Development of Cancer Chemopreventive Agents: Oltipraz as a

FEBRUARY 1999 VOLUME 12, NUMBER 2 © Copyright 1999 by the American Chemical Society

Perspective Development of Cancer Chemopreventive Agents: Oltipraz as a Paradigm Thomas W. Kensler,*,† John D. Groopman,† Thomas R. Sutter,† Thomas J. Curphey,‡ and Bill D. Roebuck§ Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205, and Department of Pathology and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received August 7, 1998

Introduction An aging population coupled with a continuing decline in mortality from cardiovascular disease will render cancer the major cause of death in the United States within the next few years. Despite notable successes in the treatment of some cancers, current treatments have not appreciably affected mortality rates for the more common cancers (1). Thus, additional strategies for reducing cancer mortality, notably, prevention, are receiving expanded attention. One aspect of cancer prevention, chemoprevention, entails the use of chemical agents, either natural or synthetic in origin, to retard, block, or even reverse the carcinogenic process that leads to the clinical manifestations of these diseases. Through extensive evaluation of chemopreventive agents in experimental models over the past decade, efficacy against a diverse array of carcinogens at multiple tissue sites has been demonstrated (2). Consequently, the prospect that chemoprevention against cancer can be achieved in humans appears promising (3). The successful development of chemopreventive agents is a largely uncharted process, for which pathways are * To whom correspondence should be addressed. Phone: (410) 9554712. Fax: (410) 955-0116. E-mail: [email protected]. † Johns Hopkins School of Hygiene and Public Health. ‡ Department of Pathology, Dartmouth Medical School. § Department of Pharmacology and Toxicology, Dartmouth Medical School.

just being developed (4-6). At present, the progress of each new agent from discovery through full clinical evaluation presents many unique challenges. This perspective seeks to highlight the scientific and regulatory considerations and constraints that have guided the development of one chemopreventive agent, oltipraz1 [4-methyl-5-(2-pyrazinyl)-3H-1,2-dithiole-3-thione], along a pathway from initial hypothesis for chemopreventive efficacy into clinical intervention trials. While this route, which is depicted in Figure 1, can hardly be considered to define a generic paradigm for agent development, it will document the major structural elements that need to be considered in unison for the development of chemopreventive agents. These elements are the “ABCs” of chemoprevention: agents, biomarkers, and cohorts. For, in addition to the selection and preclinical assessment of an agent, the process must be linked to the use of modulatable intermediate biomarkers that allow for the rapid and efficient assessment of efficacy in humans. Such biomarkers require their own, largely independent, paradigm for development and validation (7, 8). Whenever possible, the biomarkers should be applied to the 1Abbreviations: oltipraz, 4-methyl-5-(2-pyrazinyl)-3H-1,2-dithiole3-thione; AFB1, aflatoxin B1; AFM1, aflatoxin M1; AFB-N7-Gua, 8,9dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1; GST, glutathione S-transferase; DIG, 1,2-dithiole-3-thione-inducible gene; AFAR, aflatoxin aldehyde reductase; LTB4, leukotriene B4; HBV, hepatitis B virus; OR, odds ratio; AUC, area under the curve.

10.1021/tx980185b CCC: $18.00 © 1999 American Chemical Society Published on Web 01/20/1999

114 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Figure 1. Paradigm for the preclinical and clinical (shaded rectangles) development of oltipraz as a chemopreventive agent. Although presented in a linear array to highlight the timeline for milestone events, the process is highly iterative in the development and refinement of hypotheses, the expansion of mechanistic understanding of the action of oltipraz, and the incorporation of intermediate biomarkers into the paradigm. This paradigm used as its foundation the knowledge base concerning all aspects of aflatoxin carcinogenesis that have emerged since the early 1960s.

experimental anticarcinogenesis models that justify continuing enthusiasm and investment in the clinical development of an agent. Finally, these two components must be tightly integrated into the selection of study cohorts to optimize prospects for gathering meaningful information. The overall process is not, in practice, a linear one, but rather one that incorporates many loops and retracing of steps as new information is garnered and applied to the process. Each series of studies should be prospectively designed with a clear plan for how the results will be used in the overall developmental process.

A Mechanism-Based Approach for Chemoprevention: Induction of Carcinogen Detoxication Enzymes The multiple stages of carcinogenesis offer many potential strategies for prevention. However, in the majority of tests conducted in animal models, protection has been achieved by administering the chemopreventive agent prior to and/or concurrent with the exposure to the carcinogen. Given this temporal relationship between administration of anticarcinogen and carcinogen, it seems likely that these agents act, at least in part, to alter the metabolism and disposition of carcinogens, thereby altering events critical to the initial interactions of chemical carcinogens with biomolecules. Many chemicals require metabolic activation to electrophilic intermediates to exert carcinogenic activity (9).

Kensler et al.

If not detoxified, these intermediates can react with and thereby functionally modify nucleophilic moieties on critical biomolecules. Nucleophilic groups in DNA are among those targeted by electrophiles; the interaction of carcinogen metabolites with DNA can cause point mutations and other genetic lesions, which can result in activation of protooncogenes and inactivation or loss of tumor suppressor genes. The importance of metabolic activation in carcinogenesis is highlighted by the fact that target organ specificities and even species susceptibilities can be determined through the presence or absence of metabolic pathways. The processing of chemicals to proximate carcinogens typically involves an initial twoelectron oxidation. This phase 1 reaction can be catalyzed by a number of enzymes, particularly those comprising the cytochrome P450 superfamily. A second metabolic step involves the transfer or conjugation of an endogenous, water-soluble substrate to the functional group introduced during phase 1 biotransformation, thereby facilitating elimination of the carcinogen. These phase 2 reactions, which include sulfation, acetylation, glucuronidation, and conjugation with glutathione, typically lead to carcinogen detoxification. Thus, the amount of ultimate carcinogen available for interaction with its target represents, in part, a balance between competing activating and detoxifying reactions. While this balance is under genetic control, it is readily modulated by a variety of factors, including nutritional status, age, hormones, and exposure to drugs or other xenobiotics (10). In this setting, chemopreventive agents can profoundly modulate the constitutive metabolic balance between activation and inactivation of carcinogens through their actions on both phase 1 and 2 enzymes. It has been known for several decades that antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, and ethoxyquin can exert a chemopreventive effect when administered simultaneously with a carcinogen (11). Perhaps the earliest study to indicate a role for the induction of phase 2 enzymes in the protective actions of these antioxidants was that of Benson and coworkers (12). They showed that liver cytosols from butylated hydroxyanisole- or ethoxyquin-fed rats or mice exhibited much higher activities of phase 2 enzymes than controls. Moreover, cytosols prepared from the livers of the treated rodents eliminated the mutagenic activity in urine collected from mice treated with benzo[a]pyrene. In the intervening decades, substantive experimental evidence has been developed to support the view that induction of phase 2 enzymes is a critical and sufficient mechanism for engendering protection against the toxic and carcinogenic actions of reactive intermediates. This now expansive literature has been reviewed (13-15), and major elements of the supportive findings are listed in Table 1. While the evidence is most convincing in cell culture and animals models, there has been limited information in humans to corroborate these findings. Thus, a major motivation from the beginning for the use of oltipraz in experimental and clinical chemoprevention studies by our collective groups has been to rigorously test the hypothesis that induction of phase 2 enzymes is an effective means for achieving chemoprevention in humans.

Oltipraz: A Practical Choice for an Agent Current strategies for the discovery of new drugs include combinatorial chemistry, computer modeling,

Perspective Table 1. Evidence for a Major Role of Induction of Phase 2 Enzymes in Chemopreventiona many chemopreventive agents are most effective if administered prior to carcinogens treatment with chemopreventive agents profoundly alters carcinogen metabolism induced phase 2 enzymes inactivate ultimate carcinogens chemoprevention is achieved against a wide variety of carcinogens, suggesting a mechanism of low specificity enzyme induction and chemoprevention are produced by the same compounds (of many chemical classes), occur at similar doses, and have similar tissue specificities overexpression of glutathione S-transferase by cDNA transfection protects cells against carcinogen toxicity loss of expression of glutathione S-transferase activity in knockout mice enhances sensitivity to carcinogenesis monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents a

Adapted from refs 13 and 14.

rational design, and random screening with highthroughput systems (16). In only a few cases have such approaches been applied successfully to the identification of chemopreventive agents now reaching clinical trials, reflecting the long and expensive demands of preclinical evaluations that must establish both efficacy and safety. Sulforaphane, an isothiocyanate phase 2 enzyme inducer found in broccoli, is an example of the current, progressive approach (17). However, oltipraz is not. Most chemopreventive agents used in experimental models for chemoprevention in the 1970s and 1980s were selected on the basis of two criteria: being natural products, or structural analogues thereof, and availability. Oltipraz met those two criteria, but had two additional features uncommon to many agents of its era. It had a biochemical action consonant with protection against chemical carcinogenesis, and it had already been used clinically. 1,2-Dithiole-3-thiones were reported in the 1950s to be constituents of cruciferous vegetables in Czechoslovakia (18), although a more recent study failed to find the unsubstituted 3H-1,2-dithiole-3-thione in cabbage in the United States (19). Oltipraz, a substituted 1,2-dithiole3-thione, was originally developed by the pharmaceutical industry as a possible treatment for schistosomiasis and was extensively evaluated in clinical trials in the early 1980s. Field trials in Mali, Gaboon, and France, using short courses with durations of 1-5 days with total doses of 1.25-7.5 g, achieved cure rates of greater than 90%. At least 18 studies of the chemotherapy of schistosomiasis by oltipraz involving 1284 patients were conducted (20). Side effects principally related to the digestive system, namely, nausea, abdominal pain/distress, vomiting, and diarrhea, were reported in more than 10% of the study participants, as were headaches and dizziness. However, the absence of placebo groups in these studies renders attribution of these common effects solely to oltipraz problematic. Of greater concern were reports of paresthesia and fingertip pain, the severity appeared to increase after exposure to sunlight. Although all effects were reported as mild, subsided within a few days, and did not require discontinuation of the drug, the concerns regarding photosensitivity led to the abandonment of oltipraz for the treatment of schistosomiasis. This decision was surely influenced by concurrent clinical progress of less expensive, equi-effective, and less problematic drugs for the chemotherapy of schistosomiasis, such as praziquantel. But, by 1984, the major clinical problems associated with the use of oltipraz had been unmasked

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 115

and did not appear to be insurmountable in the context of its application to chemoprevention. While the ideal chemopreventive agent would be devoid of all adverse effects, allowing it to be taken with impunity, in reality, minor side effects are unavoidable. These clinical manifestations play a dominant role in defining how and where the agent might be used, i.e., high-risk populations versus the general population. While studying mechanisms of antischistosomiasis by oltipraz, Professor Ernest Bueding and colleagues initially noted that giving the drug to mice infected with Schistosoma mansoni caused a dramatic reduction in the glutathione stores of the parasite while paradoxically markedly elevating glutathione levels in many tissues of the host (21). Subsequent studies demonstrated that oltipraz and some structurally related 1,2-dithiole-3thiones were potent inducers of enzymes concerned with the maintenance of reduced glutathione pools as well as enzymes important to electrophile detoxication in multiple tissues of rats and mice (22). These results prompted Bueding to predict that oltipraz might have cancer chemopreventive properties (23). Because we were evaluating the actions of phenolic antioxidants as modulators of aflatoxin metabolism in vivo at that time, it was a simple transposition to probe the actions of oltipraz in this setting. Although the phenolic antioxidants were extremely effective inhibitors, producing substantial reductions in tumor incidence in feeding studies, they were unsettlingly lacking in potency. Dietary concentrations of 0.5-2%, which were approximately 100-fold higher that the maximum allowed for their content in any human foodstuff, were required for efficacy. The spark for our enthusiastic pursuit of oltipraz as a potential chemopreventive agent was the observation, published in 1985, that oltipraz was at least 1 order of magnitude more potent than the antioxidants as an inducer of electrophile detoxication enzymes (24). Threefold increases in the hepatic activities of glutathione S-transferases (GSTs) and UDP-glucuronosyl transferases, prototypical phase 2 enzymes, were measured after feeding rats 1000 ppm (0.1%) oltipraz.

