. 1917-1921, 1979.

49. Cha, Y-N., Martz, F., and Bueding, E. Enhancement of liver microsomeepoxide hydratase activity in rodents by treatment with 2(3Wert-butyl-4-hydroxyanisole. Cancer Res., 38 4496-4498, 1978.

50. Chang, R. L., Levin. W.. Wood. A. W.. Lehr, R. E.. Kumar, S., Yagi. H..Jerina, D. M., and Conney, A. H. Tumorigenicity of the diastereomeric bay-region benzcKe)pyrene 9,10-diol-1 1,12-epoxides in newborn mice. CancerRes., 41. 915-918, 1981.

51. Chang. R. L.. Levin. W.. Wood. A. W.. Lehr, R. E., Kumar, S., Yagi, H.,

Jerina, D.M., and Conney, A. H. Tumorigenicity of bay-region diol-epoxidesand other benzo-ring derivatives of dibenzo(a,/))pyrene and dibenzo-(a,/)pyrene on mouse skin and in newborn mice. Cancer Res., 42: 25-29,1982.

52. Chang, R. L., Levin, W., Wood, A. W., Yagi, H., Tada, M., Vyas, K. P.,Jerina, D.M., and Conney, A. H. Tumorigenicity of enantiomers of chrysene1,2-dihydrodiol and of the diastereomeric bay-region chrysene 1,2-diol-3,4-epoxides on mouse skin and in newborn mice. Cancer Res., 43: inpress, 1983.

53. Chang, R. L., Wislocki, P. G., Kapitulnik, J., Wood, A. W., Levin, W., Yagi,H.. MÃ¤h,H. D., Jerina, D. M., and Conney, A. H. Carcinogenicity of 2-hydroxybenzo(a)pyrene and 6-hydroxybenzo(a)pyrene in newborn mice.Cancer Res., 39. 2660-2664, 1979.

54. Chang, R. L., Wood. A. W.. Levin, W., MÃ¤h.H. D., Thakker, D. R., Jerina,D. M., and Conney. A. H. Differences in mutagenicity and cytotoxicity of(+)- and (â€”)-benzo(a)pyrene 4.5-oxide: a synergistic interaction of anan-tiomers. Proc. Nati. Acad. Sei. U. S. A., 76. 4280-4284. 1979.

55. Chouroulinkov, !.. Gentil, A., Grover, P. L.. and Sims, P. Tumor-initiatingactivities on mouse skin of dihydrodiols derived from benzo[a]pyrene. Br.J. Cancer, 34: 523-532, 1976.

56. Chouroulinkov. !.. Gentil. A., Tierney. B.. Grover, P., and Sims, P. Themetabolic activation of 7-methylbenz(a)anthracene in mouse skin: hightumor-initiating activity of the 3,4-dihydrodiol. Cancer Lett., 3. 247-253.1977.

57. Chouroulinkov, I., Gentil, A., Tierney, B., Grover, P. L.. and Sims, P. Theinitiation of tumours on mouse skin by dihydrodiols derived from 7,12-dimethylbenz(a)anthracene and 3-methylcholanthrene. Int. J. Cancer, 24:455-460, 1979.

58. Colbert, R. A., Bresnick, E., Levin, W., Ryan, D. E., and Thomas, P. E.Synthesis of liver cytochrome P-450b in a cell-free protein synthesizingsystem. Biochem. Biophys. Res. Commun., 91: 886-891, 1979.

59. Conney, A. H. Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev., 19: 317-366, 1967.

60. Conney, A. H. Microsomes and drug oxidations: perspectives and challenges. In: M. J. Coon, A. H. Conney, R. W. Estabrook, H. V. Gelboin, J. R.Gillette, and P. J. O'Brien (eds.), Microsomes, Drug Oxidations and Chem

ical Carcinogenesis, Vol. 2, pp. 1103-1118. New York: Academic Press,Inc., 1980.

61. Conney. A. H., Brown, R. R.. Miller, J. A., and Miller, E. C. The metabolismof methylated aminoazo dyes. VI. Intracellular distribution and propertiesof the demethylase system. Cancer Res., 77. 628-633, 1957.

62. Conney, A. H., and Burns, J. J. Stimulatory effect of foreign compounds onascorbic acid biosynthesis and on drug-metabolizing enzymes. Nature(Lond.), 784. 363-364. 1959.

63. Conney, A. H., and Burns, J. J. Physiological disposition and metabolicfate of chlorzoxazone (Paraflex) in man. J. Pharmacol. Exp. Ther., 728.340-343, 1960.

64. Conney. A. H.. and Burns, J. J. Metabolic interactions among environmentalchemicals and drugs. Science (Wash. D. C.), 778. 576-586, 1972.

65. Conney, A. H., Davison, C., Gastel, R., and Burns, J. J. Adaptive increasesin drug-metabolizing enzymes induced by phÃ©nobarbital and other drugs.J. Pharmacol. Exp. Ther., 730: 1-8, 1960.

66. Conney, A. H., and Gelboin, H. V. Antagonism and potentiation of drugaction and the mechanism of microsomal enzyme induction. In: L. Meylerand H. M. Peck (eds.), Drug Induced Diseases, Vol. 4, pp. 179-207.Amsterdam: Excerpta Medica, 1972.

67. Conney, A. H., Gillette, J. R., Inscoe, J. K., Trams, E. R., and Posner, H.S. Induced synthesis of liver microsomal enzymes which metabolize foreigncompounds. Science (Wash. D. C.), 730. 1478-1479, 1959.

