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Enzyme Induction and Dietary Chemicals as Approaches to Cancer Chemoprevention: The Seventh DeWitt S. Goodman Lecture¹

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020

	ABSTRACT

Top ABSTRACT INDUCTION OF CARCINOGEN... INHIBITORY EFFECTS OF DIETARY... INHIBITORS OF CARCINOGENESIS IN... TAILORING CANCER CHEMOPREVENTIVE... REFERENCES

 
Research on cancer chemoprevention is an important approach for decreasing both the incidence and number of deaths from cancer. The use of tamoxifen to prevent breast cancer, finasteride to prevent prostate cancer, and aspirin to prevent colon cancer are recent examples of cancer chemoprevention. This article describes research from my laboratory and related research from other laboratories on the effects of enzyme induction on chemical carcinogenesis as an approach to cancer chemoprevention, as well as studies on the inhibitory effects of curcumin, caffeine, (-)-epigallocatechin gallate (EGCG), and tea in animal models of carcinogenesis. The later substances appear to work, at least in part, by enhancing apoptosis in DNA-damaged cells or in tumors. The results of our studies and those of others provide a rationale for clinical trials on the potential chemopreventive effects of curcumin, caffeine, EGCG, and tea on the formation of cancer of the skin, mouth, esophagus, stomach, and colon in people with precancerous lesions and a high risk of developing these cancers.

It was pointed out that several compounds that are effective cancer chemopreventive agents in one experimental setting can enhance carcinogenesis in another experimental setting. These results suggest that it may be necessary to tailor the cancer chemopreventive regimen to individual subjects with known carcinogen exposures or to high cancer risk individuals with mechanistically understood pathways of carcinogenesis so that chemopreventive agents with known mechanisms of action can be better customized to the individual and selected on a more rational basis.

	INDUCTION OF CARCINOGEN-METABOLIZING ENZYMES AS AN APPROACH TO CANCER CHEMOPREVENTION

Top ABSTRACT INDUCTION OF CARCINOGEN... INHIBITORY EFFECTS OF DIETARY... INHIBITORS OF CARCINOGENESIS IN... TAILORING CANCER CHEMOPREVENTIVE... REFERENCES

 
Discovery of the Induction of Carcinogen-Metabolizing Enzymes
I joined the laboratory of James and Elizabeth Miller at the University of Wisconsin as a graduate student in September, 1952 and started work on the synthesis of a potential metabolite of ß-naphthylamine. My efforts to become a synthetic chemist resulted in two explosions and were a dismal failure. Although I didn’t know it at the time, this discouraging experience was extremely fortunate, because it led me to an exciting research program on the induction of liver microsomal enzymes as an approach to cancer chemoprevention, and to a research program on the pharmacological and toxicological significance of microsomal enzyme induction.