Efficacy of Oltipraz as a Chemopreventive Agent: Selecting the Appropriate Animal Bioassays Identification of a potent enzyme-inducing agent amenable for use in humans prompted evaluation of oltipraz in several models of experimental carcinogenesis. A partnership with Lee Wattenberg allowed Ernest Bueding to realize his prediction of chemopreventive efficacy shortly before his death (25). In their seminal study, oltipraz was administered po either 24 or 48 h before treatment with each of three structurally diverse carcinogens: diethylnitrosamine, uracil mustard, and benzo[a]pyrene. This sequence of oltipraz and carcinogen administration was repeated once a week for 4-5 weeks. Oltipraz reduced by nearly 70% the number of both pulmonary adenomas and tumors of the forestomach induced by benzo[a]pyrene. The degree of pulmonary adenoma formation induced by uracil mustard or diethylnitrosamine was also significantly reduced by oltipraz pretreatment, but to a lesser extent. With the encouragement of Professor Bueding, we concurrently initiated a 2 year bioassay to assess the chemopreventive efficacy of oltipraz against aflatoxin B1


(AFB1)-induced hepatocarcinogenesis. Our selection of the aflatoxin model was predicated on the strong mechanistic knowledge base that had already been developed around aflatoxin carcinogenesis at that time. Since the discovery of these mycotoxins in 1960, there has been an increasing concern about the possible health hazards engendered by the introduction of toxic and carcinogenic compounds into foods by toxigenic molds. There is now an enormous literature pertaining to various aspects of the aflatoxins that reflects upon their significant impact on human and animal health and the associated economic consequences (reviewed in refs 26 and 27). The extreme potency of AFB1 as an animal toxin and hepatocarcinogen, its wide distribution in the human food supply, and its implication as a liver carcinogen in humans have combined to stimulate a great deal of productive inquiry into its metabolism, its biochemical mode of action, and the procedures for monitoring human exposure. Even to this day, comparable amounts of integrated information exist for few other carcinogens. In retrospect, selection of an animal model with direct relevance to human cancer as our primary experimental tool was an extremely prudent decision as it allowed us subsequently to efficiently translate our mechanistic observations and analytic methods to the design and conduct of human intervention studies. The design of the anticarcinogensis bioassay with oltipraz in AFB1-treated rats was guided by results from short-term studies in which the modulation of the hepatic burden of putative preneoplastic lesions was evaluated (γ-glutamyltranspeptidase-positive foci). Our preliminary studies indicated inhibition of tumorigenesis over a dietary concentration range of oltipraz (100-1000 ppm) common to that for its enzyme inducing actions (28). These studies also indicated that the maximum tolerated dose (in which there was no effect on animal growth rate) was 750 ppm in the diet. Thus, for the cancer bioassay, 90 male F344 rats were randomized into either a group receiving purified diet or one with this diet supplemented with 750 ppm oltipraz. After 1 week on these diets, all animals received 25 µg of AFB1 5 days a week for 2 weeks. One week after cessation of dosing with AFB1, all animals were restored to the control diet and maintained until they became moribund or upon study termination at 2 years. This 10-dose exposure to AFB1 produced an 11% incidence of hepatocellular carcinoma (HCC) in the control animals, while an additional 9% had hyperplastic nodules in their livers (29). Unlike the typical chemoprevention models, in which tumor incidence is pushed to its fullest through the use of high doses of carcinogens, a lower incidence was desired in this model in what was, retrospectively, a successful attempt to mirror the lifetime incidence of HCC in people living in the very high-risk areas of China, Southeast Asia, and Africa. In this rat intervention study, dietary oltipraz afforded complete protection against aflatoxininduced HCC and hyperplastic nodules. Equally important, no increases in the incidence of tumors were seen in either group at extrahepatic sites, indicating that oltipraz did not serve to merely shift target organ specificity from the liver to other tissues. Protocols in which chemopreventive agents are administered before, during, and/or after the carcinogen are experimentally expedient for the identification of active agents and provide important insights regarding possible mechanisms of action, i.e., attribution of “blocking” versus

Kensler et al.

“suppressing” effects (30). Indeed, administration of oltipraz to rats following completion of AFB1 dosing demonstrated no protective effect (31). The good news was there was no enhancing or promoting of tumorigenesis either. However, such protocols largely fail to represent an accurate paradigm for interventions in human populations, where lifelong exposures to carcinogens may occur yet opportunities for chemoprevention may be more limited. Thus, we have also examined the efficacy of oltipraz in a setting that models clinical interventions. This experimental intervention strategy used a delayed, transient intervention protocol in which oltipraz was fed to rats for 2 weeks beginning 1 week after dosing with AFB1 began and ending 2 weeks before dosing with AFB1 was completed. Despite the disadvantageous bias in the dosing schedule, multiple experiments demonstrated significant reductions in the hepatic burden of preneoplastic lesions (32) and incidence of HCC (33) by the delayed, transient intervention with oltipraz. Because aflatoxin exposure proceeded unabated during the first week in this protocol, there was substantive DNA adduct formation and “initiation” of hepatocarcinogenesis in all animals. The remarkable efficacy of the delayed, transient intervention toward protecting against tumorigenesis as well as fibrosis and cirrhosis in the liver highlighted the important role that recurrent cytotoxicity likely plays in AFB1-induced hepatocarcinogenesis. Indeed, inhibition of this autopromoting component of carcinogen action may yet prove to be a significant aspect of the chemopreventive activity of oltipraz. Largely under the aegis of the drug development program of the Chemoprevention Branch of the National Cancer Institute, oltipraz has continued to undergo extensive evaluation for anticarcinogenic efficacy in animal models. As reviewed elsewhere (4, 34), oltipraz has now shown chemopreventive activity against different classes of carcinogens targeting the trachea, lung, stomach, small intestine, colon, pancreas, liver, urinary bladder, mammary gland, hematopoietic cells, and skin. The most dramatic actions of oltipraz occur in the colon and liver, where dietary administration results in significant reductions in both tumor incidence and multiplicity. Pharmacokinetic studies indicate that these two organs have among the highest tissue concentrations following oral administration of the drug (35). The use of oltipraz in combinations with agents exhibiting different mechanisms of action is also under evaluation. Combinations of oltipraz with 2-(difluoromethyl)ornithine prevents bladder carcinogenesis at doses where the single agents are inactive; protection with combinations of oltipraz with either the retinoid N-(4-hydroxyphenyl)retinamide or β-carotene have been very effective against nitrosamine-induced respiratory carcinogenesis (36, 37). Collectively, these results indicate that oltipraz has among the broadest range of activity of any chemopreventive agent currently under evaluation. We are hopeful that this broad-based action in animals can be translated into efficacy at multiple sites in humans.

Mechanisms in the 1990s: Phase 2 Enzyme Induction and More Phase 2 Enzyme Induction. As shown in Figure 2, alterations in the balance of competing pathways of the ultimate carcinogen, aflatoxin-8,9-epoxide, directly modulate the availability of the epoxide for binding to DNA.

Perspective


Figure 2. General scheme for the metabolism of AFB1. Stable metabolites and adducts of aflatoxin that are currently used as intermediate biomarkers to assess the actions of oltipraz in clinical trials are enclosed in boxes.

Anticarcinogenic concentrations of oltipraz in the diet markedly induce activities of GSTs in rat tissues to facilitate conjugation of glutathione to aflatoxin-8,9epoxide, thereby enhancing its elimination and coordinately diminishing the degree of DNA adduct formation (24). Feeding oltipraz for 1 week before exposure to AFB1 increases the initial rate of biliary elimination of the aflatoxin-glutathione conjugate by nearly 3-fold (38). Concordantly, feeding oltipraz led to 3-4-fold increases in the specific activity of rat liver GST and elevation in the levels of some R-, µ-, and π-class subunits. Quantitative HPLC analysis of GST subunits showed that levels of subunits Yb1, Yp, Yc2, and Ya2 were increased 5-10fold. In comparison, levels of subunits Yb2 and Yc1 were elevated 2-3-fold, whereas subunit Ya1 was not induced (39). Fortuitously, rat GST isozymes containing the Ya2, Yb1, or Yc2 subunits exhibit substantial conjugation activity toward the ultimate carcinogenic metabolite, aflatoxin-8,9-epoxide (40). Aflatoxin aldehyde reductase (AFAR), recently identified and purified from the livers of ethoxyquin-treated rats by Judah et al. (41), is also highly induced by oltipraz and other 1,2-dithiole-3-thiones (42). AFAR is a NADPHdependent aldo-keto reductase that catalyzes the reduction of the carbonyl groups of dialdehydes to alcohols. AFAR metabolizes aflatoxin dialdehyde, a product that can be nonenzymatically derived from the AFB-epoxide at physiological pH. While aflatoxin dialdehyde can covalently bind to basic amino acid residues of proteins, the dialcohol product of AFAR does not. Protein binding by aflatoxin dialdehyde may contribute to the cytotoxic action of AFB1, although this hypothesis has not been directly tested to date. As a correlate, induction of AFAR may contribute to protection against aflatoxin toxicity. Liu et al. (43) have described the substantial amelioration by oltipraz of AFB1-induced hepatotoxicity in the rat. Phase 1 Enzyme Inhibition. Oltipraz can also influence cytochrome P450 activities. Following oltipraz treatment in vivo, Western blotting indicates small increases in the levels of several forms of P450, especially P450

