Roles of Cytochrome P-450 Enzymes in Chemical ......rabbit P-450 1 and human P-450MP (60). As mentioned above, several forms of P-450 are found in the liver of each animal species,

[CANCER RESEARCH 48, 2946-2954, June 1, 1988]

Perspectivesin CancerResearch

Roles of Cytochrome P-450 Enzymes in Chemical Carcinogenesis and CancerChemotherapy1

F. Peter Guengerich2

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Studies involving the metabolism of chemical carcinogenswere important in the discovery and initial characterization ofthe cytochrome P-450 enzyme system. The Millers and theirassociates identified NADPH-dependent microsomal enzymesinvolved in the biotransformation of azo dyes (1, 2). Thesereactions, reduction and mixed-function oxidation, were alsofound to be important in the processing of many other carcinogens, as well as drugs and steroids. Subsequent work led tothe identification of the hemoprotein P-4503 as the terminal

oxidase involved in such microsomal mixed-function oxidations(3,4). Other early studies revealed that administration of manydifferent chemicals to animals could alter the metabolism ofcarcinogens (5-7). As we know now, many forms of P-450 existand the expression of these enzymes is influenced by the compounds which are given to the animals. Administration of thecarcinogenic polycyclic hydrocarbon 3-methylcholanthrene torats and analysis of the liver microsomes provided some of theearly evidence that multiple forms of P-450 exist (8, 9). Sincethat time, a great deal of effort has been concentrated uponunderstanding the biochemistry of these P-450 enzymes, interms of how they catalyze reactions and how their expressionis regulated. Although much of the focus regarding P-450

proteins has involved their contributions to the metabolism ofsteroids and drugs, considerable effort is still directed towardsunderstanding the roles of P-450s in carcinogen metabolism.

The characteristics of P-450 enzymes will not be reviewed atlength here.4 The microsomal enzymes are found in most tissuesbut are concentrated in liver. All have characteristic ferrous-carbon monoxide complex Soret peaks near 450 nm, havemontimene molecular weights of about 50,000, and acceptelectrons from the flavoprotein NADPH-P-450 reducÃase.Nearly 20 different P-450 proteins have been isolated from ratliver and all appear to be distinct gene products. Levels of theindividual proteins are altered by administration of or exposureto a wide variety of chemicals, many of which are carcinogensthemselves (including tumor initiators such as the polycyclichydrocarbons and tumor promoters such as phÃ©nobarbital).Mitochondrial P-450s accept electrons from ferridoxins andseem to be primarily involved in anabolic steroid metabolism,although some evidence for roles in carcinogen oxidation hasbeen presented (14).

Several reviews and monographs deal with various aspects ofthe catalytic mechanisms and regulation of the P-450 enzymes

Received 12/28/87; revised 2/10/88; accepted 2/25/88.1Research in the author's laboratory has been supported in part by USPHS

Grants ES 00267, ES 01590, ES 02205, Ã‡A30907, and Ã‡A44353.1Burroughs-Wellcome Scholar in Toxicology, 1983-1988.3The abbreviation used is: P-450, cytochrome P-450.* Individual P-450 enzymes are denoted by designations in the original litera

ture or else equated to preparations used in the author's laboratory when possible,for purposes of simplification; for reference see Refs. 10-12. Recently a tentativenomenclature for the P-450 genes has been published (13). The term "isozymes"

is not used here. Although this term has been used extensively in the past, ittechnically refers to situations in which a single reaction is catalyzed by severalproteins. Much of the attention is now focused on highly selective reactions ofindividual I' 45()s and the term is often not very appropriate.

and the reader is directed to these (15-18). This "Perspectives"

article will deal with the current state of information regardingthe metabolism of carcinogens and questions related to thegeneral importance of such processes. The pathways of P-450-mediated oxidation of carcinogens have been reviewed recentlyby Kadi uba r and Ham nions (19), and the entire subject ofmetabolism in the activation of chemical carcinogens is treatedin a comprehensive series (20) and in a number of other reviewarticles (21-31). Another area discussed here is the biotransfor-mation of cancer chemotherapeutic agents by P-450 enzymes.Although metabolism may be of considerable importance ininfluencing the efficacy and side effects of these drugs, thecurrent knowledge is rather limited.

Catalytic Specificity of P-450 Enzymes

What Do We Know about Which Carcinogens Are Activated andDetoxicated by Specific P-450 Enzymes?

The literature contains many studies regarding the catalyticspecificities of the P-450 enzymes of rats, rabbits, and otherexperimental animal species, as well as human P 450s. This"Perspectives" article is not intended to serve as a compilation

of this work. More comprehensive reviews are found elsewhere(10,11,19, 32). Table 1 includes some carcinogens which havebeen studied and representative literature references. Nearly allclasses of carcinogens have been examined to at least somedegree, and a few have been studied in great detail. Invariably,however, our knowledge is far from complete.

What are some of the ways in which the catalytic specificityof individual P-450 enzymes can be discerned? With animalmodels, some initial references can be made by comparing thein vitro rates of a particular catalytic activity using animalstreated with different inducing agents (10). In some cases sexdifferences in metabolism can be exploited. In this regard, onecan also use a series of microsomal preparations and comparethe rates of a catalytic activity under consideration with thoseof a "marker" activity which has been assigned to a particularP-450 enzyme; this approach can be used either with animalmodels (47) or with humans (48). One way of elucidatingcatalytic specificity is to purify the individual P-450 enzymesand directly measure catalytic activities in reconstituted systems. This approach gives a generally appropriate qualitativepicture of microsomal activity (49) although the unexplainedlow activity of some P-450s (50) and possible enzyme interactions (51) may complicate interpretations. A second way toaddress the problem involves insertion of cloned genes intoeukaryotic expression vectors and measurement of catalyticactivity (52).

One problem associated with the above studies is the estimation of the fraction of total activity catalyzed by a particularform of P-450. The major approach involves titration of acatalytic activity in microsomal samples with selective inhibitors. These inhibitors can be classified into three groups: Group

2946

Research. on August 15, 2017. © 1988 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

CYTOCHROME P-450 AND CANCER

1, competitive inhibitors (e.g., see Ref. 53); Group 2, noncompetitive and mechanism-based inhibitors (54); and Group 3,antibodies (55). When families of P-450 enzymes very closelyrelated in primary structure are under consideration, the establishment of specificity of these reagents can be difficult, evenwhen monoclonal antibodies are used.

Some other problems also arise. Several P-450s may allcatalyze a single reaction but with different A",,,values (44, 56).

Another problem is that the use of different end points maygenerate varying pictures of catalytic specificity. For example,the activation of the carcinogen aflatoxin H, to the putativeelectrophilic 2,3-epoxide can be considered. Different patternsof specificity have been seen with purified enzymes (and evenin rat liver microsomes) depending upon whether covalent DNAbinding (34), Salmonella typhimurium reversion (35, 36), orbacterial SOS-linked response (37) is used to monitor thereaction. Finally, caution should be exercised when attemptsare made to extrapolate catalytic specificity across species onthe basis of similarity in primary amino acid sequence. Notabledifferences exist, for example, in the warfarin 6- and 8-hydrox-ylase activities of rat P-450tfNFB and its mouse orthologuePi-450 (57), the 7-ethoxycoumarin O-deethylase activity of ratP-450pB-Band rabbit P-450LM-2(58, 59), and the 17/3-estradiol-2-hydroxylase and progesterone 21-hydroxylase activities ofrabbit P-450 1 and human P-450MP (60).

As mentioned above, several forms of P-450 are found in theliver of each animal species, and a number of lines of evidencesuggest that 10-20 P-450 proteins can exist in rat, rabbit, orhuman liver. The P-450 enzymes often display catalytic specificity. The current thought is that the chemistry involved is verysimilar with all of the enzymes but that specificity is determinedby structural features in the substrate binding site. Specificitycan be demonstrated at several levels (10): (a) different P-450s

vary in their rates of catalysis of a single reaction involving asingle substrate; (b) several P-450s can metabolize a singlesubstrate but regioselectively catalyze reactions at different sitesof the molecule; (c) enantiomeric or diastereomeric pairs of asingle substrate can be transformed at different rates by eachP-450; (d) in a pro-chiral substrate, a P-450 can selectively use

one group (namely, hydrogen) as opposed to the other. Catalyticspecificity can, then, have important consequences, since someoxidations of a single compound can render it more electrophilic (and capable of reacting with macromolecules) whileother oxidations can render it less biologically active and aid inelimination from the body. Further, the rates of reaction are afunction of the amount of each P-450 form present, which canbe regulated by environmental and genetic factors as well assexual dimorphism and development.

Evidence That Differences in P-450 Composition Can Influence

Susceptibility to Cancer

Considerable evidence has now been accumulated in supportof the view that changes in P-450 composition can affect invitro and in vivo metabolism of drugs in both animal modelsand humans. The evidence is less clear in the case of carcinogens, which cannot be dealt with so easily in humans. However,information regarding the general hypothesis does exist andcan be considered under three different headings.

