Chemoprevention by Inducersof Carcinogen Detoxication EnzymesThomas W. KenslerDepartment of Environmental Health Sciences, The Johns HopkinsUniversity, Baltimore, MarylandOne of the major mechanisms of chemical protection against carcinogenesis, mutagenesis, andother forms of toxicity mediated by electrophiles is the induction of enzymes involved in theirmetabolism, particularly phase 2 enzymes such as glutathione S-transferases (GSTs), uridinediphosphate-glucuronosyltransferases, and NAD(P)H:quinone reductase. Furthermore, inductionof phase 2 enzymes appears to be a sufficient condition for obtaining chemoprevention and can

be achieved in many target tissues by administering any of a diverse array of naturally occurringand synthetic chemical agents. One class of chemopreventive agents, 1,2-dithiole-3-thiones, was

developed on the basis of their potent activity in rodent tissues as inducers of GSTs. Asubstituted dithiolethione, oltipraz 14-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thionel, is an effectiveinhibitor of aflatoxin Bl-mediated hepatocarcinogenesis in the rat. Oltipraz produces dramaticdecreases in the levels of aflatoxin-DNA adducts in the liver as well as in the urinary levels of thedepurination product aflatoxin-N7-guanine. Corresponding increases are seen in the biliaryelimination of aflatoxin-glutathione conjugates. Administration of oltipraz results in 3- to 4-foldincreases in hepatic cytosolic GST activities and mRNA levels for some x, p and X isoforms.Nuclear run-on assays have indicated that oltipraz treatment elevates rates of transcription ofsome GST subunits. In the rat, induction of phase 2 enzymes by oltipraz is mediated, at least inpart, through the antioxidant response element in the 5' flanking region of these genes. Althougholtipraz has a very short plasma half-life, elevations in the levels of some GST isoforms can persistup to 1 week after dosing with oltipraz. Concordantly, intermittent dosing schedules (i.e., once a

week) are nearly as effective as daily interventions for inhibition of aflatoxin-mediated hepatictumorigenesis. The protective efficacy of daily and weekly administration of oltipraz to people inQidong, People's Republic of China, who are at high risk for aflatoxin exposure and subsequentdevelopment of hepatocellular carcinoma, is currently under evaluation. Environ HealthPerspect 1 05(Suppl 4):965-970 (1997)

Key words: glutathione S-transferases, NAD(P)H:quinone reductase, enzyme induction,oltipraz, 1,2-dithiole-3-thione, aflatoxin B1, biomarkers

IntroductionThere are many potential strategies forchemical protection against the multiplestages of carcinogenesis [see reviews byWattenberg (1) and De Flora and Ramel(2)]. However, in the majority of experi-mental systems, protection has beenachieved by administering the chemo-preventive agent prior to and/or concur-rent with the exposure to the carcinogen.Given this temporal relationship between

administration of anticarcinogen and car-cinogen it seems likely that these agents actprincipally by affecting the metabolism anddisposition of carcinogens, thereby alteringevents critical to the initial interactions ofcarcinogens with biomolecules. Using thisexperimental approach, it has been possibleto document protection against a diversearray of chemical carcinogens acting at dif-ferent target organ sites. Important classes of

This paper is based on a presentation at the symposium on Mechanisms and Prevention of EnvironmentallyCaused Cancers held 21-25 October 1995 in Santa Fe, New Mexico. Manuscript received at EHP 16 April1996; accepted 20 September 1996.

The author gratefully acknowledges the contributions of numerous colleagues to the oltipraz experimentaland clinical studies. Financial support for our studies on cancer chemoprevention comes from the NationalInstitutes of Health (CA39416, CA44530, CA-N01-CN-25437, and ES06052, and Center Grant ES03819).

Address correspondence to Dr. T.W. Kensler, Department of Environmental Health Sciences, JohnsHopkins School of Hygiene and Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. Telephone: (410)955-4712. Fax: (410) 955-0116. E-mail: tkensler@phnet.sph.jhu.edu

Abbreviations used: AFBj, aflatoxin B1; Ah, aryl hydrocarbon; BHA, butylated hydroxyanisole; BHT, butylatedhydroxytoluene; GST, glutathione S-transferase.

chemopreventive agents that modulate themetabolic processing of carcinogens includephenolic antioxidants, indoles, isothio-cyanates, coumarins, flavones, allyl sulfides,dithiocarbamates, and dithiolethiones.A key component in understanding the