68. Conney, A. H., Jacobson, M., Levin, W., Schneidman, K., and Kuntzman,R. Decreased central depressant effect of progesterone and other steroidsin rats pretreated with drugs and insecticides. J. Pharmacol. Exp. Ther.,754: 310-318, 1966.

69. Conney, A. H., Jacobson, M.. Schneidman, K., and Kuntzman, R. Inductionof liver microsomal Cortisol 6/3-hydroxylase by diphenylhydantoin or phÃ©nobarbital: an explanation for the increased excretion of 6-hydroxycortisol inhumans treated with these drugs. Life Sci., 4: 1091-1098, 1965.

70. Conney, A. H.. and Klutch, A. Increased activity of androgen hydroxylasesin liver microsomes of rats pretreated with phÃ©nobarbital and other drugs.J. Biol. Chem.. 238: 1611-1617, 1963.

71. Conney, A. H., and Kuntzman. R. Metabolism of normal body constituentsby drug-metabolizing enzymes in liver microsomes. In: B. B. Brodie and J.Gillette (eds.). Concepts in Biochemical Pharmacology, Handbook of Experimental Pharmacology Series, pp. 401-421. Berlin: Springer-Verlag,

1971.72. Conney. A. H.. Levin, W., Ikeda, M., Kuntzman, R.. Cooper. D. Y., and

Rosenthal, O. Inhibitory effect of carbon monoxide on the hydroxylation oftestosterone by rat liver microsomes. J. Biol. Chem., 243. 3912-3915,

1968.73. Conney. A. H., Levin, W., Jacobson, M., and Kuntzman, R. Effects of drugs

and environmental chemicals on steroid metabolism. Proceedings of theSymposium on Drugs and the Unborn Child, New York, 1973. Clin. Pharmacol. Ther., 14: 727-741. 1973.





74. Conney, A. H., Levin, W., Jacobson, M.. Kuntzman, R., Cooper, D. Y., andRosenthal, O. Specificity in the regulation of the 60-, la- and 16a-hydrox-

ylation of testosterone by rat liver microsomes. In: J. R. Gillette, A. H.Conney. G. J. Cosmides, R. W. Estabrook, J. R. Fouts, and G. J. Mannering(eds.), Microsomes and Drug Oxidations, pp. 279-302. New York: Academic Press, Inc., 1969.

75. Conney. A. H.. Levin, W., Wood, A. W., Yagi, H.. Lehr, R. E., and Jerina,D. M. Biological activity of polycyclic hydrocarbon metabolites and the bayregion theory. Advances in Pharmacology and Therapeutics, Vol. 9, pp.41-52. New York: Pergamon Press, 1978.

76. Conney, A. H., Levin, W., Wood, A. W., Yagi, H.. Thakker, D. R., Lehr, R.E., and Jerina, D. M. Metabolism of polycyclic aromatic hydrocarbons toreactive intermediates with high biological activity. In: F. Coulston and P.Shubik (eds.). Human Epidemiology and Animal Laboratory Correlations inChemical Carcinogenesis, pp. 153-183. Norwood, N. J.: Ablex PublishingCorporation, 1980.

77. Conney, A. H., Lu, A. Y. H., Levin. W.. Somogyi, A.. West, S., Jacobson,M , Ryan, 0., and Kuntzman, R. Effect of enzyme inducers on substratespecificity of the cytochrome P-450S. Proceedings of the Second International Symposium on Microsomes and Drug Oxidations, Palo Alto, California, 1972. Drug Metab. Dispos., /. 199-210, 1973.

78. Conney. A. H., Miller, E. C., and Miller, J. A. The metabolism of methylatedaminoazo dyes. V. Evidence for induction of enzyme synthesis in the rat by3-methylcholanthrene. Cancer Res.. 76. 450-459. 1956.

79. Conney, A. H., Miller, E. C., and Miller, J. A. Substrate-induced synthesisand other properties of benzpyrene hydroxylase in rat liver. J. Biol. Chem..228. 753-766, 1957.

80. Conney. A. H., Pantuck, E. J., Hsiao, K.-C., Garland, W. A., Anderson, K.E., Alvares, A. P., and Kappas, A. Enhanced phenacetin metabolism inhuman subjects fed charcoal-broiled beef. Clin. Pharmacol. Ther., 20:633-642. 1976.

81. Conney, A. H., Pantuck. E. J.. Pantuck, C. B., Buening, M., Jerina, D. M.,Fortner. J. G.. Alvares. A. P.. Anderson, K. E., and Kappas, A. Role ofenvironment and diet in the regulation of human drug metabolism. In: R. W.Estabrook and E. Lindenlaub (eds.). The Induction of Drug Metabolism, pp.583-605. New York: F. K. Schattauer Verlag, 1979.

82. Conney, A. H., Pantuck. E. J., Pantuck, C. B., Fortner, J. G., Alvares, A.P., Anderson, K. E., and Kappas. A. Variability in human drug metabolism.In: P. Turner (ed.). Clinical Pharmacology and Therapeutics, pp. 51-62.

London: Macmillan Publishers Ltd.. 1980.83. Conney, A. H., Sansur, M., Soroko, F., Koster, R., and Burns, J. J. Enzyme

induction and inhibition in studies on the pharmacological actions ofacetophenetidin. J. Pharmacol. Exp. Ther.. 151: 133-138, 1966.

84. Conney, A. H.. Trousof. N., and Burns, J. J. The metabolic fate of zoxa-zolamine (Flexin) in man. J. Pharmacol. Exp. Ther., 128. 333-339, 1960.