1A2 and 3A2 (44). Perhaps more notable, direct addition of oltipraz to rat liver microsomes inhibits AFB1 oxidation (45). Both P450 1A2 and 3A4 have been shown to catalyze the formation of the ultimate carcinogenic species, aflatoxin-8,9-epoxide. Enzyme kinetic studies on heterologously expressed human P450 1A2 indicate that oltipraz is a competitive inhibitor, with an apparent Ki of 10 µM (46), a pharmacologically achievable concentration in rats and humans (47). P450 3A4 can also be inhibited, but with an 8-fold higher Ki (46). Inhibition of P450 1A2 by oltipraz results in a diminution of the extent of AFB1 metabolism to aflatoxin M1 (AFM1) in primary cultures of human hepatocytes, and changes in the rates of AFM1 formation presumably reflect corresponding changes in 8,9-epoxide biosynthesis (46). The level of urinary excretion of AFM1 also drops dramatically immediately following oltipraz administration to aflatoxin-treated rats (48). No elevation of the level of AFM1 formation is seen in vivo in rats following oltipraz administration, suggesting that modest increases in the level of P450 1A2 protein are not exerting any enhancing effects on aflatoxin activation. Thus, in practice, both inhibition of cytochrome P450s (phase 1) and induction of electrophile detoxication enzymes (phase 2) can be envisioned to contribute to chemoprevention by oltipraz. Kinetic arguments discussed below suggest the latter could be more important than the former in rats. Influence of Dose Scheduling. A practical outcome of a mechanism of action involving enzyme induction arises from the long biological half-life of the enzyme inductive response. Although the plasma half-life of oltipraz in rodents and humans is 99% reduction in the hepatic tumor burden; remarkably, the twice-a-week and once-a-week regimens reduced the tumor burden by 97 and 95%, respectively (49). While transient micromolar concentrations of oltipraz appear to be required to trigger the induction of protective enzymes, sustained elevation of plasma levels of the drug were not necessary to achieve chemoprevention. By contrast, inhibition of P450 activities requires sustained exposure to micromolar concentrations of drug, reflecting the largely competitive nature of the inhibition and the rapid turnover rates of mammalian P450s (35, 51). While modulation of carcinogen metabolism could explain many of the actions of oltipraz in the myriad of animal bioassays, it does not explain them all. There are two notable examples. First, oltipraz blocks the action of a direct acting carcinogen (N-methyl-N-nitrosourea) in a mammary carcinogenesis model (52). Second, Rao et al. (53) observed that the protective effects of oltipraz against azoxymethane-induced colon carcinogenesis in rats was nearly equi-effective, regardless of whether oltipraz was administered during or after carcinogen administration. Moreover, dose-response experiments, as illustrated in Figure 3, indicated that reductions in DNA adduct levels often underestimated the decreases in the hepatic burden of preneoplastic foci. Collectively, these “confounding” observations prompted us to undertake a molecular genetic approach to more fully characterize genes induced by oltipraz as a means of unmasking additional mechanisms for chemoprevention. Isolation of cDNAs induced by 1,2-dithiole-3-thiones provided a means of identifying responsive genes largely independent of biases imposed by existing mechanistic hypotheses. In support of this genetic approach, Sutter et al. (54) had already found that 2,3,7,8-tetrachlorodibenzo-p-dioxin induced a number of genes not associated with xenobiotic metabolism in addition to old and new genes encoding phase 1 and 2 enzymes.

electrophiles elevation of glutathione levels (v γ-GCS) induction of phase 2 enzymes inhibition of cytochrome P450 activities induction of DNA repair free radicals elevation of glutathione levels radical scavenging altered iron homeostasis (v ferritin H and L, HO-1) induction of antioxidant enzymes (v MnSOD and catalase) inhibition of neutrophil chemotaxis and activation (v LTB4 dehydrogenase)

23 24 46 63 23 64 55, 58 56, 57 59

Induction of Other Genes. A cDNA library was prepared from liver of rats treated with the potent unsubstituted oltipraz analogue, 3H-1,2-dithiole-3-thione, and was screened by a differential hybridization method. cDNA clones for several known 3H-1,2-dithiole-3-thioneinducible genes (DIG) were isolated such as AFAR, quinone reductase, epoxide hydratase, and several subunits of GSTs (42), thereby validating the approach. cDNA clones of the heavy and light chains of ferritin, ribosomal proteins L18a and S16, and two unknown genes, termed DIG-1 and DIG-2, were also cloned through this strategy (42). Levels of mRNA recognized by each clone were increased by 2-31-fold. In independent studies, we and others have also determined that 3H-1,2dithiole-3-thione and oltipraz increased levels of mRNA and activities for γ-glutamylcysteine synthetase, the ratelimiting enzyme of glutathione biosynthesis, heme oxygenase-1, manganese-superoxide dismutase, and catalase (55-58). Nuclear run-on experiments showed that treatment with 3H-1,2-dithiole-3-thione was followed by enhanced rates of transcription of each of these genes except epoxide hydratase and catalase. Different time courses for induction as well as differing sensitivities to cyclohexamide suggest that multiple regulatory mechanisms may control the expression of these genes. Collectively, these findings offer a vastly expanded view about how oltipraz and other 1,2-dithiole-3-thiones may function as inhibitors of carcinogenesis. As listed in Table 2, in addition to modulating the fate of electrophiles, dithiolethiones can inhibit oxidative stress by modifying the toxicities of free radicals through both direct and indirect mechanisms. Protection against oxidative stress could be achieved through elevation of the levels of endogenous antioxidants and antioxidant enzymes and by a diminished level of reactive oxygen production. This latter mechanism is exemplified by the observations of increased ferritin levels (2-3-fold) in the livers of 3H-1,2dithiole-3-thione-treated rats (55). Ferritin is the major storage protein in liver for iron. Electron paramagnetic resonance spectroscopy studies document that hepatic levels of free iron diminish following treatment with 1,2dithiole-3-thiones (55). Enhanced sequestration of iron reduces the availability of Fe2+ for participating in the reduction of molecular oxygen to free radical species. Further support for modulation of oxidative stress comes from the recent identification of DIG-1 as leukotriene B4 (LTB4) dehydrogenase (59). LTB4 is a potent leukotactic molecule that signals the influx of neutrophils into sites of inflammation (60). This cytokine can also activate the respiratory burst of recruited neutrophils, yielding cyto-

Perspective

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 119 Scheme 1

toxic reactive oxygen species. The inducible LTB4 dehydrogenase catalyzes a catabolic step, producing 12-oxoLTB4, which has less than 1% of the potency of LTB4 to stimulate chemotaxis and reactive oxygen generation. Whether oltipraz or other 1,2-dithiole-3-thiones lead to the attenuation of inflammation and/or protection against oxidative stress in vivo has not been closely explored, as yet. 1,2-Dithiole-3-thiones do block the formation of lipid peroxidation and manifestation of hepatotoxicity in mice treated with either carbon tetrachloride or acetaminophen (23, 61). Clearly, there are still more inducible genes to be found and new actions to be elucidated. Using two-dimensional electrophoretic gel protein mapping, Anderson et al. (62) have determined that oltipraz changes the abundance of at least 26 proteins in the liver of F344 rats. Only a few of these proteins have been identified (e.g., GST R and AFAR); most remain unknown as are their functional contributions, if any, to chemoprevention. Molecular Mechanisms of Enzyme Induction. The molecular mechanisms regulating the induction of phase 2 enzymes by 1,2-dithiole-3-thiones and other inducers have also been investigated. Initial molecular studies indicated that increases in rat and human hepatic GST mRNA and protein levels in response to oltipraz were mediated through transcriptional activation of GST genes (65, 66). As originally proposed by Wattenberg (30), two families of phase 2 enzyme inducers exist, based upon their ability to elevate phase 1 enzymatic profiles. Prochaska and Talalay (67) have coined the terms bifunctional and monofunctional inducers to describe these families. Bifunctional inducers (e.g., polycyclic hydrocarbons, dioxins, azo dyes, and flavones) can all be characterized as large planar polycyclic aromatics and elevate phase 2 as well as selected phase 1 enzymatic activities such as aryl hydrocarbon hydroxylase. These compounds are potent ligands for the aryl hydrocarbon (Ah) receptor, and the direct participation of the Ah receptor in the activation of aryl hydrocarbon hydroxylase gene transcription has been demonstrated (68). Moreover, since the phase 2 enzyme inducibility by bifunctional inducers segregates in mice that possess functional Ah receptors, it had been presumed that these enzymes were under the direct control of the Ah receptor. Monofunctional inducers (phenols, lactones, isothiocyanates, dithiocarbamates, and 1,2-dithiole-3-thiones) elevate phase 2 enzymatic activities without significantly elevating the aforementioned phase 1 activities and do not possess an obvious defining structural characteristic. There is no evidence at this time to suggest that monofunctional inducers function through a receptormediated pathway. However, Talalay et al. (69) have identified a chemical signal present in some monofunctional inducers: the presence or acquisition of an electrophilic center. Many monofunctional inducers are Michael reaction acceptors (e.g., an olefin conjugated to

an electron-withdrawing group), and potency is generally paralleled by their efficiency as Michael reaction acceptors. These generalizations can account for the inducer activity of many types of chemopreventive agents and have led to the identification of other novel classes of inducers, including acrylates, fumarates, maleates, vinyl ketones, and vinyl sulfones. Other classes of monofunctional inducers, notably peroxides, vicinal dimercaptans, heavy metals, arsenicals, and the 1,2-dithiole-3-thiones, exhibit a common capacity for reaction with sulfhydryls by either oxidoreduction or alkylation (70). Several lines of evidence point to a critical role of thiols in the bioactivity of 1,2-dithiole-3-thiones. First, structureactivity studies demonstrate a requirement for a 1,2dithiole motif. For example, 3H-1,2-dithiole-3-thione is very active as an inducer of enzymes and inhibitor of AFB1-induced hepatocarcinogenesis, while 1,3-dithiole2-thione is inactive (71, 72). Second, in the context of chemotherapy of schistosomiasis with oltipraz, the irreversible inhibition of glutathione reductase in S. mansoni involves cleavage of the disulfide bond and formation of a mixed disulfide (73). 1,2-Dithiole-3-thiones react very rapidly with dithiols, but very slowly with monothiols such as reduced glutathione (T. J. Curphey, unpublished observations). The target molecule for these enzyme inducers has been hypothesized to contain vicinal thiols, which could be modified through oxidation or alkylation (70). A possible mechanism for the interaction of 1,2dithiole-3-thiones with thiols is presented in Scheme 1. The first step in the reaction is the addition of thiolate to S-2 of the 1,2-dithiole-3-thione 1 to form intermediate 2, as proposed by Fleury et al. (71). However, to explain the unreactivity of simple aliphatic thiols, we posit that this reaction must be reversible and the position of equilibrium must lie strongly in favor of the starting materials. In the second step, 2, probably in the form of its conjugate acid 2A, reacts with more thiolate to yield 3 and the symmetrical disulfide corresponding to the thiolate. The explanation for the large rate difference between dithiols and monothiols is that the second step is intramolecular for dithiols and therefore occurs very much faster than for monothiols. The enethiolate 3 is not likely to be stable and would be expected to be reactive toward nucleophiles, particularly amino groups such as those of lysine. Preliminary work with [14C]oltipraz suggests that covalent modification occurs with thiol rich proteins such as HIV reverse transcriptase (74). It would appear that 1,2-dithiole-3-thiones may represent a very selective class of thiol oxidizing agents, unreactive toward ordinary thiol groups, but capable of forming disulfide bonds from dithiols when the two sulfhydryl groups are in close proximity. Kim and Gates (75) have also demonstrated that oltipraz and 3H-1,2-dithiole-3-thione convert molecular oxygen to reactive oxygen species in the presence of thiols. Consonnant with such actions, modulation of the binding of transcriptional activators by