Tumor Initiation in Experimental Animal Systems. Indirectevidence for P-450 roles is provided by experiments in whichin vitro mutagenesis and DNA adduct formation are affectedby changes in the P-450 composition. Many examples exist;the changes in P-450 composition can be brought about bygenetic means (61) or by enzyme induction or reconstitution ofpurified enzymes (33).

Studies dealing with in vivo tumor initiation are more limitedand are largely restricted to the murine model of Neben andhis associates. Although increases in P-450 enzyme activityoccur in the liver and extrahepatic tissues of genetically responsive mice (21), only extrahepatic tissues show enhanced tumorformation, and these experiments have been restricted to poly-cyclic hydrocarbons, which induce the P-450 enzyme underconsideration (1V450) in the genetically responsive animals.Examples of cancers produced in these mice by polycyclichydrocarbons include lung tumors [3-methylcholanthrene], leukemias [benzo(a)pyrene, 3-methylcholanthrene], s.c. tumors[benzo(<z)pyrene, 3-methylcholanthrene] (62), brain tumors [3-

Table 1 Activation of procarcinogens by rat P-450 emymes"

ProcarcinogenRat P-450 enzymes involved in

bioactivation4Representative

referencesAaC(2-amino-9//-pyrido[2,3-e]indole)2-AcetylaminofluoreneAflatoxin H,2-Aminoanthraceneo-AminoazotoIuene4-Aminobiphenyl2-AminofluoreneBenzo(a)pyrene4,4' (Bis)mcthyk'm- chloroaniline (MOCA)1,2,3,4-Dibenzanthracene7,12-Dimethylbenz(a)anthraceneA1,,V-1>inid h ylnitn >saminuGlu-P-l(2-amino-6-methyldipyrido[l,2-a:3',2'-Â«/]imidazole)Glu-P-2(2-aminodipyrido[l,2-a:3',2'-Â«/]imidazole)IQ(2-amino-3-methylimidazo[4,5-/]quinoline)MeAaC(2-amino-3-methyl-9A/-pyrido[2,3-*lindole)MeIQ(2-amino-3,4-dimethylimidazo[4,S-/]quinoline)MeIQx(2-amino-3,8-dimethylimidazo[4,5-/]quinoline)3-MethylcholanthreneJV-Methyl-4-aminoazobenzene2-NaphthylamineTrp-P-1(3-amino-1,4-dimethyl-5//-pyrido|4,3-o]indole)Trp-P-2(3-amino-1 -methy-5//-pyrido[4,3-*|indole)

Fâ€žP-450,SF<Â¡P-450r,.â€žP-45Ã•W.B,P-450,SF-GP-450uT.A,P-45Ã•W.GP-450PB-B,P-450irr.iP-45Ã•W., P-45IW.., P-450BFJGP-45(Wo

-BMP-450flNF.B,P-450,sF-oP-45Ã•WB, P-45<W.P-45<W., P-450,SFJGP-450PB-8,P-45<W â€ž,P-450.SÂ«;P-45Ã•W.SP-450JP-450.SÂ«;P-450â€žNF..,P-4501SFJGP-45<Wâ€ž P-450.SF*P-45<W.B, P-450ISFJCP-45<W., P-450.SF-GP-45<W., P-450.SF-CP-45IW.P-45<Wâ€ž,P-450.SF.GP-450BP-GP-45Ã•W.,,,P-450.SF-G

,sF<;,P-450aNF..

333334-3733383938, 40, 4131,4239333343,4431,33,4531453333333334,46413331,33,45

"See also Ref. 19. The point was made in the text but should be reemphasized here that these are enzymes which have been shown to activate the compounds butare not necessarily the most important catalysts of oxidation. In some cases only a limited number of P-450 enzymes have been studied, sometimes the highlyinducible enzymes may not be relevant to bioactivation in uninduced animals, and sometimes other enzymes such as prostaglandin synthase or flavin-containingmonooxygenase may be more important.

2947




methylcholanthrene] (63), and lymphomas and lymphosarco-mas [7,12-dimethylbenz(a)anthracene] (64). However, caveatsmust be made because the Ah locus is a pleiotropic responseand involves changes in a number of different enzymes potentially catalyzing the biotransformation of these compounds.Further, most of the polycyclic hydrocarbons were used insingle-dose complete carcinogen bioassays, and the possibilitymust be considered that the effects might not be only oninitiation.

While some reservations exist concerning experiments involving genetic manipulation of P-450 enzymes, studies inwhich the effects of enzyme inducers on carcinogenesis havebeen examined are probably even more complex. For instance,Kouri et al. (65) reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin was an effective cocarcinogen in enhancing 3-methyl-cholanthrene-induced s.c. rodent tumors. Subsequent studiesby Poland et al. (66) indicate that the dioxin is a very potenttumor promoter and, in view of the long retention of thecompound, this effect cannot be easily isolated. Studies onenzyme induction by phÃ©nobarbitalare also complicated by theknowledge that this compound and some other barbiturates canbe tumor promoters. Thus any experiments designed to showroles of enzymes in tumor initiation must be carefully set up toshow that the enzyme inducers are truly acting as cocarcinogensand not as promoters (some barbiturates and hydantoins areinducers but not promoters). Usually the effect of enzymeinduction is to decrease tumor incidence, such as with thereduction of aflatoxin Bi-induced (67, 68) or dimethylnitrosa-rnine induced liver tumors (69-71) by phÃ©nobarbitaland reduction of aromatic amine-induced liver tumors by 3-methylchol-anthrene (72, 73). When results of this type are obtained, onemust also consider the effects of the inducer on other enzymessuch as those involved in conjugation and detoxication.

Tumor Promotion in Experimental Animal Systems. Promotion has been addressed far less than initiation in terms ofinfluences due to metabolism of carcinogens. In this article, theterm promoter is used in an operational sense. That is, promoters are chemicals which increase the number of tumorswhen administered after initiating carcinogens at doses whichby themselves do not produce tumors (and are distinguishedfrom cocarcinogens, which must be administered very close tothe time of initiation). With this loose functional definition,mechanisms are not specified, and epigenetic and genetic possibilities can be considered. Many of the classic promotingagents probably do not require biotransformation to exert theireffects; for instance, phorbol esters may exert their actions bybinding to protein kinase C (74). The characteristics and mechanism of action of individual promoting agents do vary, however; for instance, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate differs from 7,12-dimethylbenz(a)anthracene in anumber of aspects of its promoting properties (75, 76). Manychemicals are effective as complete carcinogens as well as beingdistinct initiators or promoters. Thus, these complete carcinogens should possess both initiating and promoting properties.

Of interest here is the possibility that formation of electro-philic metabolites of carcinogens can play a role in their tumor-promoting activities. The concept is not new, although it shouldagain be emphasized that for some model promoting agentsno metabolism is required. Evidence has not been generateddirectly in P-450-related systems, but the literature containsat least two relevant cases. The first is 7-bromomethylbenz-(a)anthracene, which is nearly as potent as the most activephorbol esters in promoting 7,12-dimethylbenz(a)anthracene-initiated mouse skin carcinomas and papillomas (77). The

reactivity of the molecule appears to be important, for thehydroxymethyl analogue was relatively weak as either an initiator or a promoter.

Another example comes from the work of Boberg et al. (78)with l'-hydroxysafrole and rat liver tumors. The compound

acts as a complete carcinogen in rat and mouse liver; its initiating activity in rat liver is relatively weak but the compound isa potent promoter of diethylnitrosamine-induced rat liver tumors. Both the initiating and promoting activities of l'-hy

droxysafrole are blocked by feeding pentachlorophenol, whichinhibits the sulfation of the compound to give a reactive elec-

trophile.The mechanisms by which these electrophilic promoters exert

their effects are unknown. One possibility is toxicity, perhapsthrough reaction with specific proteins, to cause a proliferativeresponse in clones of initiated cells. Alternatively, further mutations in some cells of these proliferating clones may occur(78). The cases mentioned above do not involve P-450 reactions,but the production of similar electrophiles by P-450s certainlyoccurs and the hypothesis should be considered that P-450enzymes can contribute to tumor promotion as well as initiation.

Studies in Humans. In humans, the evidence for associationof cancer risk with P-450 composition is less well developedthan in experimental animals. Although the concept seemsintuitively obvious and is the focus for many detailed investigations, there are only two major lines of evidence for theexistence of a relationship and both have associated caveats.