85. Conney, A. H., Welch. R., Kuntzman. R.. Chang. R., Jacobson. M.. Munrc-Faure, A. D., Peck, A. W.. Bye, A., Poland, A., Poppers, P. J.. Finster, M.,and Wolff, J. A. Effects of environmental chemicals on the metabolism ofdrugs, carcinogens and normal body constituents in man. Ann. N. Y. Acad.Sci.. Ã•79. 155-172. 1971.

86. Conney, A. H., Wood, A. W., Levin, W., Lu, A. Y. H.. Chang. R. L., Wislocki,P. G.. Goode, R. L., Holder, G. M., Dansette. P. M., Yagi, H.. and Jerina,D. M. Metabolism and biological activity of benzo[a]pyrene and its metabolic products. In: D. Jollow, J. Kocsis, R. Snyder, and H. Vainio (eds.),Biological Reactive Intermediates, pp. 335-356. New York: Plenum Publishing Corporation, 1977.

87. Coombs. M. M., and Bhatt, T. S. High skin tumour initiating activity of themetabolically derived frans-3.4-dihydro-3,4-diol of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one. Carcinogenesis 3.449-451. 1982.

88. Coombs, M. M., Kissonerghis, A.-M., Allen, J. A., and Vose, C. W. Identification of the proximate and ultimate forms ot the carcinogen 15,16-dihydro-11-methylcyclopenta(a]phenanthren-17-one. Cancer Res.. 39:4160-4165. 1979.

89. Coon, M. J. The 1980 Bernard B. Brodie Award Lecture. Drug metabolismby cytochrome P-450: progress and perspectives. Drug Metab. Dispos.. 9.1-4, 1981.

90. Cooper, C. S., Ribeiro, O., Hewer, A.. Walsh, C., Pal. K., Grover, P. L., andSims. P. The involvement of a 'bay-region' and a non-'bay-region' diol-

epoxide in the metabolic activation of benz[a]anthracene in mouse skinand in hamster embryo cells. Carcinogenesis (Lond.). I. 233-243. 1980.

91. Cooper. C. S.. Vigny, P., Kindts, M.. Grover. P. L., and Sims, P. Metabolicactivation of 3-methylcholanthrene in mouse skin: fluorescence spectralevidence indicates the involvement of diol-epoxides formed in the 7.8,9,10-ring. Carcinogenesis (Lond.), Ã•.855-860, 1980.

92. Cooper, D. Y., Levin, S., Narasimhulu. S., Rosenthal, O., and Estabrook,R. W. Photochemical action spectrum of the terminal oxidase of mixedfunction oxidase systems. Science (Wash. D. C.), 147: 400-402, 1965.

93. Crosby, D. G. Natural toxic background in the food of man and his animals.J. Agr. Food Chem., J 7. 532-538, 1969.

94. Cucinell. S. A., Conney, A. H.. Sansur, M., and Burns, J. J. Drug interactions in man: I. Lowering effect of phÃ©nobarbital on plasma levels ofbishydroxycoumarin (Dicumarol) and diphenylhydantoin (Dilantin). Clin.

Pharmacol. Ther., 6. 420-429, 1965.95. Daly, J. W., Jerina, D. M., and Witkop, B. Arene oxides and the NIH shirt.

The metabolism, toxicity and carcinogenicity of aromatic compounds.Experientia (Basel), 28. 1129-1164, 1972.

96. Delia Porta. G., and Terracini. B. Chemical Carcinogenesis in infant animals.In: F. Homburger (ed.), Progress in Experimental Tumor Research, pp.334-363. Basel: S. Karger AG. 1969.

97. Deutsch, J., Leutz, J.. Yang. S. K., Gelboin, H. V., Chiang, Y. L., Vatsis, K.P., and Coon. M. J. Regio- and stereoselectivity of various forms of purifiedcytochrome P-450 in the metabolism of benzo[a]pyrene and (â€”)-irans-7,8-dihydroxy-7,8-dihydrobenzo(a]pyrene as shown by product formation andbinding to DNA. Proc. Nati. Acad. Sei. U. S. A.. 75. 3123-3127, 1978.

98. Deutsch, J., Vatsis. K. P., Coon, M. J., Leutz, J. C., and Gelboin, H. V.Catalytic activity and stereoselectivity of purified forms of rabbit livermicrosomal cytochrome P-450 in the oxygÃ©nation of the (-) and (+)enantiomers of frans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Mol.Pharmacol.. 16: 1011-1018, 1979.

99. DiGiovanni, J., Diamond, L., Singer, J. M.. Daniel, F. B., Witiak, D. T., andSlaga, T. J. Tumor-initiating activity of 4-fluoro-7,12-dimethylbenz[a]an-thracene and 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in female SENCAR mice. Carcinogenesis (Lond.), 3. 651-655, 1982.

100. Dipple, A., and Nebzydoski, J. A. Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbenz[a]anthracene to DNA in cellsin culture. Chem.-Biol. Interact.. 20. 17-26. 1978.

101. DuBois, R. N., and Waterman, M. R. Effect of phÃ©nobarbital administrationto rats on the level of the in vitro synthesis of cytochrome P-450 directedby total rat liver RNA. Biochem. Biophys. Res. Commun., 90. 150-157,1979.

102. Duvaldestin, P.. Mazze, R. I., Nivoche. Y., and Desmonts, J-M. Occupational exposure to Halothane results in enzyme induction in anesthetists.Anesthesiology, 54: 57-60, 1981.