initial events of carcinogenesis was therecognition that many chemical carcino-gens are not chemically reactive per se, butmust undergo metabolic activation to formelectrophilic reactants (3). These reactivespecies can interact with nucleophilicgroups in DNA to induce point mutationsand other genetic lesions, thus leading toactivation of protooncogenes and inactiva-tion of tumor suppressor genes. Theimportance of metabolic activation in car-cinogenesis is highlighted by the fact thattarget organ specificities and even speciessusceptibilities can be determined throughthe presence or absence of metabolic path-ways. The metabolism of chemicals toproximate carcinogens often involves aninitial two-electron oxidation to a hydroxy-lated or epoxidated product and is typicallycatalyzed by the cytochrome P450 system.Collectively, the enzymes that catalyze theformation of these reactive intermediatesare termed phase 1 enzymes. Cells also havea variety of enzymatic and nonenzymaticmechanisms that protect against damage byelectrophilic metabolites. A number ofenzymes transfer or conjugate variousendogenous substrates, such as glutathione,glucuronide, and sulfate, to the products ofphase 1 metabolism. These phase 2 reac-tions, which often add large polar mole-cules to the primary metabolite, generallylimit further biotransformation by enhanc-ing elimination, thereby leading to detoxi-cation. Thus, the amount of ultimatecarcinogen available for interaction with itstarget represents, in part, a balance betweencompeting activating and detoxifying reac-tions. While this balance is under geneticcontrol, it is easily modulated by a varietyof factors including nutritional status, age,hormones, and exposure to drugs or otherxenobiotics (4). In this setting, chemopre-ventive agents can profoundly modulate theconstitutive metabolic balance betweenactivation and inactivation of carcinogensthrough their actions on both phase 1 andphase 2 enzymes. This review considers thegeneral role for inducers of electrophiledetoxication enzymes, principally the phase2 enzymes, as anticarcinogens. To illustratethe effectiveness of this strategy, adiscussion is also presented on the actions

Environmental Health Perspectives - Vol 105, Supplement 4 * June 1997 965

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by PubMed Central

T.W. KENSLER

of one class of selective inducers of phase 2enzymes, the dithiolethiones, againstaflatoxin-induced hepatocarcinogenesis.

Mechanisms of Phase 2Enzyme InductionIt has been known for several decades thatantioxidants such as butylated hydroxy-toluene (BHT), butylated hydroxyanisole(BHA), and ethoxyquin exert an anticar-cinogenic effect when given simultaneously(or prior to or both) with a carcinogen.One of the earliest studies to indicate a rolefor the induction of phase 2 enzymes, par-ticularly glutathione S-transferases (GSTs),in the protective actions of these antioxi-dants was that of Benson and co-workers(5). They showed that liver cytosols fromBHA- or ethoxyquin-fed rats or miceexhibited much higher GST activities thancontrols and that cytosols prepared fromthe livers of these rodents eliminated themutagenic activity in urine from micetreated with benzo[a]pyrene. Subsequentstudies demonstrated that dietary adminis-tration of antioxidants increased GSTactivity in extrahepatic tissues such as lung,stomach, small intestine, and kidney (6).Substantial evidence now supports the viewthat induction of phase 2 enzymes is a crit-ical and sufficient mechanism to engenderprotection against the toxic and carcino-genic actions of reactive intermediates. Theevidence includes the following:* Many chemopreventive agents are most

effective if administered prior to and/orconcurrent with carcinogens.

* Treatment with chemopreventiveagents profoundly alters carcinogenmetabolism.

* Induced phase 2 enzymes inactivateelectrophiles (ultimate carcinogens).

* chemoprevention is achieved against awide variety of carcinogens, suggestinga low specificity mechanism.

* Enzyme induction and chemopreven-tion are produced by the same com-pounds (of many chemical classes),occur at similar doses, and have similartissue specificities.

* Overexpression of glutathione S-trans-ferase by cDNA transfection protectscells against carcinogen toxicity.

* Deficiencies in the levels of expressionof glutathione S-transferases may beimportant determinants for susceptibil-ity to cancer in humans.

* Monitoring enzyme induction has ledto the recognition or isolation of novelchemopreventive agents.

Measurement of phase 2 enzymeinduction has led to the isolation of twoanticarcinogenic terpenoids, kahweol palmi-tate and cafestrol palmitate, from green cof-fee beans (7); isolation of the isothiocyanatesulforaphane as the principal and verypotent phase 2 enzyme inducer from broc-coli, and demonstration of its ability toblock dimethylbenzanthracene-inducedmammary tumorigenesis in rats (8,9); andprediction of the anticarcinogenic effects of1,2-dithiole-3-thiones, including oltipraz(4-methyl-5-(2-pyrazinyl)- 1 ,2-dithiole-3-thione), which is now in clinical cancerchemoprevention trials (10-12).

Initial studies on the possible molecularmechanisms of induction of GSTs byantioxidants were conducted by Pearson etal. (13), who observed a 20-fold increase inmRNA for the major GST in the livers ofmice several days after feeding 0.75%BHA. Benson and colleagues subsequentlyreported that significant increases inmRNA levels could be observed as early as24 hr after placing mice on the BHA-sup-plemented diet (14). More recently,Pearson et al. (15) have studied the mecha-nisms of tissue-specific induction ofmurine GST mRNAs by BHA. In thesestudies, measurements of transcriptionrates in isolated nuclei indicated thatincreased mRNA levels were due toincreased rates of transcription.