Kellerman et al. (79, 80) grouped individuals into a trimodaidistribution on the basis of their lymphocyte aryl hydrocarbonhydroxylase inducibility (in culture) and reported that smokersin the high inducibility group were more prone to develop lungtumors. These observations were originally very exciting buttechnical problems associated with the system have made theseexperiments difficult to repeat (i.e., see Ref. 81). More recently,Kouri et al. (82) have reported a relatively good correlationbetween the basal level of lymphocyte aryl hydrocarbon hydroxylase activity and the incidence of lung cancer in smokers.However, this observation does not allow one to discernwhether the high enzymatic activity is a cause or an effect oflung cancer, as the authors admit. Jaiswal et al. (83) found thatthe increased level of Pi-450 mRNA in lymphocytes was wellcorrelated with aryl hydrocarbon hydroxylase inducibility andKarki et al. (84) showed a correlation between levels of basallymphocyte and lung aryl hydrocarbon hydroxylase activity.The work of De Flora et al. (85), on the other hand, suggeststhat smoking directs pulmonary metabolism more towards detoxication than towards bioactivation. The entire body of information still does not permit definitive conclusions to be drawnabout the relationship of the lymphocyte enzyme activity tocancer risk.

The other major line of investigation deals with correlationsinvolving rates of drug oxidation in humans, particularly withregard to polymorphisms. Idle et al. (86) reported a positiverelationship between the in vivo rate of debrisoquine 4-hydrox-ylation and the incidence of liver cancer in Nigerians andsuggested an etiological role involving aflatoxin BI. However,work in the author's laboratory (87) and that of Davies (88)

argues against a role for the debrisoquine 4-hydroxylase inaflatoxin BI activation. Further, no information concerning theactual exposure of the Nigerians to aflatoxins was presented oris available.

A more intriguing study involves the observation by Ayesh etal. (89) that the debrisoquine slow metabolism phenotype could

2948




be associated with decreased incidence of lung bronchiogeniccarcinomas in smokers (and rapid hydroxylation associatedwith increased tumor incidence). No evidence exists for a metabolic basis, and, of course, the etiological factors in tobaccosmoke remain undefined. Although the results of Ayesh et al.(89) appear intriguing, a report by Drakoulis et al. (90) indicatesthat the same findings were not obtained in a similar studyinvolving a German population (i.e., no relationship was found).More recently Harris et al. (91) have found some phenotypicevidence for a positive relationship similar to that of Ayesh etal. (89) when only lung small cell tumors are considered. If arelationship does exist, its basis remains undefined and is notnecessarily metabolic. An abstract by Ritter et al. (92) alsosuggests a positive relationship between rapid debrisoquine 4-hydroxylation and carcinoma of the larynx and pharynx.

The final case involves recent work by Kaisary et al. (93)relating hydroxylation polymorphisms to bladder cancer. Patients presenting with bladder cancer were classified histologi-cally as having nonaggressive (Stages I and II) or aggressive(Stage III) bladder cancer. Aggressive bladder cancer showedan association with rapid debrisoquine 4-hydroxylation. Thisassociation was not seen when nonaggressive bladder cancercases were examined. The particular population studied apparently did not have occupational exposure to known bladdercarcinogens and the influences of ethanol consumption andsmoking were not synergistic with the extent of debrisoquine4-hydroxylation; thus, the work does not provide evidence thatthe P-450 debrisoquine 4-hydroxylase (P-450DB) is activating abladder carcinogen, although this possibility certainly still exists. Patients with nonaggressive bladder cancer had a weakerbut significant association with rapid polymorphic 4-hydroxylation of 5-mephenytoin, and this relationship was independentof a significant interaction between smoking and ethanol intake.

While other relationships might be postulated on the basisof some in vitro and in vivo knowledge (i.e., increased tumorsin alcoholics due to /V,/V-dimethylnitrosaniine oxidation, etc.),

definitive evidence is lacking. A difficulty exists in extendingstudies of this type in that, even if further evidence is obtainedfor a role of metabolic activation, identification of the chemicalsinvolved is not trivial. If there is no epidemiological reason tosuspect a particular compound or mixture, then the problem isvery difficult. Even if a crude mixture such as tobacco smoke isimplicated the problem is difficult because the active principleis unknown. However, there are approaches that could be usedto address such problems. For instance, the P-450 enzymesinvolved in activating crude cigarette smoke condensÃ¢tes tomutagenic forms could, in principle, be identified. The samephenotyping approach could be applied to some of the foodsencountered in regions of China where the local tumor incidence is high (94).

Current and Future Problems

The information discussed above supports the view thatdifferences in P-450 enzymes may contribute to variations inthe metabolism of carcinogens and to risk. What are some ofthe current problems that remain unsolved and in what directions will new research go?

1. Most of the Animal Models Remain Poorly Characterizedin Terms of Which Specific Forms of P-450 Are Involved inIndividual Biotransformation Reactions Related to Tumor Formation. In every animal species, only some of the individual P-450 enzymes have been examined for their roles in any particular reaction. Of course, in some cases, immunochemical or

other approaches may provide strong positive evidence for aprimary role of an individual enzyme and extensive furtherstudies may not be justified.

The bulk of the studies to date have dealt with rats. Even inthat species, the experimental deficiencies are manifold. Manyof the more extensive studies were done with a limited numberof individual P-450s; since then, the number of isolated formsof rat P-450 has grown. Some of these, such as lanosterol 14Â«-demethylase (95) and aromatase (96), have important roles inthe metabolism of endogenous chemicals and their roles incarcinogen biotransformation should be evaluated. Further,some of the preparations previously considered to be homogeneous have been subsequently found to be more complex [e.g.,P-450pcN-E (10, 97)]. This situation also causes problems withantibody and nucleic acid probes in establishing specificity.

Another point to be made is that many of the studies withrat (and other animal) P-450 enzymes are focused upon thoseforms which are induced dramatically. In practice these P-450smay not be encountered frequently, particularly under some ofthe conditions of chronic bioassays. Little attention has beengiven to understanding enzymology in the same strains usedfor bioassays with a particular chemical under consideration.Further, many protocols use either young animals or animalsexposed to complex promotional schemes, and little information linking enzymology to the specific conditions used duringcarcinogen exposure has been obtained. As an example, apopular model is single-dose administration of chemicals topreweanling male mice without further promotion (e.g., Ref.78); suffice it to say that we have a very vague idea of thecomplement of P-450s present to activate the carcinogens insuch animals. Any changes in P-450 composition which occurduring partial hepatectomy have likewise not been characterized.

2. What Do Extrahepatic P-450s Really Mean? We recognizethat P-450s are most concentrated in the liver but are alsofound at lower levels in nearly every other tissue in the body,chiefly lung and kidney but also all others with the apparentexceptions of striated muscle and erythrocytes. In some tissuesthe P-450s are highly localized in particular regions or celltypes so that local concentrations may approach those found inliver (98). One view is that these tissue-specific differences inP-450 can be major determinants in extrahepatic carcinogene-sis, since these P-450s can activate chemicals in close proximityto targets. On the other hand, evidence clearly exists that someof the ultimate carcinogens can migrate throughout the body(28, 99).

The importance of the extrahepatic P-450s, like the liver P-450s, probably varies with the particular enzyme and the chemical under consideration. The extrahepatic P-450s may be quiteimportant with compounds that are converted to very highlyreactive species or with chemicals that have the organ underconsideration as a port of entry. Examples of situations inwhich this latter aspect may be important include polycyclichydrocarbons on skin, airborne carcinogens in the nasal cavityand pulmonary tract, and carcinogenic foodstuffs in the gastrointestinal tract. On the other hand, hepatic P-450s maypredominate in influencing the effects of chemical carcinogensthe ingestion of which takes them through the liver and theultimate reactive forms of which are more stable (100). Examples here include aery Ionitrile (101), pyrrol i/.idine alkaloids (22,102), and 4-aminobiphenyl (103). Discernment of the importance of the extrahepatic P-450s will require detailed investigations in each particular case under consideration, and thesituation may often vary with the particular P-450 form, the

2949




chemical, and the route of administration. It should also bepointed out that in extrahepatic tissues high levels of flavin-containing monooxygenase (104, 105) and prostaglandin syn-thase (106-108) can be found; often the enzymes form the sameproducts that are encountered in P-450 reactions and cautionshould be exercised in the assignments of roles of the variousenzymes.

3. At This Time We Know Far Less about the Roles of theVarious Human P-450 Proteins in Carcinogen Metabolism ThanWe Do for the Animal Models. In recent years there has beenan explosion of interest in the enzymology of the human enzymesystems. Several of the P-450 enzymes have now been purifiedfrom human liver, and reconstitution and immunochemicaltechniques have been used to make some in vitro assessmentsof roles of individual P-450s in the metabolism of certain drugsand carcinogens (11). One of the problems encountered in thework with P-450s arising from multigene families is that antibodies are often not as selective as one would hope and maynot distinguish among closely related proteins. It is possible, atleast in principle, to develop monoclonal antibodies that recognize individual proteins and inhibit catalytic activity. Thisapproach is not trivial, however, and must be accompanied byprotein chemistry studies in the case of relatively complexsystems. Another approach involves the expression of definedgenes in chimeric systems. To date this approach has beenapplied to some of the rat and rabbit P-450s (109-111) andmore recently to human P-450Mp,5 but the systems have not

been used to look at carcinogens. It should be remembered thatsuch expression systems, like reconstitution experiments, donot provide direct quantitative information on the relativeextent to which a single enzyme contributes to a particularreaction.