103. Elshourbagy, N. A., and Guzelian, P. S. Separation, purification, andcharacterization of a novel form of hepatic cytochrome P-450 from ratstreated with pregnenolone-16a-carbonitrile. J. Biol. Chem., 255. 1279-

1285, 1980.104. Feldman. C. H.. Hutchinson. V. E., Pippenger, C. E.. Blumenfeld, T. A.,

Feldman, B. R.. and Davis. W. J. Effect of dietary protein and carbohydrateon theophylline metabolism in children. Pediatrics, 66. 956-962, 1980.

105. Fisher, G. J., Fukushima, H., and Gaylor. J. L. Isolation, purification, andproperties of a unique form of cytochrome P-450 in microsomes of isosaf-role-treated rats. J. Biol. Chem., 256. 4388-4394. 1981.

106. Fujii-Kuriyama, Y.. Mizukami, Y., Kawajiri. K., Sogawa, K.. and Muramatsu.M. Primary structure of a cytochrome P-450: coding nucleotide sequenceof phenobarbital-inducible cytochrome P-450 cDNA from rat liver. Proc.Nati. Acad. Sei. U. S. A., 79. 2793-2797, 1982.

107. Fujii-Kuriyama, Y., Taniguch, T., Mizukami. Y., Sakai, M., Tashiro, Y., andMuramatsu, M. Molecular cloning of a complementary DNA of phenobarbital-inducible cytochrome P-450 messenger RNA from the rat. Proc. Jpn.Acad., 56. 603-608. 1980.

108. Garfinkel. D. Studies on pig liver microsomes. I. Enzymic and pigmentcomposition of different microsomal fractions. Arch. Biochem. Biophys.,77. 493-509, 1958.

109. Garner. R. C.. and McLean. A. E. M. Increased susceptibility to carbontetrachloride poisoning in the rat after pretreatment with oral phenobarbi-tone. Biochem. Pharmacol.. 18: 645-650, 1969.

110. Gelboin, H. V. A microsome-dependent binding of benzo[a]pyrene to DNA.Cancer Res., 29. 1272-1276, 1969.

111. Gibson. G. G., Orton, T. C., and Tamburini. P. P. Cytochrome P-450induction by clofibraie: Purification and properties of a hepatic cytochromeP-450 relatively specific for the 12- and 11-hydroxylation of dodecanoicacid (lauric acid). Biochem. J., 203. 161-168. 1982.

112. Grover, P. L., and Sims. P. Enzyme-catalysed reactions of polycyclichydrocarbons with deoxyribonucleic acid and protein in vitro. Biochem. J.,110: 159-160, 1968.

113. Guengerich, F. P. Separation and purification of multiple forms of microsomal cytochrome P-450. Partial characterization of three apparently homogeneous cytochromes P-450 prepared from livers of phÃ©nobarbital- and3-methylcholanthrene-treated rats. J. Biol. Chem., 253. 7931-7939,1978.

114. Guzelian, P. S., Vranian, G., Boylan, J. J.. Cohn. W. J., and Blanke. R.V.Liver structure and function in patients poisoned with chlordecone (Ke-pone). Gastroenterology. 78. 206-213, 1980.

115. Hammer, W.. IdestrÃ¶m, C-M., and SjÃ¶qvist, F. Chemical control of antide-pressant drug therapy. In: S. Garattini and M. N. G. Dukes (ed.). International Congress Series, No. 122, Proceedings of the First InternationalSymposium on Antidepressant Drugs, pp. 301 -310. Amsterdam: ExcerptaMedica, 1967.

116. Hansson, R., and Wikvall. K. Hydroxylations in biosynthesis and metabolism of bile acids. Catalytic properties of different forms of cytochromeP-450. J. Biol. Chem., 255. 1643-1649, 1980.

117. Harman, A. W., Frewin, D. B., and Priestly, B. G. Induction of microsomaldrug metabolism in man and in the rat by exposure to petroleum. Br. J. Ind.Med., 38. 91-97, 1981.

118. Harman, A. W., Russell, W. J., Frewin, D. B., and Priestly, B. G. Altered

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drug metabolism in anaesthetists exposed to volatile anaesthetic agents.Anaesth. Intensive Care, 6: 210-214, 1978.

119. Harris. C. C., Trump. B. F.. Grafstrom. R., and Autrup, H. Differences inthe metabolism of chemical carcinogens in cultured human epithelial tissues and cells. In: C. C. Harris and P. Cerutti (eds.). Mechanisms ofChemical Carcinogenesis, pp. 289-298. New York: Alan R. Liss, Inc.,1982.

120. Harrison, Y. E., and West. W. L. Stimulatory effect of charcoal-broiledground beef on the hydroxylation of 3,4-benzpyrene by enzymes in rat liverand placenta. Biochem. Pharmacol., 20. 2105-2108, 1971.

121. Hart, P.. Farrell, G. C., Cooksley. W. G. E., and Powell. L. W. Enhanceddrug metabolism in cigarette smokers. Br. Med. J . 2: 147-149, 1976.

122. Harvey, R. G. Carcinogenic hydrocarbons: metabolic activation and themechanism of cancer induction. In: D. B. Walters (ed.). Safe Handling ofChemical Carcinogens, Mutagens, Teratogens and Highly Toxic Substances. Vol. 2, pp. 439-468. Ann Arbor. Mich.: Ann Arbor SciencePublishers, Inc.. 1980.

123. Hayes, J. R., and Campbell, T. C. Nutrition as a modifier of chemicalcarcinogenesis. In: T. J. Slaga (ed.), Carcinogenesis. Vol. 5, pp. 207-241.New York: Raven Press. 1980.

124. Hecht, S. S.. Hirota, N., Loy, M., and Hoffmann, D. Tumor-initiating activityof fluorinated 5-methylchrysenes. Cancer Res., 38. 1694-1698, 1978.