After failing as a synthetic chemist, the Millers and I discussed research by H.L. Richardson et al. (1) who had reported that administration of 3-methylcholanthrene inhibited the hepatocarcinogenic activity of the aminoazo dye 3'-methyl-4-dimethylaminoazobenzene in rats. We discussed the possibility that treatment of rats with 3-methylcholanthrene might protect them by altering the metabolism of the dye. In late 1952 or early 1953, I started studies on the effects of treating rats with 3-methylcholanthrene and other polycyclic aromatic hydrocarbons on the hepatic N-demethylation and azo-link reduction of aminoazo dyes, metabolic pathways that resulted in noncarcinogenic products. I was delighted to find that a single i.p. injection of 3-methylcholanthrene caused a rapid and many-fold increase in hepatic azo dye N-demethylase and azo-link reductase activities (2 , 3) , and we provided strong evidence that treatment of rats with 3-methylcholanthrene had induced the synthesis of hepatic aminoazo dye N-demethylase and azo-link reductase (3) . The induction of these enzyme systems by 3-methylcholanthrene administration, an early example of enzyme induction in mammals, is shown in Fig. 1 . Structure-activity relationship studies showed that 3-methylcholanthrene, BP,³ dibenz(a,h)anthracene, and benz(a)anthracene were potent inducers of aminoazo dye metabolism, whereas pyrene had little or no activity (2 , 3) . The effects of the different polycyclic aromatic hydrocarbons to stimulate azo dye metabolism paralleled the effects of these compounds to inhibit azo dye carcinogenesis (2, 3, 4) . Feeding 3-methylcholanthrene, BP, dibenz(a,h)anthracene, or benz(a)anthracene strongly inhibited the hepatocarcinogenicity of 3'-methyl-4-dimethylaminoazobenzene, whereas pyrene, which was inactive as an inducer of azo dye metabolism, did not inhibit azo dye carcinogenesis (Table 1 ; Ref. 4 ). Our research on the induction of azo dye-metabolizing enzymes provided a mechanistic explanation for the inhibitory effects of polycyclic hydrocarbons on azo dye carcinogenesis and provided an early example of mechanisms of cancer chemoprevention. These studies also placed into perspective the meaning of safety and benefit/risk ratio for the field of cancer chemoprevention. If I were a rat in an environment of carcinogenic aminoazo dyes that would with great certainty cause liver cancer and all that was available for protection was 3-methylcholanthrene, I would ingest the hydrocarbon, because it would save my life. Of course safer chemopreventive agents would be more desirable, and having a mechanistic understanding of cancer chemoprevention led to a search for safer and more effective chemopreventive agents.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Induction of hepatic aminoazo dye N-demethylase and reductase activities. Rats (50 g) were injected once i.p. with 1 mg of 3-methylcholanthrene (3-MC). N-Demethylase activity was determined in fortified liver homogenates by measuring the metabolism of 3-methyl-4-monomethylaminoazobenzene to 3-methyl-4-aminoazobenzene (3-methyl-AB). Reductase activity was determined by measuring the reduction of the azo linkage of 4-dimethylaminoazobenzene (DAB). Demethylase activity is expressed as µg of 3-methyl-AB formed/50 mg of liver/30 min. Reductase activity is expressed as µg of DAB reduced/30 mg of liver/30 min. Each point is the average of the activities for two or three rats. (Taken from Ref. 3 .)

View larger version (22K): [in this window] [in a new window]   Fig. 2. Induction of hepatic BP hydroxylase activity. Rats (50 g) were injected once i.p. with 0.1 or 1 mg of BP. Homogenate from 50 mg of liver was incubated with 50 µg of BP for 12 min. (Taken from Ref. 5 .)

Stimulatory Effects of Enzyme Inducers on the in Vivo Metabolism of Polycyclic Aromatic Hydrocarbons
Studies with BP.
An early and important question during the course of our studies was whether the induction of carcinogen-metabolizing enzymes was paralleled by enhanced carcinogen metabolism in vivo. The induction of BP hydroxylase activity in rats was reflected in vivo by enhanced metabolism of BP, decreased blood and tissue concentrations of this carcinogen, and enhanced biliary excretion of its metabolites (22, 23, 24) . 3-Methylcholanthrene, DMBA, and BP are potent inducers of BP hydroxylase activity, and pretreatment of rats with these compounds p.o. once daily for 2 days before an i.v. injection of tritiated BP on day 3 stimulated the disappearance of [³ H]BP from blood and decreased the concentration of [³ H]BP in various tissues (22) . The effect of pretreatment of rats with 3-methylcholanthrene, DMBA, or BP to lower the concentration of BP in blood and fat at 45 min after an i.v. injection of [³ H]BP is shown in Table 2 . Treatment of rats with pyrene or anthracene had little or no stimulatory effect on monooxygenase activity (3 , 25) or on the in vivo metabolism of [³ H]BP (Table 2) . Because the primary route of elimination of BP metabolites in rats is excretion into the bile, we investigated the effects of enzyme inducers on the metabolism of BP to biliary excretion products. Pretreatment of rats with nonradioactive BP for 2 days before an i.v. injection of [¹⁴C]BP stimulated the excretion of metabolites of [¹⁴C]BP into the bile (23) . Seven min after the i.v. injection of 300 µg of [¹⁴C]BP, the concentration of radioactive BP metabolites in the bile of pretreated rats was 20-fold higher than the concentration of [¹⁴C]BP metabolites in the bile of control rats (23) . The stimulatory effect of BP on its own metabolism is also illustrated by the decrease in tissue concentration of this compound that occurs when it is given chronically. At 24 h after a single oral dose of 1 mg of [³ H]BP to adult rats, the concentration of this hydrocarbon in fat was 249 ng/g, whereas 24 h after seven daily doses of 1 mg of [³ H]BP its concentration in fat was only 24 ng/g (22) .