Other approaches can be used in in vivo situations. Restriction fragment length polymorphism analysis of peripheral bloodcell DN A can be used to identify the molecular basis of individual deficiencies in P-450s and to develop further diagnosticassays; these approaches can be extended to individuals withcancer. Another approach involves the use of selective inhibitorsof different P-450s. For instance, quinidine inhibits P-450DB-mediated reactions (112) and sulfaphenazole inhibits tolbuta-mide methyl hydroxylation (which is mediated by a proteinclosely related to but apparently distinct from P-450Mp) (113,114). These two inhibitors have A"Â¡values of about 10~7 M in

vitro, and both can be used ethically in in vivo settings withhumans. While it is easier to envision experiments involvingmetabolism of drugs than of carcinogens, there are some possibilities with the use of industrial chemicals of low carcinogenicpotential for which finite exposure limits exist and for whichmetabolism via various routes can be estimated in a sensitivemanner (i.e., CH2C12, acrylonitrile, vinyl chloride) (115, 116).

4. Which Enzyme Reactions Are Most Relevant to ChemicalCarcinogenesis? While dealing with the question of which P-450 enzymes are involved in individual reactions, we also needto give careful evaluation to which reactions we should be mostconcerned about. Many of the most pertinent questions inchemical carcinogenesis relate to mixtures of compounds, suchas those encountered in chemical waste dumps, tobacco smoke,etc. Although presenting mixtures of chemicals to enzymes maynot be particularly satisfying from the point of view of anenzymologist, perhaps it might be more relevant in terms ofdealing with practical problems. In considering which reactionsto assay, we generally draw on knowledge from pathways de-

5W. R. Brian, D. R. Umbenhauer, R. S. Lloyd, and F. P. Guengerich,

unpublished results.

rived in experimental animal systems and select particularreactions related to bioactivation and detoxication. However,in some cases our insight is not very complete and cautionneeds to be exercised. One alternative is measurement of anend point such as in vitro formation of a specific DNA adduci.However, care must also be exercised here, for in very few caseshas unambiguous evidence for the importance of an individualDNA adduct been obtained. Another alternative is to use mu-tagenesis or a linked SOS repair response as an end point (33).This avenue has a certain appeal, although it suffers in that theprecise chemistry remains undefined. However, this approachmight be advantageous in some situations.

Another strategy involves placing individual proteins intocells and then examining the effect of a single chemical on thetransformation (of the cells) to a malignant phenotype. Addinga P-450 protein can be done in one of two ways. The purifiedprotein can be inserted using "microinjection" procedures,

either using liposomes (117) or polyethylene glycol and eryth-rocyte ghosts (118, 119). The incorporation of the protein intothe endoplasmic reticulum and the expression of functionalactivity must be monitored, of course. Another approach is toinsert a cloned chimeric gene consisting of a P-450 complementary DNA downstream from a promoter and examineexpression of this gene (16, 52). In principle, the cells shouldbecome transformed under the appropriate conditions whenexposed to a particular procarcinogen if the inserted gene codesfor a P-450 important for bioactivation of that compound. Avariation on this theme might involve examination of the effectsof "anti-sense" oligonucleotides that block translation by hybridizing to segments of the individual P-450 mRNAs alreadyexpressed constitutively in cells. A further extension of theapproach would involve expression of individual P-450 proteinsin transgenic rodents. Although these experiments are stilltechnically difficult, the approach could yield information aboutthe contribution of individual gene products to the induction oftumors by individual chemicals. Further, organ-specific expression could be achieved with the use of promoters specific totissues in which the appropriate trans-acting factors are generated.

Although this overall approach would be useful, it still suffersfrom the deficiency that the results of each individual expression, even if positive, cannot be interpreted in absolute terms.Thus, a full appreciation of the roles of the various P-450enzymes to the tumor system under consideration would requirecomparative studies with several inserted genes.

5. How Adequate Are Our Animal Models for Prediction ofEvents in Humans? Most bioassays done on chemicals involvea few select strains of rats and mice. Some other species arealso used, most often when tumors are produced specific to anorgan in that species (e.g., dog bladder in the case of 2-naph-thylamine). The general tendency is to utilize models (anddoses) where the tumorigenicity of a compound can be demonstrated relative to a background level. In many cases suchsystems involve relatively complex promotional schemes (120)or the administration of test chemicals under physiologicalconditions such that a sort of "natural promotion" occurs [e.g.,

injection of compounds into newborn or weanling mice (78,99)]. If we hypothesize that P-450s and other enzymes havemajor roles in the activation of chemicals to ultimate carcinogens, then we must ask what the similarity of the enzymesencountered in those model situations is to those of interest inhuman populations at potential risk. If we do not feel thatappropriate models exist then we need to develop better ones,with some consideration given to metabolism. On the other

2950




hand, if the prevalent animal models continue to be acceptedfor bioassay work, then more effort should be directed towardsunderstanding the enzymes encountered there (see Paragraph2).

The involvement of P-450s in promotion was mentionedabove. This aspect has not received much thought outside ofthe areas of some polycyclic hydrocarbons and estragÃ³les andshould be considered further, particularly in regard to theprevailing animal models.

6. The Metabolism of Cancer Chemotherapeutic Agents Is anArea That Deserves More Consideration. The general considerations here differ from those encountered with most substrates; toxicity is desired with these compounds and the problem is to make them selective for certain cells (121, 122). Onecan, with some insight, exploit several properties of the tumorcells. For instance, they tend to have lowered levels of P-450s,or at least the major P-450s found in normal liver (123). Tumors(at least solid tumors) also tend to have low oxygen tension(124). These and other properties can be used to design moreeffective drugs which could be inactivated by normal cells butnot by tumor cells. To date the research on the identificationof specific P-450s in the metabolism of cancer chemotherapeu-tic agents has been limited to a few instances such as cyclo-phosphamide (125, 126), procarbazine (127), and l-(2-chlo-roethyl)-3-(cyclohexyl)-l-nitrosourea and l-(2-chloroethyl)-3-(fra/Js-4-methylcyclohexyl)-l-nitrosourea(128). The knowledgeof human enzymes involved in the metabolism of these andother chemotherapeutic agents is miniscule and much morecould be done. Further, only limited information is availableconcerning the role of P-450 enzymes in the multiple drugresistance phenomenon encountered in tumor cells (129, 130).Most cancer chemotherapeutic agents produce a number of sideeffects. The rates of metabolism of these drugs are also knownto vary considerably among different individuals (131). Thus, abetter understanding of the enzyme systems that process thesecompounds would be of great use in designing therapies thatwill avoid unwanted toxicities. Also, in some cases the drugsare metabolized too rapidly and either enhanced toxicity or lackof therapeutic effectiveness can result.

7. As We Accumulate More and More Information aboutDetails of the Enzymology and Chemistry Related to the Metabolism of Each Chemical, How Can We Keep This Knowledge inPerspective? This final point is perhaps more philosophical.Already we have amassed much information but not in the caseof any compound are our data complete. If we do all of theexperiments suggested in the above sections, will we have somuch to deal with that we will be unable to put our knowledgeinto any sort of perspective?

The problem has not really been that we have collected toomuch data, for the situation is clearly the opposite. However,it can be said that a large fraction of experiments have not beendesigned to put in vitro investigations into the context of in vivoanimal work. More emphasis is needed on systems in whichtumors have been shown to be produced. Thus, perhaps someof the emphasis should be shifted away from metabolism inrabbits (where the enzymology has been excellent but tumorsare rarely studied) to mice (where the enzymology has not beenhighly developed but many bioassays have been done).

Increasing attention should be paid to the experimental human studies, for in the last analysis this is the only system inwhich tumors are of much concern to us per se. In vitro experiments establishing the roles of individual enzymes in themetabolism of carcinogens are a first priority. These can becomplemented, in the case of the chemotherapeutic agents, by

in vivo studies; e.g., if an enzyme such as P-450MP is known tometabolize mephenytoin (132) and a role in, for example,procarbazine metabolism is assigned on the basis of in vitroexperiments, will individuals (undergoing therapy and highdoses of procarbazine) who are deficient in the ability to metabolize mephenytoin also clear procarbazine at decreased rates?EpidemiolÃ³gica! links must also be strengthened through phen-otyping studies. One of the most dramatic relationships betweena P-450-linked activity and a specific tumor lies in the report

of Ayesh et al. (89), which has been discussed above; however,some controversy exists concerning the reproducibility of thebasic observation (90, 91), and to date there is no evidence thata true metabolic relationship is an etiological factor. The relationships between drug oxidation phenotype and bladder cancerincidence reported by Kaisary et al. (93) also need furtherdevelopment.