125. Hecht. S. S.. LaVoie. E. J., Bedenko, V., Pingare, L., Katayama, S.,Hoffmann, D., Sardella, D. J., Boger, E., and Lehr, R. E. Reduction oftumorigenicity and of dihydrodiol formation by fluorine substitution in theangular rings of dibenzo(a.i)pyrene. Cancer Res., 41: 4341-4345, 1981.

126. Hecht, S. S., LaVoie, E., Mazzarese, R., Amin, S., Bedenko, V., andHoffmann, D. 1,2-Dihydro-1 ^-dihydroxy-S-methylchrysene, a major activated metabolite of the environmental carcinogen 5-methylchrysene. Cancer Res., 38. 2191-2194, 1978.

127. Hecht, S. S., LaVoie. E., Mazzarese. R., Hirota. N.. Ohmori, T.. andHoffmann. 0. Comparative mutagenicity, tumor initiating activity, carcino-genicity, and in vitro metabolism of fluorinated 5-methylchrysenes. J. Nati.Cancer Inst., 63. 855-861. 1979.

128. Hecht, S. S., Rivenson, A., and Hoffmann, D. Tumor-initiating activity ofdihydrodiols formed metabolically from 5-methylchrysene. Cancer Res.,40: 1396-1399. 1980.

129. Heidelberger, C. Chemical carcinogenesis, chemotherapy: cancer's con

tinuing core challengesâ€”G. H. A. Clowes Memorial Lecture. Cancer Res.30. 1549-1569, 1970.

130. Heuman, D. M., Gallagher, E. J., Barwick. J. L., Elshourbagy, N. A., andGuzelian, P. S. Immunochemical evidence for induction of a common formof hepatic cytochrome P-450 in rats treated with pregnenolone-16a-car-bonitrile or other steroidal or non-steroidal agents. Mol. Pharmacol.. 21:753-760. 1982.

131. Holder, G., Yagi, H., Dansette, P., Jerina, D. M., Levin, W., Lu, A. Y. H.,and Conney, A. H. Effects of inducers and epoxide hydrase on the metabolism of benzo( a )py rene by liver microsomes and a reconstituted system:analysis by high pressure liquid chromatography. Proc. Nati. Acad. Sci.U. S. A., 71: 4356-4360, 1974.

132. Holder, G. M., Yagi. H., Jerina, D. M., Levin. W., Lu, A. Y. H., and Conney,A. H. Metabolism of benzo(a]pyrene. II. Effect of substrate concentrationand 3-methylcholanthrene pretreatment on hepatic metabolism by micro-somes from rats and mice. Arch. Biochem. Biophys., 170:557-566.1975.

133. Huang. M-T.. Chang. R. L.. Fortner, J. G., and Conney, A. H. Studies onthe mechanism of activation of microsomal benzo[a]pyrene hydroxylationby flavonoids. J. Biol. Chem., 256.' 6829-6836. 1981.

134. Huang, M-T., Johnson, E. F., Muller-Eberhard, U., Koop, D. R., Coon. M.J., and Conney, A. H. Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymesfrom rabbit liver microsomes. J. Biol. Chem., 256. 10897-10901, 1981.

135. Huberman, E., Sachs. L.. Yang. S. K.. and Gelboin, H. V. Identification ofmutagenic metabolites of benzo(a]pyrene in mammalian cells. Proc. Nati.Acad. Sci. U. S. A.. 73: 607-611, 1976.

136. Huggins, C., and Pataki, J. Aromatic azo derivatives preventing mammarycancer and adrenal injury from 7,12-dimethylbenz[a]anthracene. Proc.Nati. Acad. Sei. U. S. A., 53: 791-796, 1965.

137. Hunter, J. O., and Davie, M. Vitamin D metabolism in patients treated withphenytoin and phenobarbitone. In: D. Evered and G. Lawrenson (eds.),Environmental Chemicals, Enzyme Function and Human Disease, CibaFoundation Symposium 76 (new series), pp. 315-330. Amsterdam: Excerpta Medica. 1980.

138. Ikeda. M., Conney, A. H., and Burns, J. J. Stimulatory effect of phÃ©nobarbital and insecticides on warfarin metabolism in the rat. J. Pharmacol. ExpTher., f 62: 338-343, 1968.

139. Ivanovic, V.. Geacintov, N. E., Jeffrey, A. M., Fu, P. P.. Harvey. R. G., andWeinstein, I. B. Cell and microsome mediated binding of 7,12-dimethyl-benz| a Â¡anthracene to DNA studied by fluorescence spectroscopy. CancerLett.. 4: 131-140. 1978.

140. Ivanovic, V., Geacintov, N. E., Yamasaki. H., and Weinstein, I. B. DNA andRNA adducts formed in hamster embryo cell cultures exposed to benzo-[ajpyrene. Biochemistry, 17: 1597-1603. 1978.

141. Jacobson, M., Levin, W., Poppers, P. J., Wood, A. W., and Conney, A. H.

Comparison of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene in human placenta: effect of cigarette smoking.Clin. Pharmacol. Ther., Ã•6:701-710, 1974.

142. Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland,F. A., Harvey, R. G., Kasai. H., Miura, I., and Nakanishi. K. BenzolsJpyrene-nucleic acid derivative found in vivo: structure of a benzo(a]pyrenetetra-hydrodiol epoxide-guanosine adduci. J. Am. Chem. Soc., 98. 5714-5715,1976.

143. Jeffrey, A. M., Weinstein, I. B., Jennette. K. W., Grzeskowiak, K., andNakanishi, K. Structures of benzo[a]pyrene-nucleic acid adducts formed inhuman and bovine bronchial expiants. Nature (Lond.), 269: 348-350,1977.