factors affecting the redox state of protein thiols is apparently a common means of regulating gene expression (76). Several regulatory elements controlling the expression and inducibility of the Ya subunit of rodent GSTs by bifunctional and monofunctional inducers have been characterized (77, 78). A 41 bp element in the 5′-flanking region of the rat GST Ya gene, termed the “antioxidant response element” (ARE), has been identified using a series of 5′ deletion mutants fused to the chloramphenicol acetyl transferase gene and then transfected into hepatoma cells. The sequences for these cis-acting ARE enhancer regions contain two or more copies of AP-1 or AP1-like elements (79). To date, AREs have been detected in the promoters of nearly a score of genes. All share a common RTGACnnnGC core motif (80). Prestera et al. (70) have observed that many classes of monofunctional inducers stimulate expression of a reporter gene, growth hormone, through the ARE when an ARE-growth hormone construct is transfected into murine hepatoma cells. Further, when 25 dithiolethiones and related analogues were evaluated for their activities as inducers of phase 2 enzymes and as activators of the transfected ARE construct in this model system, a strong correlation was seen in the potencies of 21 active 1,2-dithiole-3-thiones to elicit the two responses (71). Moreover, no 1,2-dithiole3-thiones were inactive in only one system. The transcription factors that bind to the ARE consensus sequence have not been fully identified and are likely to vary between cell types and species. Nrf1 and Nrf2, members of the basic-leucine zipper NF-E2 family of transcription factors that regulate expression of globin genes during erythroid development (81, 82), are known to bind and activate the ARE. Overexpression of either Nrf1 or Nrf2 in human hepatoma cells enhances the basal and inducible transcriptional activity of an ARE reporter gene (83). Because other basic-leucine zipper transcription factors typically form heterodimers, Nrf1 and Nrf2 may also dimerize with other factors to activate the ARE. The tissue specific expression profiles of a number of transcription factors suggest that an Nrf2-small Maf heterodimer best mirrors the pattern for induction of phase 2 genes in vivo. Using recombinant Nrf2 and mafK proteins in an electromobility shift assay with the promoter sequence of the murine GST Ya gene, Yamamoto and colleagues (80) demonstrated binding of the heterodimer complex to this promoter. Oligonucleotides containing the ARE effectively competed for the binding of this heterodimeric complex to the GST Ya promoter. This group has also directly examined this issue by exploring the effects of disruption of the nrf2 gene in vivo on induction of phase 2 enzymes. The phenolic antioxidant BHA vigorously induced GST and other phase 2 activities and mRNA expression in several tissues of wild type and heterozygous mutant mice, but not in the homozygous mutant mice (84). Comparable effects have been seen with 3H-1,2-dithiole-3-thione (K. Itoh, T. Ishii, T. Kensler, T. Sutter, and M. Yamamoto, unpublished observations). Collectively, these results suggest that Nrf2-MafK heterodimers may be one of the key regulators of phase 2 gene expression. However, the details of how the chemical signals produced by enzyme inducers such as 1,2-dithiole-3-thiones interact with this molecular signaling pathway remain to be elucidated.

Kensler et al.

Intermediate Biomarkers: Short-Term Insights into Chemopreventive Efficacy The identification, development, and validation of modulatable intermediate biomarkers are essential to the sustained growth of the field of chemoprevention. By contrast, reliance on long-term animal bioassays or cancer end-point clinical trials ensures a glacial rate of progress in the development of new chemopreventive agents and the improved use of old ones. Use of intermediate biomarkers offers several practical advantages that streamline the efficiency of agent development. Notably, they reduce sample size requirements and the time needed to conduct studies (8). To better define the contributions of aflatoxins to the incidence of HCC in humans, considerable attention has been focused on the development of tools for assessing the exposure of individuals to AFB1. The development of these biomarkers has required analytic techniques that were sensitive, specific, and, perhaps most important, amenable to large numbers of samples. Depicted in Figure 2 are four aflatoxin biomarkers employed in our studies, namely, aflatoxin-DNA adducts, aflatoxinalbumin adducts, AFM1, and aflatoxin-mercapturic acid. Aflatoxin-DNA and -protein adducts have been of major interest because they represent direct products of (or surrogate markers for) damage to a critical macromolecular target (85-87). The primary aflatoxin-nucleic acid adduct (AFB-N7-Gua) appears to be exclusively excreted in urine following its removal from DNA. This adduct has a short biological half-life, and its measurement is thought to reflect recent exposure to aflatoxin. Albumin adducts found in serum are also derived from the 8,9-epoxide, but are thought to reflect exposures over longer time periods. This view is based upon the long (∼3 weeks) half-life of circulating albumin in humans. Other metabolites of aflatoxin found in urine also appear to be useful biomarkers for exposure and risk, namely, AFM1 (86, 88) and aflatoxin-mercapturic acid (89). All of the urinary metabolites can be measured using sequential immunoaffintiy and liquid chromatographic techniques coupled with various detection methods. Radioimmune assays or ELISAs, which are particularly amenable to large numbers of samples, are routinely employed for assays of aflatoxin-albumin adducts. Detailed reviews on the development, application, and interpretation of these biomarkers have been published (7, 90-92). A critical feature for the use of these biomarkers in chemoprevention studies is that they can be modulated by protective interventions. A variety of anticarcinogens such as ethoxyquin, BHA, and BHT as well as oltipraz significantly reduce aflatoxin-DNA adduct levels in livers of exposed animals (24). Under dosing conditions that lead to complete protection against AFB1-induced hepatocarcinogenesis, Roebuck et al. (29) observed that oltipraz pretreatment of rats led to a 67% reduction in the level of 24 h urinary excretion of AFB-N7-Gua compared to that of rats only treated with AFB1. Comparable reductions by oltipraz in levels of aflatoxinalbumin adducts in sera of rats have been observed following either single or multiple doses of AFB1 (33, 93). In a unique study, the predictive value of aflatoxinalbumin adduct measurements in defining the risk of development of HCC has been examined (93). Aflatoxin exposure was held constant, while the risk of HCC was modulated through the use of different intervention

Perspective

protocols with oltipraz. One hundred twenty-three male F344 rats were dosed with 20 µg of AFB1 daily for 5 weeks after randomization into no intervention, delayedtransient intervention (500 ppm oltipraz, weeks 2 and 3 relative to AFB1), or persistent intervention (500 ppm oltipraz, weeks -1 to 5) groups. Serial blood samples were collected from each animal at weekly intervals throughout AFB1 exposure and assayed for levels of aflatoxin-albumin adducts. Area under the curve (AUC) values for aflatoxin-albumin adducts decreased 20 and 39% in the delayed-transient and persistent oltipraz intervention groups, respectively, as compared to the value with no intervention. Total incidence of HCC dropped from 83 to 60% (p ) 0.03) and 48% (p < 0.01) in these groups, highlighting a concordance between these two end points. Overall, a significant association was seen between biomarker AUC and the risk of HCC (p ) 0.01). However, when the predictive value of aflatoxin-albumin adducts was assessed within treatment groups, there was no association between AUC and the risk of HCC (p ) 0.56). Thus, aflatoxin-albumin adducts can be useful for monitoring population-based changes induced by interventions, such as in chemoprevention trials, but have limited utility in identifying individuals destined to develop HCC. AFM1 is a major metabolite of AFB1 found in human urine, typically accounting for several percent of the ingested dose (88). Because AFM1 is formed by the same cytochrome P450 (P450 1A2) that yields the 8,9-epoxide, AFM1 may serve as a reasonable surrogate for the genotoxic potential of aflatoxin exposures in individuals. Such a possibility has been examined in residents of Fusui County, Guangxi Autonomous Region, PRC, where a high incidence of HCC has been reported. Zhu et al. (94) analyzed AFM1 concentrations in urine samples by ELISA and noted correlations between AFM1 excretion and levels of AFB1 in corn and peanut oil samples collected from different households. Using immunoaffinity and HPLC methods, Groopman et al. (95) have observed that measurements of urinary excretion of AFM1 and the labile DNA adduct AFB-N7-Gua showed strong and highly significant correlations with aflatoxin intake in this region. In a prospective, nested case-control study, Qian et al. (96) reported that the relative risk of HCC for individuals positive for urinary AFM1 was 4.4 [95% confidence interval (CI) of 2.1-9.6] compared to those not excreting this biomarker. Similarly, Yu et al. (93) reported a significant dose-response relationship between urinary AFM1 levels and the risk of HCC. The odds ratio comparing the risk of HCC in the highest with the lowest tertile of AFM1 levels was 6.0 (1.2-29.0). Thus, urinary levels of AFM1 may provide a useful index of altered risk for use in chemopreventive interventions. Scholl et al. (48) have directly examined this possibility in AFB1-exposed rats. Using a delayed, transient intervention protocol with oltipraz, it was reported that the extent of excretion of AFM1 was reduced by 77% during the active phase of the intervention, when oltipraz was added to the diet, but rapidly returned to control levels after cessation of oltipraz administration. AFM1 represents, therefore, a reversibly modulatable risk biomarker for HCC.