In summary, the entire field of relating cancer susceptibilityto alterations in P-450-related catalytic activities is still full ofpromise. It would appear that many of the necessary researchtechniques have now been developed, enabling serious andcomprehensive studies to be carried out, preferably with a focuson a few select chemicals at first. The circumstantial evidencethat the enzymatic differences are important is strong. Moredecisive experiments done in animal models and humans shouldbe forthcoming soon.

Acknowledgments

Thanks are extended to Drs. Fred F. Kadlubar and Lawrence J.Marnett and several members of my research laboratory (Drs. Lisa A.Peterson, Ghazi A. Dannan, and William R. Brian and Joan L. Cmarik)for their comments on the article. I also thank Doris Harris for herhelp in preparation of the manuscript. This "Perspective" is dedicated

to two eminent cancer research scientists who have been taken from usin the past 2 years. Lubomir S. Hnilica, my former colleague andStallimeli Professor of Cancer Research at Vanderbilt University, wasan expert in the area of eliminatili research and my good friend; hisinsight and sense of humor are still missed. Elizabeth C. Miller, of theMcArdle Laboratory for Cancer Research at the University of Wisconsin, was truly a pioneer in the field of chemical carcinogenesis. I wasalways impressed by her high scientific standards, her enthusiasm, andthe encouragement she gave to me and others.

References

1. Mueller, G. C., and Miller, J. A. The metabolism of 4-dimethylaminoazo-benzene by rat liver homogenates. J. Biol. Chem., 176: 535-544, 1948.

2. Mueller, G. C., and Miller, J. A. The metabolism of methylated aminoazodyes. II. Oxidative demethylation by rat liver homogenates. J. Biol. Chem.,202:579-587, 1953.

3. Omura, T., and Sato, R. The carbon monoxide-binding pigment of livermicrosomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239:2370-2378, 1964.

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

5. Brown, R. R., Miller, J. A., and Miller, E. C. The metabolism of methylatedamino azo dyes. IV. Dietary factors enhancing demethylation in vitro. J.Biol. Chem., 209: 211-222, 1954.

6. Conney, A. II. 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., 16: 450-459, 1956.

7. Remmer, H. The acceleration of evipan oxidation and the demethylationof methylaminopyrine by barbiturates. Naunyn-Schmiedebergs Arch. Exp.Pathol. Pharmakol., 237: 296-307, 1957.

8. Sladek, N. E., and Mannering, G. J. Evidence for a new P-450 hemoproteinin hepatic microsomes from methylcholanthrene-treated rats. Biochem.Biophys. Res. Commun., 24:668-674, 1966.

9. Alvares, A. P., Schilling, G., Levin, W., and Kuntzman, R. Studies in theinduction of CO-binding pigments in liver microsomes by phÃ©nobarbitaland 3-methylcholanthrene. Biochem. Biophys. Res. Commun., 29: 521-526, 1967.

10. Guengerich, F. P. Enzymology of rat liver cytochromes P-450. In: F. P.

2951




Guengerich (ed.), Mammalian Cytochromes P-450, Vol. l, pp. 1-54. BocaRaton, FL: CRC Press, 1987.

11. Distlerath, L. M., and Guengerich, F. P. Enzymology of human liver 41.cytochromes P-450. In: F. P. Guengerich (ed.), Mammalian CytochromesP-450, Vol. l, pp. 133-198. Boca Raton, FL: CRC Press, 1987.

12. Guengerich, F. P. Cytochrome P-450 enzymes and drug metabolism. In: S.Bridges, L. F. Chasseaud, and G. G. Gibson (eds.), Progress in Drug 42.Metabolism, Vol. 10, Chap. 1, pp. 1-54. London: Taylor and Francis, 1987.

13. Nebert, D. W., Adesnik, M., Coon, M. J., Estabrook, R. W., Gonzalez, F.J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Kernper, B., Levin,W., Phillips, I. R., Sato, R., and Waterman, M. R. The P-450 gene 43.su per famil\. Recommended nomenclature. DNA, 6:1-11, 1987.

14. Niranjan, B. G., Wilson, N. M., Jefcoate, C. R., and Avadhani, N. G.Hepatic mitochondria! cytochrome P-450 system: distinctive features ofcytochrome P-450 involved in the activation of aflatoxin B, and 44.benzo(a)pyrene. J. Biol. Chem., 259: 12495-12501, 1984.

15. Ortiz de Montellano, P. R. Cytochrome P-450: Structure, Function, andMechanism. New York: Plenum Publishing Corp., 1986. 45.

16. Whitlock, J. P., Jr. The regulation of cytochrome P-450 gene expression.Annu. Rev. Pharmacol. Toxicol., 26: 333-369, 1986.

17. Guengerich, F. P. (ed.). Mammalian Cytochromes P-450, Vols. 1 and 2.Boca Raton, FL: CRC Press, 1987. 46.

18. Nebert, D. W., and Gonzalez, F. J. P-450 genes: structure, evolution, andregulation. Annu. Rev. Biochem., 56: 945-993, 1987.

19. Kadlubar, F. F., and Hammons, G. J. Role of cytochrome P-450 in melali 47.olism of chemical carcinogens. In: F. P. Guengerich (ed.), MammalianCytochromes P-450, Vol. 2, pp. 81-130. Boca Raton, FL: CRC Press, 1987.

20. Searle, C. E. (ed.). Chemical Carcinogens, Ed. 2. ACS Monograph 182, 48.Vols. 1 and 2. Washington, DC: American Chemical Society, 1984.

21. Nebert, D. W. Genetic control of carcinogen metabolism leading to individual differences in cancer risk. Biochimie (Paris), 60:1019-1029, 1978.

22. Boyd, M. R. Effects of inducers and inhibitors on drug-metabolizing en- 49.zymes and on drug toxicity in extrahepatic tissues. In: Excerpta Medica,Environmental Chemicals, Enzyme Function, and Human Diseases (CIBAFoundation Symposium 76), pp. 43-76. Basel, Switzerland: Excerpta Medica. 1980.

23. Weisburger, E. K. Metabolism and activation of chemical carcinogens. Mol. 50.Cell Biochem., 32:95-104, 1980.

24. Wright, A. S. The role of metabolism in chemical mutagenesis and chemicalcarcinogenesis. MutÃ¢t.Res., 75: 215-241, 1980.

25. Farber, E. Chemical carcinogenesis. N. Engl. J. Med., 305: 1379-1389,1981. 51.

26. Miller, E. C., and Miller, J. A. Searches for ultimate chemical carcinogensand their reactions with cellular macro molecules. Cancer (Phila.), 47:2327-2345, 1981.

27. Weinstein, I. B. Current concepts and controversies in chemical carcinogen- 52.esis. J. Supramol. Struct. Cell. Biochem., 77:99-120, 1981.

28. Conney, A. H. Induction of microsomal enzymes by foreign chemicals andcarcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Me- 53.mortal Lecture. Cancer Res., 42:4875-4917, 1982.

29. Weisburger, J. H., and Williams, G. M. Metabolism of chemical carcinogens. In: F. F. Becker (ed.). Cancer: A Comprehensive Treatise, Vol. 1, Ed. 54.2, pp. 241-333. New York: Plenum Publishing Corp., 1982.

30. Dipple, A., Michejda, C. J., and Weisburgrr, E. K. Metabolism of chemicalcarcinogens. Pharmacol. Then, 27: 265-296,1985. 55.

31. Kato, R. Metabolic activation of mutagenic heterocyclic aromatic aminesfrom protein pyrolysates. CRC Crit. Rev. Toxicol., 16: 307-348, 1986.

32. Schwab, G. E., and Johnson, E. F. Enzymology of rabbit cytochromes P- 56.450. In: F. P. Guengerich (ed.). Mammalian Cytochromes P-450, Vol. 1,pp. 55-105, Boca Raton, FL: CRC Press, 1987.

33. Shimada, T., and Nakamura, S-I. Cytochrome P-450 mediated activationof procarcinogens and promutagens to DNA-damaging products by measuring expression of umu gene in Salmonella typhimurium TA1535/pSK 57.1002. Biochem. Pharmacol., 36: 1979-1987, 1987.

34. Guengerich, F. P. Separation and purification of multiple forms of microsomal cytochrome P-450. Activities of different forms of cytochrome P-450towards several compounds of environmental interest. J. Biol. Chem., 252:3970-3979, 1977. 58.

35. Robertson, I. G. C., Zeiger E., and Goldstein, J. A. Specificity of rat livercytochrome P-450 isozymes in the mutagenic activation of benzo(a)pyrene,aromatic amines, and aflatoxin It, Carcinogenesis (Lond.), 4:93-96, 1983. 59.