144. Jenne. J.. Nagasawa, H.. McHugh, R., MacDonald. F., and Wyse, E.Decreased theophylline half-life in cigarette smokers. Life Sci., 17: 195-198, 1975.

145. Jerina, D. M.. and Daly. J. W. Arene oxides: a new aspect of drugmetabolism. Metabolic formation of arene oxides explains many toxic andcarcinogenic properties of aromatic hydrocarbons. Science (Wash. D. C.).Â»85.573-582, 1974.

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147. Jerina, D. M., Daly, J. W., Witkop, B., Zaltzman-Nirenberg, P., and Uden-friend, S. The role of the arene oxide-oxepin systems in the metabolism ofaromatic substrates. III. Formation of 1,2-naphthalene oxide from naphthalene by liver microsomes. J. Am. Chem. Soc., 90: 6525-6527, 1968.

148. Jerina, D. M., Daly, J. W., Witkop, B., Zaltzman-Nirenberg, P., and Uden-friend, S. 1,2-Naphthalene oxide as an intermediate in the microsomalhydroxylation of naphthalene. Biochemistry, 9: 147-156, 1970.

149. Jerina, D. M., Lehr, R. E., Yagi, H., Hernandez, O.. Dansette, P. M.,Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., and Conney, A. H.Mutagenicity of benzo[a]pyrene derivatives and the description of a quantum mechanical model which predicts the ease of carbonium ion formationfrom diol epoxides. In: F. J. de Serres, J. R. Fouts, J. R. Bend, and R. M.Philpot (eds.), In Vitro Metabolic Activation in Mutagenesis Testing, pp.159-178. Amsterdam: Elsevier/North Holland BiomÃ©dical Press, 1976.

150. Jerina, D. M., Sayer, J. M., Yagi, H., Croisy-Delcey, M.. Ittah, Y., Thakker,D. R., Wood, A. W., Chang, R. L., Levin, W., and Conney, A. H. Highlytumongemc bay-region diol epoxides from the weak carcinogen benzo-[cjphenanthrene. In: R. Snyder, D. Parke, J. J. Kocsis, D. J. Jollow, G. G.Gibson, and C. Witmer (eds.). Biological Reactive Intermediatesâ€”II, PartA, pp. 501 -523. New York: Plenum Publishing Corporation, 1982.

151. Jerina, D. M., Yagi, H.. Thakker. D. R., Karle. J. M.. MÃ¤h.H. D.. Boyd, D.R.. Gadaginamath, G.. Wood. A. W., Buening. M.. Chang. R. L.. Levin, W.,and Conney, A. H. Stereoselective metabolic activation of polycyclic aromatic hydrocarbons. In: Y. Cohen (ed.), Advances in Pharmacology andTherapeutics, Vol. 9, pp. 53-62. New York: Pergamon Press. 1979.

152. Jusko, W. J., Schentag, J. J., Clark. J. H., Gardner, M., and Yurchak, A.M. Enhanced biotransformation of theophylline in marihuana and tobaccosmokers. Clin. Pharmacol. Ther., 24: 406-410. 1978.

153. Kapitulnik, J.. Levin, W., Conney. A. H., Yagi. H.. and Jerina, D.M. Benzo-[ajpyrene 7,8-dihydrodiol is more carcinogenic than benzo[a]pyrene innewborn mice. Nature (Lond.), 266. 378-380, 1977.

154. Kapitulnik, J., Levin, W., Poppers, P. J., Tomaszewski, J. E., Jerina, D.M.,and Conney, A. H. Comparison of the hydroxylation of zoxazolamine andbenzo[a Ipyrene in human placenta: effect of cigarette smoking. Clin. Pharmacol. Ther.. 20: 557-564. 1976.

155. Kapitulnik, J., Levin, W., Yagi, H., Jerina. D. M., and Conney, A. H. Lackof carcinogenicity of 4-, 5-, 6-, 7-, 8-, 9- and 10-hydroxybenzo[a]pyreneon mouse skin. Cancer Res., 36: 3625-3628, 1976.

156. Kapitulnik, J., Poppers, P. J.. Buening, M. K., Fortner, J. G., and Conney.A. H. Activation of monooxygenases in human liver by 7,8-benzoflavone.Clin. Pharmacol. Ther., 22: 475-485, 1977.

157. Kapitulnik, J., Wislocki, P. G. Levin. W., Yagi. H., Jerina, D. M., andConney. A. H. Tumorigenicity studies with diol-epoxides of benzo[a]pyrenewhich indicate that (Â±Mrans-7/i,8a-dihydroxy-9a.10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is an ultimate carcinogen in newborn mice.Cancer Res., 38: 354-358, 1978.

158. Kapitulnik, J.. Wislocki. P. G.. Levin, W., Yagi, H., Thakker. D. R., Akagi,H., Koreeda, M., Jerina, D. M., and Conney, A. H. Marked differences inthe carcinogenic activity of optically pure (+)- and (â€”Hrans-7,8-dihydroxy-7,8-dihydrobenzo(a]pyrene in newborn mice. Cancer Res., 38: 2661-2665, 1978.

159. Kappas, A., Alvares, A. P., Anderson, K. E., Pantuck. E. J., Pantuck, C. B..Chang, R., and Conney, A. H. Effect of charcoal-broiled beef on antipyrineand theophylline metabolism. Clin. Pharmacol. Ther., 23: 445-450, 1978.