The molecular mechanisms regulatingthe transcriptional activation of phase 2enzymes by antioxidants and other induc-ers have also been investigated. As origi-nally proposed by Wattenberg (1), twofamilies of phase 2 enzyme inducers exist,based upon their ability to elevate phase 1enzymatic profiles. Prochaska and Talalay(16) have coined the terms bifunctionaland monofunctional inducers to describethese compounds (Figure 1). Bifunctionalinducers (e.g., polycyclic hydrocarbons,dioxins, azo dyes, flavones) can all be char-acterized as large planar polycyclic aromat-ics; they elevate phase 2 as well as selectedphase 1 enzymatic activities, such as arylhydrocarbon hydroxylase. These com-pounds are potent ligands for the arylhydrocarbon (Ah) receptor, and the directparticipation of the Ah receptor in the acti-vation of Ah hydroxylase gene transcrip-tion has been demonstrated (17).Moreover, since phase 2 enzyme inducibil-ity by bifunctional inducers segregates inmice that possess functional Ah receptors,it had been presumed that these enzymeswere under the direct control of the Ahreceptor. Monofunctional inducers (phe-nols, lactones, isothiocyanates, dithiocarba-mates, and 1,2-dithiole-3-thiones) elevatephase 2 enzymatic activities without signif-icantly elevating phase 1 activities and donot possess an obvious defining structural

Figure 1. Mechanisms of enzyme induction by monofunctional and bifunctional inducers in the rat. SH, sulfhydrylgroup. Monofunctional inducers (e.g., oltipraz) transcriptionally activate phase genes such as GSTthrough theantioxidant response element (ARE). Bifunctional inducers, e.g., polycyclic aromatic hydrocarbons (PAHs) transcrip-tionally activate phase 1 and phase 2 genes directly through the xenobiotic response element (XRE). Some bifunc-tional inducers can also undergo metabolism to intermediates that can mediate gene expression through the ARE.Adapted from Prochaska and Talalay (16).

Environmental Health Perspectives * Vol 105, Supplement 4 * June 1997966

CHEMOPREVENTION BY PHASE 2 ENZYME INDUCERS

characteristic. Since Ah-dependent phase 1enzymes can activate procarcinogens totheir ultimate reactive forms, it is antic-ipated that monofunctional inducerswould be more desirable candidates forchemoprevention in man.

Early studies with alkyl ethers ofhydroquinones in vivo and diphenols invitro suggested that diphenols such as BHAmediate their inductive effects via a chemi-cal signal (18,19). It was also suggested thatbifunctional inducers induce phase 2enzymes in part via a metabolic cascade,wherein bifunctional inducers were metab-olized by induced phase 1 enzymes tospecies resembling monofunctional induc-ers. Talalay et al. (20) have identified thechemical signal present in some monofunc-tional inducers: the presence or acquisitionof an electrophilic center. Many com-pounds are Michael reaction acceptors (e.g.,an olefin conjugated to an electron with-drawing group) and potency is generallyparalleled by their efficiency as Michaelreaction acceptors. These generalizationscan account for the inducer activity ofmany types of chemopreventive agents andhave led to the identification of other novelclasses of inducers, including acrylates,fumarates, maleates, vinyl ketones, andvinyl sulfones. Other classes of monofunc-tional inducers, notably peroxides, vicinaldimercaptans, heavy metals, arsenicals, anddithiolethiones, exhibit a common capacityfor reaction with sulfhydryls by eitheroxidoreduction or alkylation (21).

Several regulatory elements controllingthe expression and inducibility of the Yasubunit of murine and rat GSTs by bifunc-tional and monofunctional inducers havebeen characterized (22,23). A 41-bp ele-ment in the 5'-flanking region of the ratGST Ya gene, termed the antioxidantresponse element, and a homologous ele-ment in 5'-region of the murine Ya gene,designated the electrophile response ele-ment, have been identified using a series of5' deletion mutants fused to the chloram-phenicol acetyl transferase gene and thentransfected into hepatoma cells. Prestera etal. (21) have observed that many classes ofmonofunctional inducers stimulate expres-sion of a reporter gene through this 41-bpelectrophile-antioxidant enhancer element.DNA footprinting and gel shift assays haverecently established specific interactionsbetween nuclear proteins and the elec-trophile-antioxidant regulatory element;however, the identity and exact role of theseproteins in the induction pathway remainsto be elucidated (24).

Assessment of Phase 2Enzyme InductionA number of approaches have been used toassess phase 1 enzyme induction and inhi-bition in humans, including measurementof the pharmacokinetics of drug probesand determination of changes in the dispo-sition of endogenous substrates for thecytochrome P450s of interest (25). Theseapproaches have been less useful for assess-ing phase 2 induction, in part because therate-limiting step in their overall metabo-lism is often the phase 1 enzyme compo-nent. Nonetheless, pharmacologic anddietary manipulations have been shown tomodify the phase 2 metabolism ofantipyrine, phenacetin, oxazepam, andacetaminophen in humans (25,26).Acetaminophen undergoes three conjuga-tion reactions: glucuronidation, sulfation,and glutathione addition. Measurement ofthe fractional clearances to these metabo-lites provides a simultaneous, three-waymeasure of drug conjugation. Miners et al.(27) observed that treatment with sulphin-pyrazone or anticonvulsant drugs selec-tively enhanced both glucuronidation andmercapturic acid formation from aceta-minophen. The mercapturic acid is derivedfrom the initial glutathione adduct.Increases in urinary thioether excretion(mercapturic acid and other products of glu-tathione conjugation) have been observedfollowing exposure of humans to cigarettesmoke, cancer chemotherapeutic drugs, andindustrial chemicals (28). However, thequantitative relations between enzymeinduction and thioether excretion have notbeen defined and the overall utility of thisapproach remains to be established. Directmeasurements of phase 2 enzyme activitiesin blood cells and serum have also been usedto assess enzyme induction. In a recentlyreported clinical study, small but significantincreases in plasma levels of a-class GSTwere observed in volunteers consuming adiet enriched in brussels sprouts (29).Earlier studies in mice fed BHA indicatedthat plasma levels of GST and quinonereductase correlated with, but underesti-mated, increased activity of these enzymesin liver (30). Quinone reductase activity isinduced in human peripheral bloodlymphocytes by several classes of mono-functional inducers, including dithiolethi-ones (31); ongoing phase I clinical studieswith oltipraz indicate that some phase 2enzyme mRNA levels and activities areincreased in peripheral lymphocytes of indi-viduals receiving the drug (12,32). Anintriguing approach for assessing the