36. Ishii, K., Maeda, K., Kamataki, T., and Kato, R. Mutagenic activation ofaflatoxin B, by several forms of purified cytochrome P-450. MutÃ¢t.Res.,774:85-88, 1986.

37. Shimada, T., Nakamura, S-I., Imaoka, S., and Funae, Y. Genotoxic and 60.mutagenic activation of aflatoxin B, by constitutive forms of cytochrome P-450 in rat liver microsomes. Toxicol. Appi. Pharmacol., 91: 13-21, 1987.

38. Kawano, S., Kamataki, T., Maeda, K., Kato, R., Nakao, T., and Mizoguchi,I. Activation and inactivation of a variety of mutagenic compounds by the 61.reconstituted system containing highly purified preparations of cytochromeP-450 from rat liver. Fundam. Appi. Toxicol., 5:487-498, 1985.

39. Guengerich, F. P., Butler, M. A., Macdonald, T. L., and Kadlubar, F. F.Oxidation of carcinogenic aryl amines by cytochrome P-450 enzymes. In:C. M. King, L. J. Romano, and D. Schuetzle (eds.), Carcinogenic and 62.Mutagenic Responses to Aromatic Amines and Nitroarenes, pp. 89-95.New York: Elsevier Scientific Publishing Co., Inc., 1987.

40. Frederick, C. B., Mays, J. B., Ziegler, D. M., Guengerich, F. P., andKadlubar, F. F. Cytochrome P-450 and flavin-containing monooxygenase-

2952

catalyzed formation of the carcinogen JV-hydroxy-2-aminofluorene, and itscovalent binding to nuclear DNA. Cancer Res., 42: 2671-2677, 1982.Hammons, G. J., Guengerich, F. P., Weis, C. C., Beland, F. A., andKadlubar, F. F. Metabolic oxidation of carcinogenic arylamines by rat, dog,and human hepatic microsomes and by purified flavin-containing and cytochrome P-450 monooxygenases. Cancer Res., 45: 3578-3585, 1985.Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D.M., and Conney, A. H. Metabolism of benzo(a)pyrene and benzo(a)pyrenederivatives to mutagenic products by highly purified hepatic microsomalenzymes. J. Biol. Chem., 251:4882-4890, 1976.Levin, W., Thomas, P. E., Oldfield, N., and Ryan, D. E. /V-Demethylationof .V-iiiirosiidimethyluniine catalyzed by purified rat hepatic microsomalcytochrome P-450: isozyme specificity and role of cytochrome b,. Arch.Biochem. Biophys., 248: 158-165, 1986.Patten, C. J., Ning, S. M., Lu, A. Y. H., and Yang, C. S. Acetone-induciblecytochrome P-450: purification, catalytic activity, and interaction with cytochrome *5. Arch. Biochem. Biophys., 251:629-638, 1986.Yamazoe, Y., Shimada, M., Maeda, K., Kamataki, T., and Kato, R. Specificity of four forms of cytochrome P-450 in the metabolic activation ofseveral aromatic amines and benzo(a)pyrene. Xenobiotica, 14: 549-552,1984.Kimura, T., Kodama, M., Nagata, C., Kamataki, T., and Kato, R. Comparative study on the metabolism of JV-methyl-4-aminoazobenzene by two formsof cytochrome P-448. Biochem. Pharmacol., 34: 3375-3377, 1985.Shimada, T., and Guengerich, F. P. Participation of a rat liver cytochromeP-450 induced by pregnenolone 16a-carbonitrile and other compounds inthe 4-hydroxylation of mephenytoin. Mol. Pharmacol., 28: 215-219, 1985.Beaune, P., Kremers, P. G., Kaminsky, L. S., De Graeve, J., and Guengerich,F. P. Comparison of monooxygenase activities and cytochrome P-450isozyme concentrations in human liver microsomes. Drug Metab. Dispos.,7*437-442, 1986.Kaminsky, L. S., Guengerich, F. P., Dannan, G. A., and Aust, S. D.Comparisons of warfarin metabolism by liver microsomes of rats treatedwith a series of polybrominated biphenyl congeners and by the component-purified cytochrome P-450 isozymes. Arch. Biochem. Biophys., 225: 398-404, 1983.Guengerich, F. P., Martin, M. W., Beaune, P. H., Kremers, P., Wolff, T.,and Waxman, D. J. Characterization of rat and human liver microsomalcytochrome P-450 forms involved in nifedipine oxidation, a prototype forgenetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 261:5051-5060, 1986.Kaminsky, L. S., and Guengerich, F. P. Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reducÃaseconcentrations as factors in microsomal metabolism of warfarin. Eur. J.Biochem., 149: 479-489, 1985.Zuber, M. X., Simpson, E. R., and Waterman, M. R. Expression of bovine17a-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1)cells. Science (Wash. DC), 234: 1258-1261, 1986.Hall, S. D., Guengerich, F. P., Branch, R. A., and Wilkinson, G. R.Characterization and inhibition of mephenytoin 4-hydroxylase activity inhuman liver microsomes. J. Pharmacol. Exp. Ther., 240: 216-222, 1987.Ortiz de Montellano, P. R., and Correia, M. A. Suicidal destruction ofcytochrome P-450 during oxidative drug metabolism. Annu. Rev. Pharmacol. Toxicol., 23:481-503, 1983.Fujino, T., Park, S. S., West, D., and Gelboin, H. V. Phenotyping ofcytochromes P-450 in human tissues with monoclonal antibodies. Proc.Nati. Acad. Sci. USA, 79: 3682-3686, 1982.Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G., Wilkinson,G. R., and Guengerich, F. P. Purification and characterization of the humanliver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phen-acetin O-deethylation, two prototypes for genetic polymorphism in oxidativedrug metabolism. J. Biol. Chem., 260:9057-9067, 1985.Kaminsky, L. S., Dannan, G. A., and Guengerich, F. P. Composition ofcytochrome P-450 isozymes from hepatic microsomes of C57BL/6 andDBA/2 mice assessed by warfarin metabolism, immunoinhibition, and ini-munoelectrophoresis with unii (nu cytochrome P-450). Eur. J. Biochem.,141:141-148, 1984.Guengerich, F. P. Preparation and properties of highly-purified cytochromeP-450 and NADPH-cytochrome P-450 reducÃasefrom pulmonary microsomes of untreated rabbits. Mol. Pharmacol., 13:911-923, 1977.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.Umbenhauer, D. R., Martin, M. V., Lloyd, R. S., and Guengerich, F. P.Cloning and sequence determination of a complementary DNA related tohuman liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase.Biochemistry, 26:1094-1099, 1987.Levitt, R. C., Pelkonen, O., Okey, A. B., and Nebert, D. W. Geneticdifferences in metabolism of polycyclic aromatic carcinogens and aromaticamines by mouse liver microsomes. Detection by DNA binding of metabolites and by mutagenicity in histidine-dependent Salmonella typhimuriumin vitro. J. Nati. Cancer Inst., 62:947-955, 1979.Nebert, D. W., Atlas, S. A., Guenthner, T. M., and Kouri, R. E. The Ahlocus: genetic regulation of the enzymes which metabolize polycyclic hydrocarbons and the risk for cancer. In: P. O. P. Ts'o and H. V. Gelboin (eds.),

Polycyclic Hydrocarbons and Cancer: Chemistry, Molecular Biology andEnvironment, Vol. 2, pp. 345-390. New York: Academic Press, 1978.




63. Levitt, R. C., Fysh, J. M., Jensen, N. M., and Nebert, D. W. The Ah locus:biochemical basis for genetic differences in brain tumor formation in mice.Genetics, 92: 1205-1210, 1979.

64. Nebert, D. W. Genetic differences in susceptibility to chemically inducedmyelotoxicity and leukemia. Environ. Health Perspect., 39:11-22, 1981.

65. Kouri, R. E., RÃ¼de,T. H., Joglekar, R., Dansette, P. M., Jerina, D. M.,Atlas, S. A., Owens, I. S., and Nebert, D. W. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: cocarcinogen which enhances 3-methyIcholanthrene-initiated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. CancerRes., 38: 2777-2783, 1978.

66. Poland, A., Palen, D., and Glover, E. Tumour production by TCDD in skinof HRS/J hairless mice. Nature (Lond.), 300: 217-273, 1982.

67. McLean, A. E. M., and Marshall, A. Reduced carcinogenic effects ofaflatoxin in rats given phenobarbitone. Br. J. Exp. Palhol.. 52: 322-329,1971.

68. Swenson, D. H., Lin, J-K., Miller, E. C., and Miller, J. A. Aflatoxin B,-2,3-oxide as a probable intermediate in the covalent binding of aflatoxinsBI and B2 to rat liver DNA and ribosomal RNA in vivo. Cancer Res., 37:172-181, 1977.