View larger version (13K): [in this window] [in a new window]   Fig. 3. Metabolism of procarcinogens to inactive and active metabolites by Phase I and Phase II enzymes. Phase I enzymes (predominantly the CYP family of enzymes) metabolize procarcinogens to inactive and active metabolites. Phase II enzymes inactivate electrophilic active metabolites formed by Phase I enzymes, and they also conjugate inactive metabolites (e.g., by glucuronidation, sulfonation, etc.) thereby enhancing their excretion in urine and bile.

Treatment of rodents with inducers of microsomal monooxygenases provided protection from the carcinogenic effects of BP (39, 40, 41, 42) , DMBA (31 , 32 , 42, 43, 44, 45, 46) , 2-acetylaminofluorene (4 , 47 , 48) , 4-dimethylaminostilbene (49) , urethane (50) , aflatoxin B₁ (51 , 52) , diethylnitrosamine (48 , 53) , and aminoazo dyes (1 , 2 , 4 , 48 , 54 , 55) . Examples of inducers of liver microsomal monooxygenases that inhibit chemical carcinogenesis include 3-methylcholanthrene and other polycyclic aromatic hydrocarbons (1 , 2 , 4 , 49) , ß-naphthoflavone (41 , 44 , 52) , indole-3-carbinol (42 , 56 , 57) , TCDD (39 , 40) , polychlorinated biphenyls (48) , 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT; Ref. 46 ), phenobarbital (47 , 51) , and spironolactone (45) . Phenothiazine and some closely related derivatives are potent inducers of hepatic and extrahepatic BP hydroxylase activity, and they inhibit DMBA-induced adrenal necrosis (58) . The inhibitory effect of 3-methylcholanthrene administration on the carcinogenicity of 2-acetylaminofluorene was associated with an increased ratio of the metabolism of 2-acetylaminofluorene to inactive ring hydroxylated products relative to the formation of the carcinogenic N-hydroxylated metabolite in vivo as measured by the urinary excretion of these metabolites (59) . In another study, rats were injected with 3-methylcholanthrene once daily for 2 days before the oral administration of carbon 14-labeled IQ. Pretreatment with 3-methylcholanthrene before administration of IQ markedly decreased the formation of IQ-DNA adducts in the liver, colon, small intestine, kidneys, bladder, heart, and lung (60) . The results of this study suggested that 3-methylcholanthrene had induced CYP1A1/1A2 and had enhanced the ring hydroxylation of IQ (inactivation pathway) to a greater extent than the N-hydroxylation activation pathway.

Because many chemical carcinogens are metabolized by CYP enzymes to noncarcinogenic as well as to proximate and ultimate carcinogenic metabolites, the effects of inducers of these enzymes on the carcinogenicity of a chemical will depend on the effects of the inducer on the ratio of metabolism of the carcinogen to inactive and active metabolites by Phase I enzymes as well as on the levels of Phase II enzymes (e.g., GST, epoxide hydrolase, NAD(P)H:quinone reductase, and UDP-glucuronosyl transferase) that inactivate chemically reactive metabolites. Because treatment of rodents with appropriate inducers of monooxygenases usually inhibits the carcinogenicity of chemical carcinogens in vivo, it is likely that these inducers enhance the in vivo detoxification of carcinogens to a greater extent than their activation.