160. Kappas, A., Anderson, K. E., Conney, A. H., and Alvares, A. P. Influenceof dietary protein and carbohydrate on antipyrine and theophylline metabolism in man. Clin. Pharmacol. Ther.. 20: 643-653, 1976.

161. Kato, R. Modificato quadro farmacologico di alcuni tarmaci in animalipretrattatiâ€”48 ore primaâ€”con altri tarmaci. Atti Soc. Lombarda Se. Med-Biol., ) 4. 783-786, 1959.

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170. Klingenberg, M. Pigments of rat liver microsomes. Arch. Biochem. Bio-phys., 75. 376-386, 1958.

171. Kolmodin, B., Azarnoff, D. L, and SjÃ¶qvist, F. Effect of environmentalfactors on drug metabolism: decreased plasma half-life of antipyrine inworkers exposed to chlorinated hydrocarbon insecticides. Clin. Pharmacol.Ther., 10.638-642, 1969.

172. Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A. H., Yagi,H., and Jerina, D. M. Binding of benzo(a]pyrene 7.8-diol-9,10-Â«poxides toDNA, RNA and protein of mouse skin occurs with high stereoselectivity.Science (Wash. D. C.), Â»99.778-781, 1978.

173. Krampl, V., and KontsekovÃ¡, M. Effect of polychlorinated biphenyls on theelimination rate of antipyrine from plasma of rats and man. Bull. Environ.Contam. Toxicol., 20. 191-198, 1978.

174. Kuntzman, R., Jacobson. M., Schneidman, K., and Conney, A. H. Similarities between oxidative drug-metabolizing enzymes and steroid hydroxyl-ases in liver microsomes. J. Pharmacol. Exp. Ther., Â»46.280-285, 1964.

175. Kuntzman, R., Levin, W., Jacobson, M., and Conney, A. H. Studies onmicrosomal hydroxylation and the demonstration of a new carbon monoxidebinding pigment in liver microsomes. Life Sci., 7: 215-224, 1968.

176. Kuntzman, R., Mark, L. C., Brand, L., Jacobson, M., Levin, W., and Conney.A. H. Metabolism of drugs and carcinogens by human liver enzymes. J.Pharmacol. Exp. Ther.. Â»52.151-156. 1966.

177. Kuntzman, R., Sansur, M., and Conney, A. H. Effect of drugs and insecticides on the anesthetic action of steroids. Endocrinology, 77. 952-954,1965.

178. Kunz, W., Schaude, G.. and Thomas, C. The effect of phÃ©nobarbital andhalogenated hydrocarbons on nitrosamine carcinogenesis. Z. Krebs-forsch., 72. 291-304, 1969.

179. Kupfer, D. Endogenous substrates of monooxygenases: fatty acids andprostaglandins. In: J. B. Schenkman and D. Kupfer (eds.), Hepatic Cyto-chrome P-450 Monooxygenase System, pp. 157-187. New York: Perga-rr.on Press, 1982.

180. Larsen, P. R., Atkinson, A. J., Jr., Wellman. H. N., and Goldsmith, R. E.The effect of diphenylhydantoin on thyroxine metabolism in man. J. Clin.Invest., 49: 1266-1279, 1970.

181. Lasker, J. M., Huang, M-T., and Conney, A. H. In vivo activation ofzoxazolamine metabolism by flavone. Science (Wash. D. C.), 2Ã•6: 1419-1421. 1982.

182. LaVoie, E. J., Amin, S., Hecht, S. S.. Furuya. K., and Hoffmann, D. Tumourinitiating activity of dihydrodiols of benzo[b]fluoranthene, benzofj]fluoran-thene, and benzo(/<]fluoranthene. Carcinogenesis (Lond.). 3. 49-52,1982.

183. LaVoie, E. J., Tulley-Freiler, L.. Bedenko, V., and Hoffmann, D. On themetabolic activation of methylated phenanthrenes. Proc. Am. Assoc. Cancer Res., 22:94, 1981.

184. Levin, W. Purification of liver microsomal cytochrome P-450: Hopes andpromises. In: V. Ullrich, I. Roots, A. Hildebrandt, R. W. Estabrook, and A.H. Conney (eds.), Microsomes and Drug Oxidations, Proceedings of theThird International Symposium, pp. 735-747. New York: Pergamon Press,1977.

185. Levin. W., Buening, M. K., Wood, A. W., Chang, R. L.. Kedzierski, B.,Thakker, D. R., Boyd, D. R., Gadaginamath, G. S., Armstrong, R. N., Yagi,H., Karle, J. M., Slaga, T. J., Jerina. D. M.. and Conney, A. H. Anenantiomeric interaction in the metabolism and tumorigenicity of {+)- and(-H>enzo{a]pyrene 7.8-oxide. J. Biol. Chem.. 255. 9067-9074, 1980.

186. Levin, W., Buening, M. K.. Wood. A. W., Chang, R. L., Thakker, D. R.,Jerina, D. M., and Conney. A. H. Tumorigenic activity of 3-methylcholanthrene metabolites on mouse skin and in newborn mice. Cancer Res., 39.3549-3553, 1979.

187. Levin. W., and Conney, A. H. Stimulatory effect of polycyclic hydrocarbonsand aromatic azo derivatives on the metabolism of 7,12-dimethylbenz[a]anthracene. Cancer Res., 27: 1931-1938, 1967.

188. Levin, W.. Conney. A. H.. Alvares. A. P., Merkatz, I., and Kappas, A.Induction of benzo(a]pyrene hydroxylase in human skin. Science (Wash.D. C.). Â»76:419-420. 1972.