pharmacodynamic action of enzyme induc-ers is highlighted by the recent work ofSreerama et al. (33), who reported that lev-els of GST and quinone reductase activitywere elevated in the saliva of subjects whocontinually ingested large quantities of cof-fee or broccoli. Saliva may provide an easilyobtained medium for assessing the enzymeinductive potential of various diets anddrugs and for establishing the optimal doseand schedule for chemopreventive interven-tions. Nonetheless, despite the expandingattention to the identification and utiliza-tion of phase 2 enzyme inducers, muchdevelopmental work is still required for theaccurate and facile assessment of theirconstitutive and inducible levels in humans.

Inhibition of AflatoxinHepatocarcinogenesisby DithiolethionesExperimental hepatocarcinogenesis inrodents can be inhibited by a number ofantioxidants and is particularly suited formechanistic studies. A brief discussion ofthe impact of dithiolethiones on aflatoxin-induced liver cancers will illustrate some ofthe enzyme-inducing and anticarcinogenicproperties of this class of chemopreventiveantioxidants. Dithiolethiones are five-mem-bered cyclic sulfur-containing compoundswith radioprotective, chemopreventive,chemotherapeutic, and antiviral activities(11,12). For example, the drug oltiprazshows significant antischistosomal activityin experimental animals and in humans.During studies on the mechanisms ofschistosomicidal activity of oltipraz,Bueding et al. (10) noted that admin-istration of this drug, as well as severalanalogues, to mice resulted in marked ele-vations of the activities of phase 2 enzymesin hepatic and extrahepatic tissues. Thesefindings led Bueding to predict that dithio-lethiones such as oltipraz might be excel-lent candidate compounds for cancerchemoprevention studies (10). As recentlysummarized elsewhere, oltipraz has subse-quently proven to be an effective anticar-cinogen in breast, colon, pancreas, lung,forestomach, skin, bladder, and liver tumormodels (11). As a consequence, oltiprazhas undergone phase I clinical trial in theUnited States (32,34).

Aflatoxin B1 (AFB1) is a potent hepato-toxin and carcinogen in a wide variety ofanimals and is linked epidemiologicallywith a high incidence of primary hepato-cellular carcinoma in humans (35).Elimination of aflatoxins from the human

Environmental Health Perspectives * Vol 105, Supplement 4 * June 1997 967

T.W. KENSLER

food supply throughout the world will beextremely difficult and chemopreventionoffers an attractive alternative for popula-tions at high risk for aflatoxin-induced dis-eases. A number of classic chemopreventiveagents, notably BHA, BHT, and ethoxy-quin, inhibit AFB1 carcinogenesis in ratswhen fed simultaneously with the carcino-gen (36,37). A search for protective agentsmore amenable for use in man led to theevaluation of oltipraz in this rat model..After feeding male F344 rats either purifieddiet or diet supplemented with 0.075%oltipraz for 1 week, the animals receivedAFB1 5 days a week for 2 weeks. One weekafter cessation of dosing, all animals wererestored to the control diet and maintaineduntil they became moribund or upon studytermination at 23 months. This 10-doseexposure to AFB1 produced an 11% inci-dence of hepatocellular carcinomas in thecontrol animals, while an additional 9% ofthese rats had hyperplastic nodules in theirlivers (38). This incidence of hepatic dis-ease mirrors the lifetime incidence of hepa-tocellular carcinoma in humans in high-riskareas of China, Southeast Asia, and Africa(6,39). In the rat intervention study,dietary oltipraz afforded complete protec-tion against aflatoxin-induced hepatocellu-lar carcinomas and hyperplastic nodules.Further, no tumors were seen in eithergroup at extrahepatic sites, indicating thatoltipraz did not serve to merely shift targetorgan specificity from the liver to other tis-sues. No protection is observed if oltipraz isadministered after exposure to AFB1 (40).