Sometimes inducers of detoxifying enzymes also inhibit metabolic activation pathways. Treatment of rainbow trout with high doses of ß-naphthoflavone, indole-3-carbinol, or with the condensation products of indole-3-carbinol induces the CYP1A family thereby enhancing the hydroxylation of aflatoxin B₁ to aflatoxin M₁ (inactivation pathway), but these inducers also inhibit the 8,9-epoxidation of aflatoxin B₁ (activation pathway; Refs. 61 , 62 ). The inhibitory effect of low dose levels of the inducers on the action of aflatoxin B₁ in rainbow trout is predominantly by inhibition of the epoxidation pathway rather than by induction of enhanced detoxification (61 , 62) . Although inducers of Phase I enzymes usually inhibit chemical carcinogenesis, the stimulatory effect of phenobarbital administration on the hepatocarcinogenicity of safrole (63) is an example of an inducer of Phase I enzymes that enhances carcinogenesis.

The complexities of the effects of microsomal enzyme inducers on the carcinogenicity of chemicals was pointed out in studies with a pharmacogenetic model for transplacental carcinogenesis in noninducible or inducible pregnant mice where half of the fetuses were susceptible to induction of polycyclic hydrocarbon metabolism by CYP1A1 inducers and the other half were refractory to induction (64 , 65) . Although pretreatment of inducible pregnant mice with ß-naphthoflavone (a noncarcinogenic inducer of CYP1A1) protected the fetuses from 3-methylcholanthrene-induced lung tumors observed later in the offspring, pretreatment of noninducible pregnant mice with ß-naphthoflavone increased the carcinogenicity of 3-methylcholanthrene but only in the inducible fetuses (65) . The results indicate that inducibility in both the mother and the fetus are important factors for determining whether an inducer of polycyclic hydrocarbon metabolism inhibits or enhances transplacental carcinogenesis by 3-methylcholanthrene (reviewed in Ref. 66 ).

Inhibition of Phase I monooxygenases can also inhibit chemical carcinogenesis apparently by inhibiting the metabolic activation of certain procarcinogens to a greater extent than their metabolic detoxification (67, 68, 69, 70) . The importance of Phase I enzymes for the metabolic activation and carcinogenicity of BP was emphasized recently by the studies of Shimizu et al. (71) . These investigators demonstrated that mice lacking the aryl hydrocarbon receptor did not have detectable basal or BP-inducible levels of CYP1A1 gene expression, and these mice were also refractory to BP-induced s.c. or epidermal carcinogenesis presumably because they were unable to metabolically activate BP (71) .

In an additional study, Uno et al. (72) found that CYP1A1 homozygous knockout mice treated with BP had impaired elimination of BP from the serum and higher levels of BP-DNA adducts in the liver than CYP1A1 heterozygous knockout mice treated with BP. These investigators also showed that TCDD pretreatment of mice with either genotype enhanced the elimination of BP from the serum and lowered the levels of hepatic BP-DNA adducts. These results indicate that enzymes other than CYP1A1 are induced by TCDD and can play a role in the detoxification and elimination of BP.

Polycyclic aromatic hydrocarbons in cigarette smoke and ß-napthoflavone are potent inducers of cytochromes P450 (CYP1A1/1A2) that hydroxylate aflatoxin B₁ to aflatoxin M₁, (an inactivation pathway; Refs. 73, 74, 75 ). Feeding rats ß-naphthoflavone, which stimulates the hepatic metabolism of aflatoxin B₁ to aflatoxin M₁, inhibits the hepatocarcinogenic activity of aflatoxin B₁ (52) . In accord with these observations, epidemiology studies suggest that cigarette smokers who are heavily exposed to aflatoxin B₁ in their daily diet have a decreased risk of liver cancer when compared with nonsmokers who are also exposed to aflatoxin B₁ (76) . If these studies are confirmed, it may be possible to isolate from tobacco smoke potent nontoxic inducers of aflatoxin B₁ detoxification that would be useful chemopreventive agents in people exposed to high levels of aflatoxin B_1. Alternatively, other inducers of the CYP1A family such as indole-3-carbinol, ß-naphthoflavone, ß-apo-8'-carotenal, astaxanthin, or canthaxanthin could be considered for additional studies. The later three compounds are of interest because of a recent report that indicates that treatment of rats with these carotenoids induced increased levels of hepatic CYP1A1/1A2, increased the hepatic hydroxylation of aflatoxin B₁ to aflatoxin M₁, and inhibited aflatoxin B₁-induced DNA damage and the formation of preneoplastic foci (77) . Although the above studies suggest that an elevated level of CYP1A1/1A2 protects animals and possibly humans from the hepatocarcinogenic effects of aflatoxin B₁, additional studies are needed to determine whether or not an increased level of CYP1A1/1A2 is associated with decreased carcinogenicity of aflatoxin B₁ in humans.