189. Levin, W., Kuntzman, R., and Conney, A. H. Stimulatory effect of phÃ©nobarbital on the metabolism of the oral contraceptive 17a-ethynyl-estradiol-3-methyl ether (mestranol) by rat liver microsomes. Pharmacology, Â»9:249-255, 1979.

190. Levin, W., Lu. A. Y. H., Ryan, D., Wood, A. W.. Kapitulnik, J.. West, S..Huang, M-T., Conney, A. H., Thakker, D. R., Holder, G., Yagi, H., andJerina, D. M. Properties of the liver microsomal monooxygenase systemand epoxide hydrase: factors influencing the metabolism and mutagenicityof benzo[a]pyrene. In: H. H. Hiatt. J. D. Watson, and J. A. Winsten (eds.),Origins of Human Cancer, pp. 659-682. Cold Spring Harbor, N. Y.: ColdSpring Harbor Laboratory, 1977.

191. Levin. W., Thakker, D. R., Wood. A. W.. Chang, R. L.. Lehr. R. E.. Jerina.D. M., and Conney. A. H. Evidence that benzo[a]anthracene 3,4-diol-1,2-epoxide is an ultimate carcinogen on mouse skin. Cancer Res., 38:1705-

1710, 1978.192. Levin, W., Welch, R. M., and Conney, A. H. Effect of chronic phÃ©nobarbital

treatment on the liver microsomal metabolism and uterotropic action of17/8-estradiol. Endocrinology, 80. 135-140, 1967.

193. Levin. W., Welch, R. M., and Conney. A. H. Decreased uterotropic potencyof oral contraceptives in rats pretreated with phÃ©nobarbital. Endocrinology,83: 149-156. 1968.

194. Levin, W., Welch, R. M., and Conney, A. H. Effect of phÃ©nobarbital andother drugs on the metabolism and uterotropic action of estradiol-17ÃŸ andestrone. J. Pharmacol. Exp. Ther., Â»59:362-371, 1968.

195. Levin, W., Welch, R. M., and Conney, A. H. Inhibitory effect of phÃ©nobarbitalor chlordane pretreatment on the androgen-induced increase in seminalvesicle weight in the rat. Steroids, Â»3.155-161, 1969.

196. Levin, W., Welch, R. M., and Conney. A. H. Increased liver microsomalandrogen metabolism by phÃ©nobarbital: correlation with decreased andro-gen action on the seminal vesicles of the rat. J. Pharmacol. Exp. Ther.,Â»88:287-292, 1974.

197. Levin, W., Wood, A. W., Chang, R. L., Ittah, Y., Croisy-Delcey, M., Yagi,H., Jerina, D. M., and Conney, A. H. Exceptionally high tumor-initiatingactivity of benzo(c)phenanthrene bay-region diol-epoxides on mouse skin.Cancer Res., 40: 3910-3914, 1980.

198. Levin, W., Wood, A. W., Chang, R., Ryan, D., Thomas, P., Yagi, H.,Thakker, D., Vyas, K., Boyd, C., Chu, S-Y., Conney, A. H., and Jerina, D.M. Oxidative metabolism of polycyclic aromatic hydrocarbons to ultimatecarcinogens. Drug Metab. Rev.. Â»3:555-580, 1982.

199. Levin, W., Wood, A. W., Chang. R. L., Slaga, T. J., Yagi, H., Jerina, D. M.,and Conney. A. H. Marked differences in the tumor-initiating activity ofoptically pure (+)- and (-)-(rans-7.8-dihydroxy-7,8-dihydrobenzo[a]py-rene on mouse skin. Cancer Res.. 37. 2721 -2725, 1977.

200. Levin, W., Wood, A. W., Chang, R. L.. Yagi, H., MÃ¤h.H. D., Jerina. D. M.,and Conney, A. H. Evidence for bay region activation of chrysene-1,2-dihydrodiol to an ultimate carcinogen. Cancer Res., 38:1831 -1834,1978.

201. Levin, W.. Wood, A. W., Wislocki, P. G., Kapitulnik, J., Yagi. H., Jerina, D.M., and Conney, A. H. Carcinogenicity of benzo-ring derivatives ofbenzo(a)pyrene on mouse skin. Cancer Res., 37: 3356-3361. 1977.

202. Levin, W., Wood. A. W., Yagi, H., Dansette, P. M., Jerina, D. M., andConney. A. H. Carcinogenicity of benzo[a]pyrene 4.5-, 7.8-, and 9.10-oxides on mouse skin. Proc. Nati. Acad. Sci. U.S. A.. 73. 243-247, 1976.

203. Levin, W., Wood, A. W., Yagi, H.. Jerina. D. M., and Conney. A. H. (Â±)-(rans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene: a potent skin carcinogenwhen applied topically to mice. Proc. Nati. Acad. Sei. U. S. A., 73: 3867-

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208. Lu, A. Y. H., Kuntzman, R.. West, S., and Conney, A. H. Reconstituted livermicrosomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. I. Determination of substrate specificity by the cytochrome P-450 and P-448 fractions. Biochem. Biophys. Res.Commun., 42. 1200-1206, 1971.

209. Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney. A. H.Reconstituted liver microsomal enzyme system that hydroxylates drugs,other foreign compounds and endogenous substrates. II. Role of thecytochrome P-450 and P-448 fractions in drug and steroid hydroxylations.J. Biol. Chem., 247: 1727-1734, 1972.

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DECEMBER 1982 4917



1982;42:4875-4917. Cancer Res Allan H. Conney Clowes Memorial LectureCarcinogenesis by Polycyclic Aromatic Hydrocarbons: G. H. A. Induction of Microsomal Enzymes by Foreign Chemicals and

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