69. Tawfic, H. N. Studies on ear duct tumors in rats. Part II: Inhibitory effectsof methylchiilanthrvnc and 1,2-benzanthracene on tumor formation by 4-dimethylaminostilbene. Acta Pathol. Jpn., IS: 255-260, 1965.

70. Kunz, W., Schande, G., and Thomas, C. The effect of phÃ©nobarbitalandhalogenated hydrocarbons on nitrosamine carcinogenesis. Z. Krebsforsch.,72:291-304, 1969.

71. Uchida. E., and H irono, I. Effect of phÃ©nobarbitalon induction of liver andlung tumors by dimethylnitrosamine in newborn mice, ("Â¡Â¡inn,70:639-644,

1979.72. Richardson, H. l,., Stier, A. R., and Borsos-Nachtnebel, E. Liver tumor

inhibition and adrenal histologie responses in rats to which 3'-methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene were simultaneouslyadministered. Cancer Res., 12: 356-361, 1952.

73. Miller, E. C., Miller, J. A., Brown, R. R., and MacDonald, J. C. On theprotective action of certain polycyclic aromatic hydrocarbons against carcinogenesis by aminoazo dyes and 2-acetylaminofluorene. Cancer Res., 7*:469-477, 1958.

74. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishi-zuka, Y. Direct activation of calcium-activated, phospholipid-dependentprotein kinase by tumor-promoting phorbol esters. J. Biol. ("hem., 257:7847-7851, 1982.

75. Verma, A. K., Conrad, E. A., and Boutwell, R. K. Differential effects ofretinole acid and 7,8-benzoflavone on the induction of mouse skin tumorsby the complete carcinogenic process and by the initation-promotion regimen. Cancer Res., 42:3519-3525, 1982.

76. Verma, A. K., Garcia, C. T., Ashendel, C. L., and Boutwell, R. K. Inhibitionof 7-bromoethylbenz(a)anthracene-promoted mouse skin tumor formationby retinoic acid and dexamethasone. Cancer Res., 43:3045-3049, 1983.

77. Scribner, J. D., Scribner, N. K., Mcknight, B., and Mottet, N. K. Evidencefor a new model of tumor progression from carcinogenesis and tumorpromotion studies with 7-bromoethylbenz(a)anthracene. Cancer Res., 43:2034-2041, 1983.

78. Boberg, E. W., Liem, A., Miller, E. C., and Miller, J. A. Inhibition bypentachlorophenol of the initiating and promoting activities of 1'-hydrox-ysafrole for the formation of enzyme-altered foci and tumors in rat liver.Carcinogenesis (Lond.), 8: 531-539, 1987.

79. Kellermann, G., Luyten-Kellermann, M., and Shaw, C. R. Genetic variationof aryl hydrocarbon hydroxylase in human lymphocytes. Am. J. Hum.Genet., 25: 327-331, 1973.

80. Kellermann, G., Shaw, C. R., and Luyten-Kellermann, M. Aryl hydrocarbonhydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med.,29Â«:934-937, 1973.

81. Paigen, B., Ward, E., Rei Ih, A., Gurtoo, H. L., Minowada, J., Steenland,K., Havens, M. B., and Sartori, P. Seasonal variation of aryl hydrocarbonhydroxylase activity in human lymphocytes. Cancer Res., 41: 2757-2761,1981.

82. Kouri, R. E., McKinney, C. E., Slomiany, D. J., Snodgress, D. R., Wray,N. P., and McLemore, T. L. Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopre-served lymphocytes. Cancer Res., 42: 5030-5037, 1982.

83. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. Human P.-450 genesequence and correlation of mRNA with genetic differences in benzo-(a)pyrene metabolism. NucÃ.Acids Res., 13:4503-4520, 1985.

84. KÃ¤rki,N. T., Pokela, R., Nuutinen, L., and Pelkonen, O. Aryl hydrocarbonhydroxylase in lymphocytes and lung tissue from lung cancer patients andcontrols. Int. J. Cancer, 39: 565-570, 1987.

85. De Flora, S., Petruzzelli, S., Camoirano, A., Bennicelli, C., Romano, M.,Rindi, M., Ghelarducci, L., and Giuntini, C. Pulmonary metabolism ofmutagens and its relationship with lung cancer and smoking habits. CancerRes., 47:4740-4745, 1987.

86. Idle, J. R., Mahgoub, A., Sloan, T. P., Smith, R. L., Mbanefo, C. O., andBababunmi, E. A. Some observations on the oxidation phenotype status ofNigerian patients presenting with cancer. Cancer Lett., 77: 331-338, 1981.

87. Wolff, T., Distlerath, L. M., Worthington, M. T., Groopman, J. D.,Mammons, G. J., Kudlubar, F. F., Prough, R. A., Martin, M. V., andGuengerich, F. P. Substrate specificity of human liver cytochrome P-450debrisoquine 4-hydroxylase probed using immunochemical inhibition andchemical modeling. Cancer Res., 45: 2116-2122, 1985.

88. Plummer, A., Boobis, A. R., and Davies, D. S. Is the activation of aflatoxin

B, catalyzed by the same form of cytochrome P-450 as that 4-hydroxylatingdebrisoquine in rat and/or man? Arch. Toxicol., 58: 165-170, 1986.

89. Ayesh, R., Idle, J. R., Ritchie, J. C., Crothers, M. J., and Hetzel, M. R.Metabolic oxidation phenotypes as markers for susceptibility to lung cancer.Nature (Lond.), 312: 169-170, 1984.

90. Drakoulis, N., Minks, T., Ploch, M., Otte, F., Heinemeyer, G., Kampf, D.,Loddenkemper, R., and Roots, I. Questionable association of debrisoquinehydroxilator [sic] phenotype and risk for bronchial carcinoma. Acta Phar-macol. Toxicol., 59 (Suppl. 5): 220, 1986.

91. Harris, C. C., Weston, A., Willey, J. C., Trivers, G. E., and Mann, D. L.Biochemical and molecular epidemiology of human cancer: indicators ofcarcinogen exposure, DNA damage, and genetic predisposition. In: D. J.Birkett (ed.), Microsomes and Drug Oxidations (Proceedings of the SeventhInternational Symposium). London: Taylor and Francis, in press, 1988.

92. Ritter, J., Somasundaram, R., Heinemeyer, G., and Roots, I. The debrisoquine hydroxilation [\i'c| phenotype and the acetylator phenotype as genetic

risk factors for the occurrence of larynx and pharynx carcinoma. ActaPharmacol. Toxicol., 59 (Suppl. 5): 221, 1986.

93. Kaisary, A., Smith, P., Jaczq, E., McAllister, C. B., Wilkinson, G. R., Ray,W. A., and Branch, R. A. Genetic predisposition to bladder cancer: abilityto hydroxylate debrisoquine and mephenytoin as risk factors. Cancer Res.,47: 5488-5493, 1987.

94. Yang, C. S. Research on esophageal cancer in China: a review. Cancer Res.,Â¥0:2633-2644, 1980.

95. Trzaskos, J., Kawata, S., and Gaylor, J. C. Microsomal enzymes of cholesterol biosynthesis: purification of lanosterol 14a-methyl demethylase cytochrome P-450 from hepatic microsomes. J. Biol. Chem., 261:14651-14657,1986.

96. Kellis, J. T., Jr., and Vickery, L. E. Purification and characterization ofhuman placenta! aromatase cytochrome P-450. J. Biol. Chem., 262: 4413-4420, 1987.

97. Graves, P. E., Kaminsky, L. S., and Halpert, J. Evidence for functional andstructural multiplicity of pregnenolone-16a-carbonitrile-inducible cytochrome P-450 isozymes in rat liver microsomes. Biochemistry, 26: 3887-3894, 1987.

98. Baron, J., Voigt, J. M., Whitter, T. B., Kawabata, T. T., Knapp, S. A.,Guengerich, F. P., and Jakoby, W. B. Identification of intratissue sites forxenobiotic activation and detoxication. Adv. Exp. Med., 797:119-144. NewYork: Plenum Publishing Corp., 1986.

99. 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 (+) frans-7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tet-rahydrobenzo[a]pyrne is an ultimate carcinogen in newborn mice. CancerRes., 38: 354-358, 1978.

100. Guengerich, F. P., and Liebler, D. C. Enzymatic activation of chemicals totoxic metabolites. CRC Crit. Rev. Toxicol., 14: 259-307, 1985.

101. Hogy, L. L., and Guengerich, F. P. In vivo interaction of acrylonitrile and2-cyanoethylene oxide with DNA in rats. Cancer Res., 46: 3932-3938,1986.

102. Mattocks, A. R., and Driver, H. E. Metabolism and toxicity of anacrotine,a pyrrolizidine alkaloid, in rats. <'hem. Biol. Interact., 63: 91-104, 1987.