Study Cohorts: Where To Test the Hypothesis Quantifiable, well-characterized risk factors can be used to define cohorts for chemopreventive interventions (8). These risk markers fall into five broad categories: (1) carcinogen exposure, (2) carcinogen effect, (3) genetic predisposition, (4) precancerous lesions, and (5) previous cancer. Given the strong association between aflatoxin exposure and HCC, individuals regularly consuming aflatoxins are at high risk for liver cancer and form a useful cohort for evaluating the efficacy of oltipraz as a chemopreventive agent and for further probing its mechanisms of action. HCC is the fifth leading cause of cancer death worldwide and is one of the most common cancers in China where there are an estimated 200 000 deaths annually. HCC is the leading cause of cancer death in Qidong County in eastern Jiangsu Province, PRC, and accounts for up to 10% of all adult deaths in some of the rural townships (98, 99). Moreover, the median age of death from HCC in this region is 50 years. Case-control studies indicate that chronic infection with hepatitis B virus (HBV) is an important risk factor. However, while the percentage of individuals infected with HBV is constant throughout Jiangsu Province, the incidence of HCC increases more than 10-fold over a 100 km west-east gradient near the mouth of the Yangtze River (99). It has been postulated that exposure to aflatoxins in the diet and algal toxins in the drinking water also contribute to the extraordinarily high risk of HCC in Qidong (98, 99). Climatic conditions in Qidong, featuring high humidity and average summer temperatures of >30 °C, coupled with primitive storage facilities, are very conducive to mold spoilage of foods and therefore aflatoxin contamination. Food surveys indicate persistent contamination of dietary staples such as rice, corn, peanuts, and soy with aflatoxin (100). An ongoing nested case-control study in nearby Shanghai has demonstrated a multiplicative interaction between HBV and aflatoxins in the risk of HCC (96, 101). A significant increase in the odds ratio (OR) was observed for those cases of HCC where aflatoxins were detected in urine (OR ) 3.4, 95% CI of 1.110.0). Similarly, the OR for people who tested positive for HBV surface antigen was elevated (OR ) 7.3, 95% CI of 2.2-24.4). Remarkably, individuals with both aflatoxin-positive urine and HBV seropositivity had an OR for developing HCC of 59.4 (95% CI of 16.6-212.0). Unlike measurements of urinary excretion of aflatoxins, which measure very recent exposures to aflatoxins, the determination of serum levels of aflatoxin-albumin adducts appears to provide a more integrated assessment of aflatoxin exposures over a period of several months. A longitudinal survey of 120 residents of Daxin Township, Qidong, conducted in 1993 indicated that a majority of the participants tested positive for serum aflatoxinalbumin adducts throughout a 12 month period (102). A nested case-control study in a prospective cohort of individuals in Qidong who are seropositive for hepatitis B surface antigen has indicated that the OR for HCC among individuals also positive for aflatoxin-albumin adducts was 2.4 (95% CI of 1.2-4.7) (103). Similarly, in Taiwan, Wang et al. (104) observed an OR of 2.8 (95% CI of 0.9-9.1) for detectable compared to nondetectable aflatoxin-albumin adducts in HBsAg seropositive men. Molecular studies also suggest a role for aflatoxin in the


etiology of HCC. Characterization of the mutational spectra in the p53 tumor suppressor gene in HCC from Qidong demonstrated a high frequency (>50%) of AGG f AGT transversion mutations on the noncoding strand at codon 249 (105). These mutations are not observed in liver cancers from low-aflatoxin exposure regions of China. Consistent with these findings, exposure of human liver cell lines to AFB1 leads to preferential G f T transversion mutations of the third base in codon 249 (106). The synergism between virus and environmental carcinogen for the development of HCC suggests that reduction in either risk factor will have important public health consequences. Strategies for the primary prevention of HCC in Qidong include HBV vaccination programs, improved water quality and crop control, and diminished consumption of corn (98, 99). However, to break the cycle that begins with HBV infection at birth, universal vaccination must be carried out for several generations. As a consequence, even under ideal circumstances, the desired effect of reducing HCC may take some time to emerge (107). Cost also greatly restricts the use of HBV vaccines. The extent of aflatoxin contamination in foods is a function of the ecology of molds and is not completely preventable, nor is adequate control economically feasible in much of the world. Thus, in practice, additional strategies need to be developed to have immediate, significant worldwide impact upon mortality rates of HCC. Secondary prevention programs, such as chemoprevention, may be useful in this context.

Clinical Trials Phase I clinical trials are designed to characterize the pharmacokinetics and tolerableness of the chemopreventive agent (6). Doses and the schedule of administration are based on achieving plasma drug levels that are very likely to be safe and likely to show effectiveness based upon preclinical studies in in vivo and in vitro models. Single-dose Phase I studies with oltipraz indicated that administration of 500 mg orally would produce a peak plasma concentration of about 20 µM while 125 mg produced a peak of only 2 µM (47). Dose escalation studies with repeated administration suggested that 125 mg of oltipraz was close to the maximum tolerated dose following administration daily for 6 months (108). Although the steady state concentrations of oltipraz are rather low, reflecting the rapid clearance of the drug from the body, the peak concentrations following administration of 125-500 mg of oltipraz/day are comparable to those required to induce phase 2 enzyme expression in rodent and human cell culture models. Gupta et al. (47) reported a doubling in the specific activity of GST in peripheral lymphocytes obtained from Phase I study participants 10 h after administration of 125 mg of oltipraz. Elevations in the levels of glutathione were also observed. In a dose-finding study with 125, 250, 500, or 1000 mg/m2 oltipraz as a single oral dose, increases in GST activities were seen in peripheral mononuclear cells and colon mucosa biopsies at the lower, but not higher, doses (50). Increases of 4-5-fold in the level of mRNA transcripts for γ-glutamylcysteine synthetase and quinone reductase were seen in colon mucosa at 250 mg/ m2. Higher doses were not more effective. mRNA content increased after dosing to reach a peak on day 2 and declined to baseline levels over the subsequent week.

Kensler et al.

Collectively, these results demonstrate that oltipraz triggers the expression of phase 2 enzymes in humans. To more directly test the hypothesis that oltipraz can modulate the metabolism of carcinogens in humans, we conducted a Phase IIa intervention trial with oltipraz. The primary goals of Phase IIa studies, in addition to establishing the general feasibility of conducting biomarker measurements, are to characterize the dose response of biomarker modulation, the tolerance or loss of the effect of biomarker modulation over time, and the drug toxicity with chronic administration (6). Study participants for this Phase IIa trial were recruited from residents of Daxin Township, Qidong, PRC, the site of our earlier longitudinal analysis of aflatoxin biomarkers (102). This trial with oltipraz was a randomized, placebocontrolled, double-masked study. Two hundred forty adults in good general health without any history of major chronic illnesses and with detectable serum aflatoxin-albumin adduct levels at baseline were randomized into one of three intervention arms: (A) placebo, (B) 125 mg of oltipraz administered daily, or (C) 500 mg of oltipraz administered weekly. The methods, participant characteristics, compliance, and adverse events as well as initial results on modulation of biomarkers from this trial have been reported (108-111). Urine samples were collected at 2 week intervals throughout the active 8 week intervention period as well as during the 8 week followup period. To date, aflatoxin metabolites have been assayed in urine samples from one cross section in time, after the first month on the active intervention (111). Sequential immunoaffinity and liquid chromatography coupled to mass spectrometry and fluorescence detection were used to identify and quantify the phase 1 metabolite, AFM1, and the phase 2 metabolite, aflatoxin-mercapturic acid, in these urine samples. As depicted in Figure 4, 1 month of weekly administration of 500 mg of oltipraz led to a significant (p ) 0.030) 51% decrease in median levels of AFM1 excreted in urine compared to the results with the placebo, but had no effects on levels of aflatoxin-mercapturic acid (p ) 0.772). By contrast, daily intervention with 125 mg of oltipraz led to a significant (p ) 0.017) 2.6-fold increase in the median levels of aflatoxin-mercapturic acid excretion, but had no pronounced effect on excreted AFM1 levels. Thus, sustained low-dose oltipraz increased the extent of phase 2 conjugation of aflatoxin, yielding higher levels of mercapturic acid, but did not appreciably affect P450 1A2-mediated formation of AFM1. Intermittent, high-dose oltipraz inhibited the phase 1 activation of aflatoxin, as reflected by a lowered level of excretion of aflatoxin M1. Potential effects of induction of phase 2 enzymes, i.e., GSTs, in this arm appear to be masked by the inhibition of aflatoxin-8,9-epoxide formation. Indeed, Langoue´t et al. (46) have reported 2-4-fold increases in the protein levels of alpha and mu classes of GSTs in primary human hepatocytes treated with 50 µM oltipraz, but found this inductive effect was not associated with an increased level of formation of aflatoxin-glutathione conjugates because it was overridden by the inhibitory effect of oltipraz on AFB1 activation by P450 1A2. As previously noted in experimental models, it would appear that both mechanisms are likely to contribute to reduced genotoxicity and other chemopreventive actions of this drug. In addition to the cross-sectional measurements of effects of oltipraz on urinary aflatoxin metabolites,

Perspective

Figure 4. Median values for the excretion of AFM1 (left) and aflatoxin-mercapturic acid (AFB-NAC) (right) after 4 weeks of the Phase IIa oltipraz intervention trial. Overnight urine samples from 72, 57, and 60 participants in the placebo (P), 125 mg of oltipraz daily (125), and 500 mg of oltipraz weekly (500) groups, respectively, were collected and then assayed by sequential immunoaffinity and liquid chromatography with fluorescence detection. Wilcoxon rank sum tests were used to compare levels of metabolites in each of the two treatment arms to the results in the placebo arm. P values are two-tailed. Adapted from ref 111.

longitudinal analyses of effects on the slopes of aflatoxinalbumin adducts have been conducted (110). There were no consistent changes in albumin-adduct levels in the placebo arm, or in the 125 mg of oltipraz daily arm over the 16 week observation period. However, individuals receiving 500 mg of oltipraz once a week for 8 weeks showed a triphasic response to oltipraz. No effect was observe during the first month of the intervention, whereas a significant (p ) 0.001) diminution in adduct levels was observed during the second month of active intervention and during the first month of followup. A partial rebound in adduct levels toward baseline values was observed during the second month of followup. Linear regression models up to week 13 confirmed a significant (p ) 0.008) weekly decline in biomarker levels in this group. Because modulation of aflatoxin-albumin adducts and diminution of AFM1 levels were both observed in the 500 mg weekly arm, comparisons of the albumin adduct slopes with levels of AFM1 were made. Individuals ranked in the lowest tertile of AFM1 levels showed the greatest decline in aflatoxin-albumin adduct levels (p ) 0.078). This moderate correlation suggests that inhibition of cytochrome P450 activity could contribute to the observed decline in the levels of albumin adducts.