These protective actions of oltipraz (aswell as the food antioxidants) are thoughtto result primarily from an altered balancebetween the activation and detoxication ofaflatoxin in the hepatocyte. In the case ofaflatoxin, alterations in the balance of com-peting pathways of the ultimate carcinogenaflatoxin-8,9-oxide directly modulate theavailability of the epoxide for binding toDNA (Figure 2). Anticarcinogenic concen-trations of oltipraz in the diet markedlyinduce activities of GSTs in rat tissues tofacilitate conjugation of glutathione toaflatoxin-8,9-oxide, thereby enhancing itselimination and coordinately diminishingDNA adduct formation (41). Feedingoltipraz for 1 week before exposure to AFB1increases the initial rate of biliary elimina-tion of the aflatoxin-glutathione conjugatenearly 3-fold. Concordantly, feedingoltipraz led to 3- to 4-fold increases in thespecific activity of rat liver GST and eleva-tion in the levels of some a-, p-, and i-classsubunits. Quantitative high-performance

liquid chromatography analysis of GSTsubunits showed that levels of subunitsYb1, Yp, Yc2 and Ya2 were increased 5- to10-fold. In comparison, levels of subunitsYb2 and Yc1 were elevated 2- to 3-fold,whereas subunit Ya1 was not induced (42).Fortuitously, rat GST isozymes containingthe Ya2, Yb1, or Yc2 subunits exhibit sub-stantial conjugation activity toward theultimate carcinogenic metabolite aflatoxin-8,9-oxide (43). Molecular studies indicatethat initial increases in hepatic GSTmRNA and protein levels in response tooltipraz were mediated through transcrip-tional activation of GST genes (44) andappear to be mediated by the antioxidantresponse element (45). Induction of GSTsby oltipraz in primary cultures of humanhepatocytes has also been observed (46). Asignificant attribute of oltipraz is theresponsiveness of many tissues to itsenzyme inductive actions.

Oltipraz can also influence cytochromeP450 activities. Western blotting indicatessmall increases in several forms of P450 fol-lowing oltipraz treatment in vivo, especiallyCYP1A2 and CYP3A2 (47). Perhaps morenotable, direct addition of oltipraz to ratmicrosomes inhibits AFB1 oxidation (48).Inhibition of CYP1A2 and CYP3A4 byoltipraz results in the reduction of aflatoxinmetabolism to aflatoxin M1 and the 8,9-oxide in primary cultures of humanhepatocytes (49). Urinary excretion of afla-toxin M1 also drops dramatically immedi-ately following oltipraz administration to

Hydroxylationproducts

(AFM1, AFP1, AFQ1)

aflatoxin-treated rats (50). Thus, bothinhibition of cytochrome P450s and induc-tion of electrophile detoxication enzymesare likely to contribute to chemopreven-tion by oltipraz, although kinetic argu-ments suggest the latter could be moreimportant than the former.A practical outcome of a mechanism of

action involving enzyme induction arisesfrom the long biological half-life of theenzyme inductive response. Although thehalf-life of oltipraz in rodents and man is< 6 hr, the inductive effects on some phase2 enzymes persist for more than 1 week.Thus, intermittent dosing schedules mayoffer advantages (fewer side effects, greatercompliance) while maintaining efficacy(enhanced carcinogen detoxication). Withthis view in mind, the effect of dose sched-uling on inhibiting aflatoxin-inducedtumorigenesis has been recently evaluated.Rats were treated with AFB1 daily for 28consecutive days and received oltipraz daily,twice weekly, once weekly, or not at allthroughout this period. Daily treatmentwith oltipraz engendered > 99% reductionin hepatic tumor burden; remarkably, thetwice- and once-weekly regimens reducedtumor burden by 97 and 95%, respectively(42). While transient micromolar concen-trations of oltipraz appear to be required totrigger the induction of protective enzymes,sustained elevation of plasma levels of thedrug were not necessary to achieve chemo-prevention. By contrast, inhibition of P450activities requires sustained exposure to

-*<~ P450Phase 1

ibits

S--S

N

Oltipraznduces

AFB,-8,9-epoxi

GSTo- Phase 2 tran

OH +fGS

0<AFB,-glutathione

0 DNAadducts

ide

+ H20 (epoxide hydrolase?)

sferaseOH

HO

0A

AFB1-diol

Figure 2. Effect of oltipraz on the metabolism of aflatoxin Bl.

Environmental Health Perspectives * Vol 105, Supplement 4 - June 1997968

CHEMOPREVENTION BY PHASE 2 ENZYME INDUCERS

micromolar concentrations of drug, reflect-ing the largely competitive nature of theinhibition and the rapid turnover rates ofmammalian P450s.A phase II clinical trial with oltipraz is

underway in Qidong, Jiangsu Province,People's Republic of China, under the aus-pices of the Shanghai Cancer Institute andthe Qidong Liver Cancer Institute. Qidongis located at the mouth of the Yangtze Riverand has a population of more than one mil-lion. Hepatocellular carcinoma is the lead-ing cause of cancer death in Qidong with amortality rate of 55 per 100,000 per year(39). Major risk factors for liver cancer inthis region are infection with hepatitis Bvirus and exposure to aflatoxins. Approx-imately 10% of the population of Qidongis positive for hepatitis B surface antigen.Aflatoxins are consistent contaminants ofthe food supply; the prevalence of residentstesting positive for aflatoxin biomarkersexceeds 90% (51). Using a nestedcase-control study design, Qian et al. (52)reported highly significant associationsbetween the presence of urinary aflatoxins,

serum hepatitis B surface antigen positivity,and risk of hepatocellular carcinoma.Particularly striking was a marked synergis-tic interaction between viral and chemicalrisk factors. Collectively, these studies high-light the importance of both large-scalehepatitis B virus vaccination programs andlimitation of exposure and toxicities of afla-toxins as important strategies to decreasethe incidence of liver cancer.