103. Â«allindar,F. F., Dooley, K. L., Benson, R. W., Roberts, D. W., Butler, M.A., Teitel, C. H., Bailey, J. R., and Young, J. F. Pharmacokinetic model ofaromatic amine-induced urinary bladder carcinogenesis in beagle dogs administered 4-aminobiphenyl. In: C. M. King, L. J. Romano, and D. Sehnetzie (eds.). Carcinogenic and Mutagenic Responses to Aromatic Amines andNitroarenes, pp. 173-180. New York: Elsevier Scientific Publishing Co.,Inc., 1987.

104. Dannan, G. A., and Guengerich, F. P. Immunochemical comparison andquantitation of microsomal flavin-containing monooxygenases in varioushog, mouse, rat, rabbit, dog, and human tissues. Mol. Pharmacol., 22:787-794, 1982.

105. Williams, D. E., Ziegler, D. M., Nordin, D. J., Hale, S. E., and Masters, B.S. S. Rabbit lung flavin-containing monooxygenase is immunochemicallyand catalytically distinct from the liver enzyme. Biochem. Biophys. Res.Commun., 125: 116-122, 1984.

106. Krauss, R. S., and Eling, T. E. Arachidonic acid-dependent cooxidation: apotential pathway for the activation of chemical carcinogens in vivo.Biochem. Pharmacol., 33: 3319-3324, 1984.

107. Battista, J. R., and Mameli, L. J. Prostaglandin H synthase-dependentepoxidation of aflatoxin B,. Carcinogenesis (Lond.), 6: 1227-1229, 1985.

108. Maraett, L. J. Arachidonic acid metabolism and tumor initiation. In: L. J.Marnett (ed.), Arachidonic Acid Metabolism and Tumor Initiation, pp. 39-82. The Hague: Martinus Nihoff Publishing, 1985.

109. Sakaki, T., Oeda, K., Miyoshi, M., and Ohkawa, H. Characterization of ratcytochrome P-450Mc synthesized in Saccharomyces cerevisiae. J. Biochem.(Tokyo), 9Â«:167-175, 1985.

110. Imai, Y. Cytochrome P-450 related to P-4504 from phÃ©nobarbitaltreatedrabbit liver: molecular cloning of cDNA and characterization of cytochromeP-450 obtained by its expression in yeast cells. J. Biochem. (Tokyo), 101:1129-1139,1987.

111. Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J.Rat testosterone 7a-hydroxylase: isolation, sequence, and expression ofcDNA and its developmental regulation and induction by 3-methylcholan-threne. J. Biol. Chem., 262; 2787-2793, 1987.

112. Guengerich, F. P., Mu1er-linoci), D., and Blair, I. A. Oxidation of quinidineby human liver cytochrome P-450. Mol. Pharmacol., 30: 287-295, 1986.

113. Knodell, R. G., Hall, S. D., Wilkinson, G. R., and Guengerich, F. P. Hepatic

2953



CYTOCHROME P-Â»50AND CANCER

metabolism of tolbutamide: characterization of the form of cytochrome P-450 involved in methyl hydroxylation and relationship to in vivodisposition.J. Pharmacol. Exp. Ther., 241:1112-1119, 1987.

114. Miners, J. (>., Robson, R. A., Smith, K. J., McManus, M. E., and Birkett,D. J. Relationship between tolbutamide hydroxylation and other cytochromeI' 450 dependent oxidations in human liver microsomes. In: D. J. Birkett(ed.), Microsomes and Drug Oxidations (Seventh International Symposium). London: Taylor and Francis, in press, 1988.

115. Peterson, J. E. Modeling the uptake, metabolism, and excretion of dichlo-romethane by man. Am. Ind. Hyg. Assoc. J., 39:41-47, 1978.

116. Buchter, A., Bolt, H. M., Filser, J. G., Goergens, H. W., Laib, R. J., andBolt, W. Pharmakokinetik und Karzinogenese von Vinylchlorid. Verh.Dtsch. Ges. Arbeitsmed., 18:111-124,1978.

117. Straubinger, R. M., Hong, K., Friend, D. S., and Papahadjopoulos, D.Endocytosis of liposomes and intracellular fate of encapsulated molecules:encounter with a low pH compartment after internalization in coatedvesicles. Cell, 32: 1069-1079, 1983.

118. Docherty, P. A., and Aronson, N. N., Jr. Injection of proteins into primaryrat hepatocytes by erythrocyte-mediated techniques. Biochem. Biophys. Res.Commun., 109: 527-532,1982.

119. McElligott, M. A., and Dice, J. F. Erythrocyte-mediated microinjection, atechnique to study protein degradation in muscle cells. Biochem. J., 276:559-566, 1983.

120. Goldsworthy, T. I... Hanigan, M. II., and Pitot, H. C. Models of hepato-carcinogenesis in the ratâ€”contrastsand comparisons. CRC Crii. Rev. Tox-icol., Ã•7; 61-89, 1986.

121. Cline, M. J., and Haskell, C. M. Factors affecting the usefulness of drugs.Cancer Chemotherapy, Ed. 3, Chap. 2, pp. 13-30. Philadelphia: W. B.SaundersCo., 1980.

122. Erlichman, C., and Kerr, I. G. Antineoplastic drugs. In: H. Kant, W. H. E.Roschlau, and E. M. Sellers (eds.), Principles of Medical Pharmacology,Ed. 4, Chap. 59, pp. 699-710. Toronto, Canada: University of TorontoPress, 1985.

123. El Mouelhi, M., Didolkar, M. S., Elias, E. G., Guengerich, F. P., andKauffman, F. C. Hepatic drug metabolizing enzymes in primary and sec

ondary tumors of human liver. Cancer Res., 47:460-466, 1987.124. Kennedy, K. A., Teicher, B. A., Rockwell, S., and Sartore-Ili, A. C. Chema

therapeutic approaches to cell populations of tumors. In: A. C. Salterelli,J. S. Lazo, and J. R. Benino (eds.), Molecular Actions and Targets forCancer Chemotherapeutic Agents, Bristol Myers Cancer Symposium 3, pp.85-101. New York: Academic Press, 1981.

125. MÂ¡trincilo.A. J., Bansal, S. K., Paul, B., Koser, P. L., Love, J., Struck, R.F., and Gurtoo, H. L. Metabolism and binding of cyclophosphamide andits metabolite acrolein to rat hepatic microsomal cytochrome P 450. CancerRes., 44:4615-4621, 1984.

126. Struck, R. F., Kari, P., Kalin, J., Montgomery, J. A., Mannello, A. J.,Love, J., Bansal, S. K., and Gurtoo, H. L. Metabolism of cyclophosphamideby purified cytochrome P-450 from microsomes of phenobarbital-treatedrats. Biochem. Biophys. Res. Commun., 120: 390-396, 1984.

127. Prough, R. A., Brown, M. I., DamiÃ¡n,G. A., and Guengerich, F. P. Majorisozymes of rat liver microsomal cytochrome P-450 involved in the N-oxidation of Ar-isopropyl-a-(2-methylazo)-p-toluamide, the azo derivative ofprocarbazine. Cancer Res., 44: 543-548, 1984.

128. Potter, D. W., Levin, W., Ryan, D. E., Thomas, P. E., and Reed, D. J.Stereoselective monooxygenation of carcinostatic l-(2-chloroethyl)-3-(cy-clohirxyI)-1-nitrosourea and 1-(2-diloroet hyI) -3-(frun.v-4-methyIcyclohcxy I)-1-nitrosourea by purified cytochrome P-450 isozymes. Biochem. Pharmacol., 33:609-613, 1984.

129. Cowan, K. H., Batist, G., Tulpule, A., Sinha, B. K., and Myers, C. E.Similar biochemical changes associated with multidrug resistance in humanbreast cancer cells and carcinogen-induced resistance to xenobiotics in rats.Proc. Nati. Acad. Sci. USA, S3:9328-9332, 1986.

130. Carr, B. I. Pleiotropic drug resistance in hepatocytes induced by carcinogensadministered to rats. Cancer Res., 47: 5577-5583, 1987.

131. Colvin, M., and Grochow, L. B. Clinical pharmacokinetics of cyclophosphamide. Clin. Pharmacokinet., 4: 380-394, 1979.

132. Shimada, T., MÂ¡sono,K. S., and Guengerich, F. P. Human liver microsomalcytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism: purification and characterizationof two similar forms involved in the reaction. J. Biol. ( 'hem., 261:909-921,

1986.

2954



1988;48:2946-2954. Cancer Res F. Peter Guengerich Carcinogenesis and Cancer ChemotherapyRoles of Cytochrome P-450 Enzymes in Chemical

Updated version

http://cancerres.aacrjournals.org/content/48/11/2946.citation

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications


http://cancerres.aacrjournals.org/content/48/11/2946.citation

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


Documents

Roles of Cytochrome P-450 Enzymes in Chemical ......rabbit P-450 1 and human P-450MP (60). As mentioned above, several forms of P-450 are found in the liver of each animal species,