Proof of Principle and Future Prospects The 15-year-old question of whether oltipraz can induce phase 2 enzymes or other genes in humans has been addressed in human cells in culture and in the Phase I and IIa clinical trials. The answer is yes in all systems. Oltipraz clearly induces GSTs in primary


cultures of human hepatocytes. Elevation of glutathione concentrations and GST activity in lymphocytes and increased levels of mRNA transcripts for quinone reductase and γ-glutamylcysteine synthetase in colon mucosa of patients receiving oltipraz are indicative of pharmacodynamic action in vivo as well. The finding of significantly elevated levels of aflatoxin-mercapturic acid in the residents of Daxin taking 125 mg of oltipraz daily forms the capstone to the answer. Clearly, a proof of principle has been established. But there are now a whole round of new questions to be addressed. Clinical trials rarely are definitive, particularly those probing the initial manifestations of efficacy. For example, are all phase 2 enzymes induced? And what about the other 1,2-dithiole3-thione-inducible genes? Are they induced in humans, and how might they contribute to chemoprevention? The preclinical and clinical results have also established that oltipraz is an effective inhibitor of aflatoxin activation. Can the dual mechanisms be teased apart and the contribution of each component identified? The bigger question of whether modulation of carcinogen metabolism, either by enzyme inhibition or by enzyme induction, can substantively reduce the risk of cancer in individuals at high risk for exposure to environmental carcinogens remains open. A Phase IIb intervention trial with oltipraz in Qidong is scheduled to begin in early 1999. This trial will evaluate the efficacy of 250 or 500 mg of oltipraz given weekly to modulate levels of aflatoxin biomarkers over a 1 year period in comparison to a placebo group. Biomarkers for exploring the multiple mechanisms of action of oltipraz will be used. The Phase IIb study should serve as a foundation for selecting a safe and effective dose for a Phase III trial. Phase III trials are used to actually establish the efficacy of the drug in chemoprevention and, unless the biomarker is a strong predictor of cancer prevention, rely on reduced incidence of disease as the end point. In a sense, oltipraz is a drug of expedience for chemoprevention; it possesses some strengths and many weaknesses. Is it the drug of choice for modifying carcinogen metabolism? Perhaps, but only for the moment. Oltipraz, which stood at the front of the class 10 years ago, may no longer be the best option. Chemopreventive 1,2dithiole-3-thiones at least 1 order of magnitude more potent than oltipraz in vivo have been developed (71, 112). Moreover, structurally unrelated agents with 1001000-fold greater potency as phase 2 enzyme inducers have been identified with cell culture screening assays (16, 17, 113). Some of these agents are found in foods, making them potentially much more accessible and acceptable to the general population. The major challenge in the process is to bring together the best of the ABCs (agents, biomarkers, and cohorts) at one point in time. Success in meeting the challenge requires continual reevaluation and upgrading of the research tools and refinement of the experimental questions as new knowledge confirms or refutes underlying assumptions.

Acknowledgment. Financial support has been provided by the National Cancer Institute (CA39416, CA44530, CA77130, and NO1CN25437) and the National Institute of Environmental Health Sciences (ES03819 and ES06052). Numerous colleagues have contributed to many aspects of our mechanistic and clinical work with oltipraz for which we offer our heartfelt thanks. Two of our colleagues have lost their lives to cancer. It is to them


we dedicate this Perspective. Ernest Bueding, M.D. (1910-1986), was a dedicated scholar who devoted the later years of his illustrious career to studies on cancer chemoprevention. He made the seminal observations on the enzyme inductive actions of oltipraz, predicted its chemopreventive efficacy, and gently but persistently encouraged us to probe its action and efficacy in model systems. Hans J. Prochaska, M.D., Ph.D. (1958-1998), was an imaginative and enthusiastic young scientist who provided novel insights into the mechanisms of regulation of phase 2 enzymes (monofunctional vs bifunctional inducers), developed rapid, facile screening assays for the identification of new inducers, and participated as a physician and scientist in the Phase IIa oltipraz intervention trial in Qidong, PRC, in 1995. We miss these friends who did much to bring this story to where it now stands.

Kensler et al.

(16) (17)

(18)

(19)

(20) (21)

References (1) Bailar, J. C., and Gornik, H. L. (1997) Cancer undefeated. N. Engl. J. Med. 336, 1569-1574. (2) Steele, V. E., Moon, R. C., Lubet, R. A., Grubbs, C. J., Reddy, B. S., Wargovich, M., McCormick, D. L., Perira, M. A., Crowell, J. A., Bagheri, D., Sigman, C. C., Boone, C. W., and Kelloff, G. J. (1994) Preclinical efficacy evaluation of potential chemopreventive agents in animal carcinogenesis models: methods and results from the NCI chemoprevention drug development program. J. Cell. Biochem. 20S, 32-54. (3) Sporn, M. B. (1996) The war on cancer. Lancet 347, 1377-1381. (4) Kelloff, G. J., Crowell, J. A., Boone, C. W., Steele, V. E., Lubet, R. A., Greenwald, P., Alberts, D. S., Covey, J. M., Doody, L. A., Knapp, G. G., Nayfield, S., Parkinson, D. R., Prasad, V. K., Prorok, P. C., Sausville, E. A., and Sigman, C. C. (1994) Strategy and planning for chemopreventive drug development: clinical development plans. J. Cell. Biochem. 20S, 55-299. (5) Kelloff, G. J., Crowell, J. A., Hawk, E. T., Steele, V. E., Lubet, R. A., Boone, C. W., Covey, J. M., Doody, L. A., Omenn, G. S., Greenwald, P., Hong, W. K., Parkinson, D. R., Bagheri, D., Baxter, G. T., Blunden, M., Doeltz, M. K., Eisenhauer, K. M., Johnson, K., Knapp, G. G., Longfellow, D. G., Malone, W. F., Nayfield, S. G., Seifried, H. E., Swall, L. M., and Sigman, C. C. (1996) Strategy and planning for chemopreventive drug development: clinical development plans II. J. Cell. Biochem. 26S, 54-315. (6) Kelloff, G. J., Johnson, J. R., Crowell, J. A., Boone, C. W., DeGeorge, J. J., Steele, V. E., Mehta, M. U., Temeck, J. W., Schmidt, W. J., Burke, G., Greenwald, P., and Temple, R. J. (1995) Approaches to the development and marketing approval of drugs that prevent cancer. Cancer Epidemiol. Biomarkers Prev. 4, 1-10. (7) Groopman, J. D., and Kensler, T. W. (1993) Molecular biomarkers for human chemical carcinogen exposures. Chem. Res. Toxicol. 6, 764-770. (8) Kelloff, G. J., Boone, C. W., Crowell, J. A., Nayfield, S. G., Hawk, E., Malone, W. F., Steele, V. E., Lubet, R. A., and Sigman, C. C. (1996) Risk biomarkers and current strategies for cancer chemoprevention. J. Cell. Biochem. 25S, 1-14. (9) Miller, E. C., and Miller, J. A. (1985) Some historical perspectives on the metabolism of xenobiotic chemicals to reactive intermediates. In Bioactivation of Foreign Compounds (Anders, M. W., Ed.) pp 1-28, Academic Press, New York. (10) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (11) Wattenberg, L. W. (1972) Inhibition of carcinogenic and toxic effects of polycyclic hydrocarbons by phenolic antioxidants and ethoxyquin. J. Natl. Cancer Inst. 48, 1425-1430. (12) Benson, A. M., Batzinger, R. P., Ou, S.-Y. L., Bueding, E., Cha, Y.-N., and Talalay, P. (1978) Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo[a]pyrene by dietary antioxidants. Cancer Res. 38, 4486-4495. (13) Talalay, P., DeLong, M. J., and Prochaska, H. J. (1987) Molecular mechanisms in protection against carcinogenesis. In Cancer Biology and Therapeutics (Cory, J. G., and Szentivanyi, A., Eds.) pp 197-216, Plenum, New York. (14) Kensler, T. W. (1997) Chemoprevention by inducers of carcinogen detoxication enzymes. Environ. Health Perspect. 105S4, 965-970. (15) Hayes, J. D., McLeod, R., Ellis, E. M., Pulford, D. J., Ireland, L. S., McLellan, L. I., Judah, D. J., Manson, M. M., and Neal, G. E.

(22) (23)

(24)

(25)

(26)

(27)

(28)

(29)

(30) (31)

(32)

(33)

(34)

(35)

(36)

(37)

(1996) Regulation of glutathione S-transferases and aldehyde reductase by chemoprotectors: studies of mechanisms responsible for inducible resistance to aflatoxin B1. In Principles of Chemoprevention (Stewart, B. W., McGregor, D., and Kleihues, P., Eds.) pp 175-187, International Agency for Cancer Research, Lyon, France. Pezutto, J. M. (1997) Plant-derived anticancer agents. Biochem. Pharmacol. 53, 121-133. Zhang, Y., Talalay, P., Cho, C.-G., and Posner, G. H. (1992) A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. Natl. Acad. Sci. U.S.A. 89, 2399-2403. Jirousek, L., and Starka, L. (1958) Uber das vorkommen von trithionen (1,2-dithiocyclopent-4-en-3-thione) in Brassicapflanzen. Naturwissenschaften 45, 386-387. Marks, H. S., Leichtweis, H. C., and Stoewsand, G. S. (1991) Analysis of a reported organosulfur, carcinogenesis inhibitor: 1,2dithiole-3-thione in cabbage. J. Agric. Food Chem. 39, 893-895. Archer, S. (1985) The chemotherapy of schistosomiasis. Annu. Rev. Pharmacol. 25, 485-508. Bueding, E., Dolan, P., and Leroy, J.-P. (1982) The antischistosomal activity of oltipraz. Res. Commun. Chem. Pathol. Pharmacol. 37, 293-303. Ansher, S. S., Dolan, P., and Bueding, E. (1986) Biochemical effects of dithiolthiones. Food Chem. Toxicol. 24, 405-415. Ansher, S. S., Dolan, P., and Bueding, E. (1983) Chemoprotective effects of two dithiolthiones and of butylhydroxyanisole against carbon tetrachloride and acetaminophen toxicity. Hepatology 3, 932-935. Kensler, T. W., Egner, P. A., Trush, M. A., Bueding, E., and Groopman, J. D. (1985) Modification of aflatoxin B1 binding to DNA in vivo in rats fed phenolic antioxidants, ethoxyquin and a dithiolthione. Carcinogenesis 6, 759-763. Wattenberg, L. W., and Bueding, E. (1986) Inhibitory effects of 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on carcinogenesis induced by benzo[a]pyrene, diethylnitrosamine and uracil mustard. Carcinogenesis 7, 1379-1381. Eaton, D. L., and Groopman, J. D., Eds. (1994) The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance, Academic Press, New York. International Agency for Research on Cancer (1993) Some naturally occurring substances: food items and constituents, heteroxyclic aromatic amines and mycotoxins. IARC Monogr. Eval. Carcinog. Risk Chem. Man 56, 245-395. Kensler, T. W., Egner, P. A., Dolan, P. M., Groopman, J. D., and Roebuck, B. D. (1987) Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol3-ones. Cancer Res. 47, 4271-4277. Roebuck, B. D., Liu, Y.-L., Rogers, A. R., Groopman, J. D., and Kensler, T. W. (1991) Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry. Cancer Res. 51, 5501-5506. Wattenberg, L. W. (1985) Chemoprevention of cancer. Cancer Res. 45, 1-8. Maxuitenko, Y. Y., MacMillan, D. L., Kensler, T. W., and Roebuck, B. D. (1993) Evaluation of the post-initiation effects of oltipraz on aflatoxin B1-induced preneoplastic foci in a rat model of hepatic tumorigenesis. Carcinogenesis 14, 2423-2425. Bolton, M. G., Munõz, A., Jacobson, L. P., Groopman, J. D., Maxuitenko, Y. Y., Roebuck, B. D., and Kensler, T. W. (1993) Transient intervention with oltipraz protects against aflatoxininduced hepatic tumorigenesis. Cancer Res. 53, 3499-3504. Kensler, T. W., Gange, S. J., Egner, P. A., Dolan, P. M., Munõz, A., Groopman, J. D., Rogers, A. E., and Roebuck, B. D. (1997) Predictive value of molecular dosimetry: Individual versus group effects of oltipraz on aflatoxin-albumin adducts and risk of hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev. 6, 603-610. Kensler, T. W., and Helzlsouer, K. J. (1995) Oltipraz: clinical opportunities for chemoprevention. J. Cell. Biochem. 22S, 101107. Heuse, D., Marland, M., Bredenbac, J., Decouvelaere, B., Leroy, J. P., Bieder, A., and Jumeau, H. (1985) Disposition of 14C-oltipraz in animals. Arzneim.-Forsch. 35, 1431-1436. Moon, R. C., Kelloff, G. J., Detrisac, C. J., Steele, V. E., Thomas, C. F., and Sigman, C. C. (1994) Chemoprevention of OH-BBN induced bladder cancer in mice by oltipraz, alone or in combination with 4-HPR and DFMO. Anticancer Res. 14, 5-11. Moon, R. C., Rao, K. V. N., Detrisac, C. J., Kelloff, G. J., Steele, V. E., and Doody, L. A. (1994) Chemoprevention of respiratory