The randomized, placebo-controlledtrial has examined the effects of daily(125 mg) and weekly (500 mg) doses ofoltipraz on levels of two independent bio-markers: aflatoxin-N7-guanine adductsexcreted into urine and aflatoxin-albuminadducts in serum. These two biomarkershave been extensively validated throughecological and prospective epidemiologicalstudies (51-53). While these biomarkersreflect exposures to aflatoxins, their pres-ence also signals increased risk for livercancer. Levels of these biomarkers can bemarkedly attenuated in rats by interventionwith oltipraz during periods of aflatoxinexposure (38,54). As a consequence, blood

and urine samples have been collectedthroughout a 2-month intervention periodas well as during a 2-month postinterven-tion follow-up period to fully determinethe dynamics of potential changes in phase2 enzyme activities and biomarker levels.With 80 participants in each of the treat-ment arms, the clinical trial has the powerto determine small decreases in the levels ofthe urinary and/or serum biomarkers (55).The availability of intermediate biomarkersreflecting the modulation of biologicallyeffective doses of environmental carcino-gens as study end points allows the designand conduct of efficient clinical trials. Wehope that results from such trials witholtipraz will provide insights into the util-ity of phase 2 enzyme induction as a useful,mechanism-based approach to achievelarge-scale reductions in the incidence ofhepatocellular carcinoma in populations athigh risk for unavoidable exposures to afla-toxins. Further, such studies may serve astemplates for chemopreventive interven-tions targeting individuals at high risk forother environmentally induced diseases.

REFERENCES

1. Wattenberg LW. Chemoprevention of cancer. Cancer Res45:1-8 (1985).

2. De Flora S, Ramel C. Mechanisms of inhibitors of mutagenesisand carcinogenesis. Classification and overview. Mutat Res202:285-306 (1988).

3. Miller EC, Miller JA. Some historical perspectives on themetabolism of xenobiotic chemicals to reactive electrophiles. In:Bioactivation of Foreign Compounds (Anders MW, ed). NewYork:Academic Press, 1985;1-28.

4. Conney AH. Induction of microsomal enzymes by foreignchemicals and carcinogenesis by polycyclic aromatic hydrocar-bons: GHA Clowes Memorial Lecture. Cancer Res42:4875-4917 (1982).

5. Benson AM, Batzinger RP, Ou S-YL, Bueding E, Cha Y-N,Talalay P. Elevation of hepatic glutathione S-transferase activi-ties and protection against mutagenic metabolites ofbenzo[a]pyrene by dietary antioxidants. Cancer Res38:4486-4495 (1978).

6. Benson AM, Cha Y-N, Bueding E, Heine HS, Talalay P.Elevation of extrahepatic glutathione S-transferases and epoxidehydratase activities by 2(3)-tert-butyl-4-hydroxyanisole. CancerRes 39:2971-2977 (1979).

7. Lam LKT, Sparnins VL, Wattenberg LW. Isolation and identi-fication of kahweol palmitate and cafestrol palmitate as activeconstituents of green coffee beans that enhance glutathioneS-transferase activity in the mouse. Cancer Res 42:1193-1198(1982).

8. Zhang Y, Talalay P, Cho C-G, Posner GH. A major inducer ofanticarcinogenic protective enzymes from broccoli: isolation andelucidation of structure. Proc Natl Acad Sci USA89:2399-2403 (1992).

9. Zhang Y, Kensler TW, Cho C-G, Posner GH, Talalay P.Anticarcinogenic activities of sulforaphane and structurallyrelated synthetic norbornyl isothiocyanates. Proc Natl Acad SciUSA 91:3147-3150 (1994).

10. Bueding E, Dolan P, Leroy JP. The antischistosomal activity ofoltipraz. Res Commun Chem Pathol Pharmacol 37:293-303(1982).

11. Kensler TW, Helzlsouer KJ. Oltipraz: clinical opportunities forcancer chemoprevention. J Cell Biochem 22S:101-107 (1995).

12. Kelloff GJ, Crowell JA, Boone CW, Steele VE, Lubet RA,Greenwald P, Alberts DS, Covey JM, Doody LA, Knapp GG etal. Clinical development plan: oltipraz. J Cell Biochem20S:205-218 (1994).

13. Pearson WR, Windle JJ, Morrow JF, Benson AM, Talalay P.Increased synthesis of glutathione S-transferase in response toanticarcinogenic antioxidants. Cloning and measurement ofmessenger RNA. J Biol Chem 258:2052-2062 (1983).

14. Benson AM, Hunkeler MJ, Morrow JF. Kinetics of glu-tathione transferase messenger RNA, and reduced nicoti-namide adenine dinucleotide (phosphate):quinone reductaseinduction by 2(3)-tert-butyl-4-hydroxyaniso[e in mice. CancerRes 44:5256-5261 (1984).

15. Pearson WR, Reinhart J, Sisk RC, Anderson KS, Adler PN.Tissue-specific induction of murine glutathione transferasemRNAs by butylated hydroxyanisole. J Biol Chem263:13324-13332 (1988).

16. Prochaska HJ, Talalay P. Regulatory mechanisms of monofunc-tional and bifunctional anticarcinogenic enzyme inducers inmurine liver. Cancer Res 48:4776-4782 (1988).