Many clinically useful drugs are inducers of CYP monooxygenases in humans, and it is likely that these drugs will also influence the metabolism and carcinogenicity of environmental carcinogens. Examples of inducers of oxidative drug metabolism in humans include phenobarbital (inducer of the CYP2B and 3A families), rifampicin (inducer of the CYP3A and 2C families), clotrimazole (inducer of CYP3A4), omeprazole (inducer of the CYP1A family), phenytoin (inducer of the CYP2C family), ethanol (inducer of CYP2E1), and the herbal antidepressant St. Johns wort (inducer of CYP3A4). Epidemiology studies are needed to determine whether individuals who are heavily exposed to environmental carcinogens have an altered risk when they are also taking enzyme-inducing drugs.

Although the CYP monooxygenases metabolize chemical carcinogens to biologically inactive metabolites and to chemically reactive ultimate carcinogens, both kinds of metabolites can undergo additional metabolism by Phase II enzymes such as GST, NAD(P)H: quinone reductase (DT-diaphorase), epoxide hydrolase, and UDP-glucuronosyltransferase resulting in the inactivation and elimination of chemically reactive ultimate carcinogens so that elevated levels of Phase II enzymes help protect organisms from chemical carcinogens and from other toxic effects of electrophiles. The importance of GST for the detoxification of polycyclic aromatic hydrocarbons was emphasized by a study indicating enhanced DMBA-induced skin carcinogenesis in mice lacking the class of GST when compared with DMBA-induced skin carcinogenesis in wild-type mice (78) . The importance of GST was also pointed out by a study indicating that the (+)-7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-DNA adduct level in the lungs of smokers is correlated with the GSTM1*O genotype, and this genotype appears to be a risk factor (79) . Although many individuals may have sufficient GST and other Phase II enzymes to inactivate ultimate carcinogens that are formed during the metabolism of procarcinogens by Phase I enzymes, some individuals may benefit from increased levels of the Phase II enzymes.

Effects of Inducers of Phase II Enzymes on Chemical Carcinogenesis
Shortly after the discovery of the induction of hepatic monooxygenases and azo dye reductase, it was found that some inducers of Phase I enzymes such as polycyclic aromatic hydrocarbons and phenobarbital also induced increased levels of a Phase II enzyme (UDP-glucuronosyltransferase; Refs. 80, 81, 82 ). In accord with these observations, treatment of humans with phenobarbital decreased the serum level of unconjugated bilirubin (a substance eliminated from the body by glucuronidation), and jaundice was ameliorated in children with hyperbilirubinemia who were treated with phenobarbital (83, 84, 85) . Another early example of the induction of a Phase II enzyme was provided by Huggins and his colleagues who demonstrated the induction of hepatic menadione reductase [NAD(P)H: quinone reductase] in rats by a variety of polycyclic aromatic hydrocarbons and aromatic azo compounds (30 , 33 , 34) . The induction of hepatic menadione reductase in rats by the various compounds was associated with inhibitory effects of these compounds on DMBA-induced adrenal necrosis and mammary cancer (30 , 33 , 34) . Subsequent studies have shown that many inducers of Phase I enzymes also induce Phase II enzymes.

During the 1960s and 1970s, Lee Wattenberg (86 , 87) pioneered in research that identified several classes of inhibitors of chemical carcinogenesis, and some of the inhibitors enhanced the oxidative metabolism of carcinogens. It was shown by Ulland et al. (88) and Wattenberg and his colleagues (89, 90, 91, 92, 93) that treatment of rodents with several polyphenolic antioxidants present in our diet inhibited chemical carcinogenesis.