Perspective

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

(50)

(51)

(52)

(53)

(54)

(55)

(56)

tract neoplasia in the hamster by oltipraz, alone and in combination. Int. J. Oncol. 4, 661-667. Kensler, T. W., Davidson, N. E., Egner, P. A., Guyton, K. Z., Groopman, J. D., Curphey, T. J., Liu, Y.-L., and Roebuck, B. D. (1991) Protection against aflatoxin-induced hepatocarcinogenesis by dithiolethiones. In Mycotoxins, Cancer and Health (Bray, G. A., and Ryan, D. H., Eds.) pp 359-371, LSU Press, Baton Rouge, LA. Meyer, D. J., Harris, J. M., Gilmore, K. S., Coles, B., Kensler, T. W., and Ketterer, B. (1993) Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole3-thiones. Carcinogenesis 14, 567-572. Raney, K. D., Meyer, D. J., Ketterer, B., Harris, T. M., and Guengerich, F. P. (1992) Glutathione conjugation of aflatoxin B1 exo- and endo-epoxides by rat and human glutathine S-transferases. Chem. Res. Toxicol. 5, 470-478. Judah, D. J., Hayes, J. D., Yang, J.-C., Lian, L.-Y., Roberts, G. C. K., Farmer, P. B., Lamb, J. H., and Neal, G. E. (1993) A novel aldehyde reductase with activity towards a metabolite of aflatoxin B1 is expressed in rat liver during carcinogenesis and following administration of an antioxidant. Biochem. J. 292, 13-18. Primiano, T., Gastel, J. A., Kensler, T. W., and Sutter, T. R. (1996) Isolation of cDNAs representing dithiolethione-responsive genes. Carcinogenesis 17, 2297-2303. Liu, Y.-L., Roebuck, B. D., Yager, J. D., Groopman, J. D., and Kensler, T. W. (1988) Protection by 5-(2-pyrazinyl)-4-methyl-1,2dithiol-3-thione (oltipraz) against the hepatotoxicity of aflatoxin B1 in the rat. Toxicol. Appl. Pharmacol. 93, 442-451. Buetler, T. M., Gallagher, E. P., Wang, C., Stahl, D. L., Hayes, J. D., and Eaton, D. L. (1995) Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. Toxicol. Appl. Pharmacol. 135, 45-57. Putt, D. A., Kensler, T. W., and Hollenberg, P. (1991) Effect of three chemopreventive antioxidants, ethoxyquin, oltipraz, and 1,2dithiole-3-thione on cytochrome P450 levels and aflatoxin B1 metabolism. FASEB J. 5, A1517. Langoue¨t, S., Coles, B., Morel, F., Becquemont, L., Beaune, P., Guengerich, F. P., Ketterer, B., and Guillouzo, A. (1995) Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Res. 55, 5574-5579. Gupta, E., Olopade, O. I., Ratain, M. J., Mick, R., Baker, T. M., Berezin, F. K., Benson, A. B., III, and Dolan, M. E. (1995) Pharmacokinetics and pharmacodynamics of oltipraz as a chemopreventive agent. Clin. Cancer Res. 1, 1133-1138. Scholl, P., Musser, S. M., Kensler, T. W., and Groopman, J. D. (1996) Inhibition of aflatoxin M1 excretion in rat urine during dietary intervention with oltipraz. Carcinogenesis 17, 1385-1388. Primiano, T., Egner, P. A., Sutter, T. R., Kelloff, G. J., Roebuck, B. D., and Kensler, T. W. (1995) Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxininduced tumorigenesis and induction of glutathione S-transferases. Cancer Res. 55, 4319-4324. O’Dwyer, P. J., Szarka, C. E., Yao, K.-S., Halbherr, T. C., Pfeiffer, G. R., Green, F., Gallo, J. M., Brennan, J., Frucht, H., Goosenberg, E. B., Hamilton, T. C., Litwin, S., Balshem, A. M., Engstrom, P. F., and Clapper, M. L. (1996) Modulation of gene expression in subjects at risk for colorectal cancer by the chemopreventive dithiolethione oltipraz. J. Clin. Invest. 98, 1210-1217. Shiraki, H., and Guengerich, F. P. (1984) Turnover of membrane proteins: kinetics of induction and degradation of seven forms of rat liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydrolase. Arch. Biochem. Biophys. 235, 86-96. Moon, R. C. (1988) Evaluation of chemopreventive agents by in vivo screening assays. Prepared for National Cancer Institute by IIT Research Institute under Contract N01-CN-55448-04. Final Report. Rao, C. V., Rivenson, A., Katiwalla, M., Kelloff, G. J., and Reddy, B. S. (1993) Chemopreventive effect of oltipraz during different stages of experimental colon carcinogenesis induced by azoymethane in male F344 rats. Cancer Res. 53, 2502-2506. Sutter, T. R., Guzman, K., Dodd, K. M., and Greenlee, W. F. (1991) Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science 254, 415-418. Primiano, T., Kensler, T. W., Kuppusamy, P., Zweier, J. L., and Sutter, T. R. (1996) Induction of hepatic heme oxygenase-1 and ferritin in rats by cancer chemopreventive dithiolethiones. Carcinogenesis 17, 2291-2296. Otieno, M., Guyton, K., and Kensler, T. W. (1998) Induction of manganese superoxide dimutase (MnSOD) and catalase mRNA by 1,2-dithiole-3-thione in vivo. Proc. Am. Assoc. Cancer Res. 39, 88.

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 125 (57) Antras-Ferry, J., Maheó, K., Chevanne, M., Dubois, M.-P., Morel, F., Guillouzo, A., Cillard, P., and Cillard, J. (1997) Oltipraz stimulates the transcription of the manganese superoxide gene in rat hepatocytes. Carcinogenesis 18, 2113-2117. (58) Prestera, T., Talalay, P., Alam, J., Ahn, Y. I., Lee, P. J., and Choi, A. M. (1995) Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophile and antioxidants: regulation by upstream antioxidant-responsive elements (ARE). Mol. Med. 1, 827-837. (59) Primiano, T., Li, Y., Kensler, T. W., Trush, M. A., and Sutter, T. R. (1998) Identification of dithiolethione-inducible gene-1 as a leukotriene B4 12-dehydrogenase: implications for chemoprevention. Carcinogenesis 19, 999-1005. (60) Ford-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E., and Smith, M. J. H. (1980) Leukotriene B4, a potent chemotactic and aggregating substance released from polymorphonuclear leukocytes. Nature 286, 264-265. (61) Mansuy, D., Sassi, A., Dansette, P. M., and Plat, M. (1986) A new potent inhibitor of lipid peroxidation in vitro and in vivo, the heptoprotective drug anisyldithiolthione. Biochem. Biophys. Res. Commun. 135, 1015-1021. (62) Anderson, L., Steele, V. K., Kelloff, G. J., and Sharma, S. (1995) Effects of oltipraz and related chemoprevention compounds on gene expression in rat liver. J. Cell. Biochem. 22S, 108-116. (63) O’Dwyer, P. J., Johnson, S. W., Khater, C., Kruger, A., Matsumoto, Y., Hamilton, T. C., and Yao, K.-S. (1997) The chemopreventive agent oltipraz stimulates repair of damaged DNA. Cancer Res. 57, 1050-1053. (64) Christen, M.-O. (1995) Anethole Dithiolethione: biochemical considerations. Biothiols Part B. Glutathione and Thioredoxin, Thiols in Signal Transduction and Gene Regulation. Methods Enzymol. 252, 316-340. (65) Davidson, N. E., Egner, P. A., and Kensler, T. W. (1990) Transcriptional control of glutathione S-transferase gene expression by the chemoprotective agent 5-(2-pyrazinyl)-4-methyl-1,2dithiole-3-thione (oltipraz) in rat liver. Cancer Res. 50, 22512255. (66) Morel, F., Fardel, O., Meyer, D. J., Langoue¨t, S., Gilmore, K. S., Meunier, B., Tu, C.-P. D., Kensler, T. W., Ketterer, B., and Guillouzo, A. (1993) Preferential increase of glutathione Stransferase class alpha transcripts in cultured human hepatocytes by phenobarbital, 3-methylcholanthrene and dithiolethiones. Cancer Res. 53, 231-234. (67) Prochaska, H. J., and Talalay, P. (1988) Regulatory mechanisms of monofunctional and bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res. 48, 4776-4782. (68) Fisher, J. M., Wu, L., Denison, M. S., and Whitlock, J. P., Jr. (1990) Organization and function of a dioxin-responsive enhancer. J. Biol. Chem. 265, 9676-9681. (69) Talalay, P., DeLong, M. J., and Prochaska, H. J. (1988) Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 85, 8261-8265. (70) Prestera, T., Holtzclaw, W. D., Zhang, Y., and Talalay P. (1993) Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc. Natl. Acad. Sci. U.S.A. 90, 2965-2969. (71) Egner, P. A., Kensler, T. W., Prestera, T., Talalay, P., Libby, A. H., Joyner, H. H., and Curphey, T. J. (1994) Regulation of phase 2 enzyme induction by oltipraz and other dithiolethiones. Carcinogenesis 15, 177-181. (72) Maxuitenko, Y. Y., Curphey, T. J., Kensler, T. W., and Roebuck, B. D. (1996) Protection against aflatoxin B1-induced hepatic toxicity as a short term screen for cancer chemopreventive dithiolethiones. Fundam. Appl. Toxicol. 32, 250-259. (73) Fleury, M.-B., Largeron, M., and Martins, T. (1991) Toward an understanding of the schisosomicidal effect of 4-methyl-5-(2pyrazinyl)-1,2-dithiole-3-thione (oltipraz). Biochem. Pharmacol. 41, 361-367. (74) Chavan, S. J., Bornmann, W. G., Flexner, C., and Prochaska, H. J. (1996) Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct. Arch. Biochem. Biophys. 324, 143-152. (75) Kim, W., and Gates, K. S. (1997) Evidence for thiol-dependent production of oxygen radicals by 4-methyl-5-pyrazinyl-3H-1,2dithiole-3-thione (oltipraz) and 3H-1,2-dithiole-3-thione: possible relevance to the anticarcinogenic properties of 1,2-dithiole-3thiones. Chem. Res. Toxicol. 10, 296-301. (76) Primiano, T., Sutter, T. R., and Kensler, T. W. (1997) Redox regulation of genes that protect against carcinogens. Comp. Biochem. Physiol. 118B, 487-497. (77) Friling, R. S., Bensimon, A., Tichauer, Y., and Daniel, V. (1990) Xenobiotic-inducible expression of murine glutathione S-trans-