17. Fisher JM, Wu L, Denison MS, Whitlock JP Jr. Organizationand function of a dioxin-responsive enhancer. J Biol Chem265:9676-9681 (1990).

18. Prochaska HJ, Bregman HS, DeLong MJ, Talalay P. Specificityof induction of cancer protective enzymes by analogues of tert-butyl-4-hydroxyanisole (BHA). Biochem Pharmacol34:3909-3914 (1985).

19. Prochaska HJ, DeLong MJ, Talalay P. On the mechanisms ofinduction of cancer-protective enzymes: a unifying proposal.Proc Natl Acad Sci USA 82:8232-8236 (1985).

Environmental Health Perspectives * Vol 105, Supplement 4 * June 1997 969

T.W. KENSLER

20. Talalay P, DeLong MJ, Prochaska HJ. Identification of a com-mon chemical signal regulating the induction of enzymes thatprotect against chemical carcinogenesis. Proc Natl Acad SciUSA 85:8261-8265 (1988).

21. Prestera T, Holtzclaw WD, Zhang Y, Talalay P. Chemical andmolecular regulation of enzymes that detoxify carcinogens. ProcNatl Acad Sci USA 90:2965-2969 (1993).

22. Friling RS, Bensimon A, Tichauer Y, Daniel V. Xenobiotic-inducible expression of murine glutathione S-transferase Ya sub-unit gene is controlled by an electrophile-responsive element.Proc Natl Acad Sci USA 87:6258-6262 (1990).

23. Rushmore TH, King RG, Paulson KE, Pickett CB. Regulationof glutathione S-transferase Ya subunit gene expression: identifi-cation of a unique xenobiotic-responsive element controllinginducible expression by planar aromatic compounds. Proc NatlAcad Sci USA 87:3826-3830 (1990).

24. Jaiswal AK. Antioxidant response element. Biochem Pharmacol48:439-444 (1994).

25. Park BK, Kitteringham NR. Assessment of enzyme inductionand enzyme inhibition in humans: toxicological implications.Xenobiotica 20:1171-1185 (1990).

26. Pantuck EJ, Pantuck CB, Garland WA, Min BH, WattenbergLW, Anderson KE, Kappas A, Conney AH. Stimulatory effectof brussels sprouts and cabbage on human drug metabolism.Clin Pharmacol Ther 25:88-95 (1979).

27. Miners JO, Attwood J, Birkett DJ. Determinants of aceta-minophen metabolism: effect of inducers and inhibitors of drugmetabolism on acetaminophen's metabolic pathways. ClinPharmacol Ther 35:480-486 (1984).

28. Chasseaud LF. Determination of thioethers as biochemicalmarkers for monitoring exposure to potential toxicants. In:Glutathione Conjugation (Anders *MW, ed). NewYork:Academic Press, 1988;391-413.

29. Bogaards JJP, Verhagen H, Willems MI, van Poppel G, vanBladeren PJ. Consumption of brussels sprouts results in elevatedac-class glutathione S-transferase levels in human blood plasma.Carcinogenesis 15:1073-1075 (1994).

30. Prochaska HJ, Fernandes CL. Elevation of serum phase IIenzymes by anticarcinogenic enzyme inducers: markers for achemoprotected state? Carcinogenesis 14:2441-2445 (1993).

31. Gordon GB, Prochaska HJ, Yang LY-S. Induction ofNAD(P)H:quinone reductase in human peripheral blood lym-phocytes. Carcinogenesis 12:2393-2396 (1991).

32. Gupta E, Olopade OL, Ratain MJ, Mick R, Baker TM, BerezinFK, Benson AB III, Dolan ME. Pharmacokinetics and pharma-codynamics of oltipraz as a chemopreventive agent. Clin CancerRes 1:1133-1138 (1995).

33. Sreerama L, Hedge MW, Sladek NE. Identification of a class 3aldehyde dehydrogenase in human saliva and increased levelsof this enzyme, glutathione S-transferases, and DT-diaphorasein the saliva of subjects who continually ingest large quantitiesof coffee or broccoli. Clin Cancer Res 1:1153-1163 (1995).

34. Benson AB III. Oltipraz: a laboratory and clinical review. J CellBiochem 17F:278-291 (1993).

35. Busby WF Jr, Wogan GN. Aflatoxins. In: ChemicalCarcinogens. 2nd ed (Searle CE, ed). Washington:AmericanChemical Society, 1984;945-1136.

36. Cabral JRP, Neal GE. The inhibitory effects of ethoxyquin onthe carcinogenic action of aflatoxin B1 in rats. Cancer Lett19:125-132 (1983).

37. Williams GM, Tanaka T, Maeura Y. Dose-related inhibition ofaflatoxin B1 induced hepatocarcinogenesis by the phenolicantioxidants, butylated hydroxytoluene and butylated hydroxy-anisole. Carcinogenesis 7:1043-1050 (1986).

38. Roebuck BD, Liu Y-L, Rogers AE, Groopman JD, Kensler TW.Protection against aflatoxin B1-induced hepatocarcinogenesis inrats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-hione (oltipraz):predictive role for short-term molecular dosimetry. Cancer Res51:5501-5506 (1991).

39. Zhu Y-R, Chen J-G, Huang X-Y. Hepatocellular carcinoma inQidong County. In: Primary Liver Cancer (Tang Z-Y, Wu N-C,Xia S-S, eds). Beijing:China Academic Publishers, 1988;204-222.

40. Maxuitenko YY, MacMillan DL, Kensler TW, Roebuck BD.Evaluation of the post-initiation effects of oltipraz on aflatoxinBI-induced preneoplastic foci in a rat model of hepatic tumori-genesis. Carcinogenesis 14:2423-2425 (1993).

41. Kensler TW, Egner PA, Dolan PM, Groopman JD, RoebuckBD. Mechanism of protection against aflatoxin tumorigenicityin rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione(oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones. Cancer Res 47:4271-4277 (1987).

42. Primiano T, Egner PA, Sutter TR, Kelloff GJ, Roebuck BD,Kensler TW. Intermittent dosing with oltipraz: relationshipbetween chemoprevention of aflatoxin-induced tumorigenesisand induction of glutathione S-transferases. Cancer Res55:4319-4324 (1995).

43. Raney KD, Meyer DJ, Ketterer B, Harris TM, Guengerich FP.Glutathione conjugation of aflatoxin B1 exo- and endo-epoxidesby rat and human glutathione S-transferases. Chem Res Toxicol5:470-478 (1992).

44. Davidson NE, Egner PA, Kensler TW. Transcriptional controlof glutathione S-transferase gene expression by the chemopro-tective agent 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione(oltipraz) in rat liver. Cancer Res 50:2251-2255 (1990).

45. Egner PA, Kensler TW, Prestera T, Talalay P, Libby AH,Joyner HH, Curphey TJ. Regulation of phase 2 enzyme induc-tion by oltipraz and other dithiolethiones. Carcinogenesis15:177-181 (1994).

46. Morel F, Fardel 0, Meyer DJ, Langouet S, Gilmore KS,Meunier B, Tu C-PD, Kensler TW, Ketterer B, Guillouzo A.Preferential increase of glutathione S-transferase class alphatranscripts in cultured human hepatocytes by phenobarbital,3-methylcholanthrene and dithiolethiones. Cancer Res53:231-234 (1993).

47. Buetler TM, Gallagher EP, Wang C, Stahl DL, Hayes JD,Eaton DL. Induction of phase I and phase II drug-metabolizingenzyme mRNA, protein, and activity by BHA, ethoxyquin, andoltipraz. Toxicol Appl Pharmacol 135:45-57 (1995).

48. Putt DA, Kensler TW, Hollenberg P. Effect of three chemopre-ventive antioxidants, ethoxyquin, oltipraz, and 1,2-dithiole-3-thione on cytochrome P450 levels and aflatoxin B1 metabolism.Fed Am Soc Exp Biol J 5:A1517 (1991).

49. Langouet S, Coles B, Morel F, Becquemont L, Beaune P,Guengerich FP, Ketterer B, Guillouzo A. Inhibition of CYP1A2and CYP3A4 by oltipraz results in reduction of aflatoxin B1metabolism in human hepatocytes in primary culture. CancerRes 55:5574-5579 (1995).

50. Scholl P, Musser SM, Kensler TW, Groopman JD. Inhibitionof aflatoxin Ml excretion in rat urine during dietary interven-tion with oltipraz. Carcinogenesis 17:1385-1388 (1996).

51. Wang J-S, Qian G-S, Zarba A, He Z, Zhu Y-R, Zhang B-C,Jacobson L, Gange SJ, Muuioz A, Kensler TW et al. Temporalpatterns of aflatoxin-albumin adducts in hepatitis B surfaceantigen positive and negative residents of Daxin, QidongCounty, People's Republic of China. Cancer EpidemiolBiomarkers Prev 5:253-262 (1996).

52. Qian G-S, Ross RK, Wu MC, Yuan J-M, Gan Y-T, HendersonBE, Wogan GN, Groopman JD. A follow-up study of urinarymarkers of aflatoxin exposure and liver cancer risk in Shanghai,People's Republic of China. Cancer Epidemiol Biomarkers Prev3:3-10 (1994)

53. Groopman JD, Zhu J, Donahue PR, Pikul A, Zhang L-S, ChenJ-S, Wogan GN. Molecular dosimetry of urinary aflatoxin DNAadducts in people living in Guangxi Autonomous Region,People's Republic of China. Cancer Res 52:45-51 (1992).

54. Egner PA, Gange SJ, Dolan PM, Groopman JD, Mufioz A,Kensler TW. Levels of aflatoxin-albumin biomarkers in ratplasma are modulated by both long-term and transient interven-tions with oltipraz. Carcinogenesis 16:1769-1773 (1995).

55. Jacobson LP, Zhang B-C, Zhu Y-R, Wang J-B, Wu Y, ZhangQ-N, Yu Y-L, Qian G-S, Kuang S-Y, Li Y-F et al. Oltiprazchemoprevention trial in Qidong, People's Republic of China:study design and clinical outcomes. Cancer EpidemiolBiomarkers Prev 6:257-265 (1997).

970 Environmental Health Perspectives - Vol 105, Supplement 4 * June 1997