(78)

(79) (80) (81) (82)

(83)

(84)

(85)

(86)

(87)

(88)

(89) (90) (91)

(92) (93)

(94)

(95)

(96)

ferase Ya subunit gene is controlled by an electrophile-responsive element. Proc. Natl. Acad. Sci. U.S.A. 87, 6258-6262. Rushmore, T. H., King, R. G., Paulson, K. E., and Pickett, C. B. (1990) Regulation of glutathione S-transferase Ya subunit gene expression: Identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. Proc. Natl. Acad. Sci. U.S.A. 87, 3826-3830. Jaiswal, A. K. (1994) Antioxidant response element. Biochem. Pharmacol. 48, 439-444. Wasserman, W. W., and Fahl, W. E. (1997) Functional antioxidant responsive elements. Proc. Natl. Acad. Sci. U.S.A. 94, 5361-5366. Chan, J. Y., Han, X., and Kan, Y. W. (1993) Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast. Proc. Natl. Acad. Sci. U.S.A. 90, 11366-11370. Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y. W. (1993) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the β-globin locus control region. Proc. Natl. Acad. Sci. U.S.A. 91, 9926-9930. Venugopal, R., and Jaiswal, A. K. (1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expresion of NAD(P)H:quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. U.S.A. 93, 14960-14965. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through anitoxidant response elements. Biochem. Biophys. Res. Commun. 236, 313-322. Groopman, J. D., Donahue, P. R., Zhu, J., Chen, J.-S., and Wogan, G. N. (1985) Aflatoxin metabolism in humans: detection of metabolites and nucleaic acid adducts in urine by affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 82, 6492-6496. Gan, L.-S., Skipper, P. L., Peng, X., Groopman, J. D., Chen, J.S., Wogan, G. N., and Tannenbaum, S. R. (1988) Serum albumin adducts in the molecular epidemiology of aflatoxin carcinogenesis: correlation with aflatoxin B1 intake and urinary excretion of aflatoxin M1. Carcinogenesis 9, 1323-1325. Wild, C. P., Hudson, G. J., Sabbioni, G., Chapot, B., Hall, A. J., Wogan, G. N., Whittle, H., Montesano, R., and Groopman, J. D. (1992) Dietary intake of aflatoxins and the level of alfumin-bound aflatoxin in peripheral blood in The Gambia, West Africa. Cancer Epidemiol. Biomarkers Prev. 1, 229-234. Cheng, Z.-Q., Root, M., Pan, W., Chen, J.-S., and Campbell, T. C. (1997) Use of an improved method for analysis of urinary aflatoxin M1 in a survey of mainland China and Taiwan. Cancer Epidemiol. Biomarkers Prev. 6, 523-529. Scholl, P. F., Musser, S. M., and Groopman, J. D. (1997) Synthesis and characterization of aflatoxin B1 mercapturic acids and their identification in rat urine. Chem. Res. Toxicol. 10, 1144-1151. Skipper, P. L., and Tannenbaum, S. R. (1990) Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis 11, 507-518. Kensler, T. W., Groopman, J. D., and Wogan, G. N. (1996): Carcinogen-DNA and protein adduct biomarkers for cohort selection and as modifiable endpoints in chemoprevention trials. In IARC Scientific Publication No. 139: Principles of Chemoprevention (Stewart, B. W., McGregor, D., and Kleihues, P., Eds.) pp 237-248, International Agency for Research on Cancer, Lyon, France. Groopman, J. D., and Kensler, T. W. (1999) The light at the end of the tunnel for chemical-specific biomarkers: daylight or headlight? Carcinogenesis 20, 1-11. Egner, P. A., Gange, S. J., Dolan, P. M., Groopman, J. D., Munõz, A., and Kensler, T. W. (1995) Levels of aflatoxin-albumin biomarkers in rat plasma are modulated by both long-term and transient interventions with oltipraz. Carcinogenesis 16, 17691773. Zhu, J.-Q., Zhang, L.-S., Hu, X., Xiao, Y., Chen, J.-S., Xu, Y.-C., Fremy, J., and Chu, F.-S. (1987) Correlation of dietary aflatoxin B1 levels with excretion of aflatoxin M1 in human urine. Cancer Res. 47, 1848-1852. Groopman, J. D., Zhu, J.-Q., Donahue, P. R., Pikul, A., Zhang, L.-S., Chen, J.-S., and Wogan, G. N. (1992) Molecular dosimetry of urinary aflatoxin-DNA adducts in people living in Guangxi Autonomous Region, People’s Republic of China. Cancer Res. 52, 45-52. Qian, G.-S., Ross, R. K., Yu, M. C., Yuan, J.-M., Gao, Y.-T., Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994) A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People’s Republic of China. Cancer Epidemiol. Biomarkers Prev. 3, 3-10.

Kensler et al. (97) Yu, M.-W., Lien, J.-P., Chiu, Y.-H., Santella, R. M., Liaw, Y.-F., and Chen, C.-J. (1997) Effect of aflatoxin metabolism and DNA adduct formation on hepatocellular carcinoma among chronic hepatitis B carriers in Taiwan. Hepatology 27, 320-330. (98) Zhu, Y.-R., Chen, J.-G., and Huang, X. Y. (1989) Hepatocellular carcinoma in Qidong County. In Primary Liver Cancer (Tang, Z. Y., Ed.) pp 204-222, China Academic Publisher and SpringerVerlag, Beijing, PRC. (99) Yu, S.-Z. (1995) Primary prevention of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 10, 674-682. (100) Zarba, A., Qian, G.-S., Zhu, Y.-R., Munõz, A., Kensler, T. W., and Groopman, J. D. (1995) Assessment of nutrient intake among carriers and non-carriers of hepatitis B surface antigen in Daxin Township, Qidong County, PRC. Proc. Am. Assoc. Cancer Res. 36, 277. (101) Ross, R. K., Yuan, J.-M., Yu, M. C., Wogan, G. N., Qian, G.-S., Tu, J.-T., Groopman, J. D., Gao, Y.-T., and Henderson, B. E. (1992) Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 339, 943-946. (102) Wang, J.-S., Qian, G.-S., Zarba, A., He, X., Zhu, Y.-R., Zhang, B.-C., Jacobson, L., Gange, S. J., Munõz, A., Kensler, T. W., and Groopman, J. D. (1996) Temporal patterns of aflatoxin-albumin adducts in hepatitis B surface antigen-positive and antigennegative residents of Daxin, Qidong County, People’s Republic of China. Cancer Epidemiol. Biomarkers Prev. 5, 253-261. (103) Kuang, S.-Y., Fang, X., Lu, P.-X., Zhang, Q.-N., Wu, Y., Wang, J.-B., Zhu, Y.-R., Groopman, J. D., Kensler, T. W., and Qian, G.-S. (1996) Aflatoxin-albumin adducts and risk for hepatocellular carcinoma in residents of Qidong, People’s Republic of China. Proc. Am. Assoc. Cancer Res. 37, 252. (104) Wang, L.-Y., Hatch, M., Chen, C.-J., Levin, B., You, S.-L., Lu, S.-N., Wu, M.-H., Wu, W.-P., Wang, L.-W., Wang, Q., Huang, G.-T., Yang, P.-M., Lee, H.-S., and Santella, R. M. (1996) Aflatoxin exposure and the risk of hepatocellular carcinoma in Taiwan. Int. J. Cancer 67, 620-625. (105) Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855-4878. (106) Aguilar, F., Hussain, S. P., and Cerutti, P. (1993) Aflatoxin B1 induces the transversion of G to T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 8586-8590. (107) Chang, M. H., Chen, C. J., Lai, M. S., Hsu, H. M., Wu, T. C., Kong, M. S., Liang, D. C., Shau, W. Y., and Chen, D. S. (1997) Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group. N. Engl. J. Med. 336, 1855-1859. (108) Benson, A. B., III (1993) Oltipraz: a laboratory and clinical review. J. Cell. Biochem. 17F, 278-291. (109) Jacobson, L. P., Zhang, B.-C., Zhu, Y.-R., Wang, J.-B., Wu, Y., Zhang, Q.-N., Yu, L.-Y., Qian, G.-S., Kuang, S.-Y., Li, Y.-F., Fang, X., Zarba, A., Chen, B., Enger, C., Davidson, N. E., Gorman, M. B., Gordon, G. B., Prochaska, H. J., Egner, P. A., Groopman, J. D., Munõz, A., Helzlsouer, K. J., and Kensler, T. W. (1997) Oltipraz chemoprevention trial in Qidong, People’s Republic of China: study design and clinical outcomes. Cancer Epidemiol. Biomarkers Prev. 6, 257-265. (110) Kensler, T. W., He, X., Otieno, M., Egner, P. A., Jacobson, L. P., Chen, B., Wang, J.-S., Zhu, Y.-R., Zhang, B.-C., Wang, J.-B., Wu, Y., Zhang, Q.-N., Qian, G.-S., Kuang, S.-Y., Fang, X., Li, Y.-F., Yu, L.-Y., Prochaska, H. J., Davidson, N. E., Gordon, G. B., Gorman, M. B., Zarba, A., Enger, C., Munõz, A., Helzlsover, K. J., and Groopman, J. D. (1998) Oltipraz chemoprevention trial in Qidong, People’s Republic of China: modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol. Biomarkers Prev. 7, 127-134. (111) Wang, J.-S., Shen, X., He, X., Zhu, Y.-R., Zhang, B.-C., Wang, J.-B., Qian, G.-S., Kuang, S.-Y., Zarba, A., Egner, P. A., Jacobson, L. J., Munõz, A., Helzlsouer, K. J., Groopman, J. D., and Kensler, T. W. (1999) Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J. Natl. Cancer Inst. (in press). (112) Maxiutenko, Y. Y., Curphey, T. J., Libby, A. H., Joyner, H. H., MacMillan, D. L., Kensler, T. W., and Roebuck, B. D. (1998) Identification of dithiolethiones with better chemopreventive properties than oltipraz. Carcinogenesis 19, 1609-1615. (113) Prochaska, H. J., Santamaria, A. B., and Talalay, P. (1992) Rapid detection of inducers of enzymes that protect against carcinogens. Proc. Natl. Acad. Sci. U.S.A. 89, 2394-2398.

TX980185B

Development of Cancer Chemopreventive Agents: Oltipraz as a

Recommend Documents