During 1978–1980, the laboratories of Paul Talalay and Ernst Bueding reported on an important and novel type of induction of xenobiotic metabolism by certain antioxidants (94, 95, 96, 97) . They found that treatment of mice with butylated hydroxyanisole, butylated hydroxytoluene, and/or ethoxyquin resulted in increased levels of hepatic GST, epoxide hydrolase, and NAD(P)H: quinone reductase (94, 95, 96, 97) . These were the first reports on the induction of Phase II enzymes by antioxidants. Subsequent research by Talalay and his colleagues identified a large number of synthetic and dietary chemicals that are strong inducers of Phase II enzymes (98 , 99) , and many of these substances are electrophilic Michael reaction acceptors (99 , 100) . Many inducers of Phase II enzymes that inactivate ultimate carcinogens also induce enzymes that result in increased cellular levels of antioxidants (e.g., glutathione and other substances) that protect cells from oxidative stress, and this response may be an important component of the effect of Phase II enzyme inducers to protect animals from chemical carcinogens (98 , 101) . Additional evidence for the protective effects of increased levels of Phase II enzymes against oxidants and electrophiles is discussed in a recent review by Talalay et al. (102) .

The importance of Phase II enzymes for inactivating chemical carcinogens was highlighted in a study with nrf2 transcription factor-deficient mice (103) . Nrf2 binds to the antioxidant response element and is necessary for the induction of Phase II enzymes. Constitutive hepatic and gastric activities of GST and NAD(P)H:quinone reductase, and the induction of these enzymes by oltipraz were markedly reduced in nrf2-deficient mice when compared with wild-type mice (103) . These results are in agreement with in vivo studies indicating that nrf2-deficient mice are more sensitive to BP-induced gastric tumors than wild-type mice, and they are refractory to oltipraz-induction of Phase II enzymes and to oltipraz-induced inhibition of BP-induced gastric neoplasms (103) . It would be of interest to determine whether induction of cytochromes P450 by TCDD, or a polycyclic hydrocarbon in nrf2-deficient mice or in GST-deficient mice would inhibit BP and DMBA-induced carcinogenesis as was observed in wild-type mice. Recent studies indicate that sulfhydryl groups of Keap1 are sensors regulating the induction of Phase II enzymes and that inducers of Phase II enzymes disrupt the cytoplasmic complex between actin-bound protein Keap1 and nrf2, thereby releasing nrf2 to migrate to the nucleus and stimulate the transcription of genes for Phase II enzymes (104) .

Two of the most widely studied inducers of Phase II enzymes are oltipraz (an antischistosomal drug) and sulforaphane (an isothiocyanate in broccoli; Refs. 105, 106, 107, 108 ). Pretreatment of rats with sulforaphane inhibited DMBA-induced mammary cancer (108) , and treatment of rats with oltipraz inhibited aflatoxin B₁-induced liver cancer (109) . Oltipraz has undergone extensive evaluation as a cancer chemopreventive agent, and it was shown to be an effective anticarcinogen in >12 animal models (110) . Oltipraz-induced decreases in aflatoxin B₁-induced liver cancer in rats was associated with decreased levels of aflatoxin-N-guanine adducts in the liver and urine, and this provided a useful biomarker (110) .

Although sulforaphane and oltipraz are inducers of Phase II enzymes, both of these compounds may modulate carcinogen metabolism by other mechanisms. The administration to rats of certain isothiocyanates related to sulforaphane inhibits some CYP enzymes, whereas others are induced (111 , 112) . Both oltipraz and sulforaphane are strong in vitro inhibitors of human cytochromes P450 1A2 and 3A4 (113 , 114) , and oltipraz has also been reported to induce increased levels of CYP1A2-associated dealkylation of methoxyresorufin in rat liver (115) . In normal volunteers, administration of a single 125 mg dose of oltipraz inhibited CYP1A2 activity as measured by studies on the in vivo metabolism of caffeine (116) . Although many literature reports speak of monofunctional Phase II enzyme inducers, most inducers of Phase II enzymes also inhibit or induce Phase I CYP enzymes.

A recent intervention study in people heavily exposed to aflatoxin B₁ in China indicated that treatment of these individuals with 125 mg of oltipraz daily for 4 weeks increased the urinary excretion of aflatoxin B₁-mercapturic acid (glutathione conjugate of aflatoxin B₁), which is an inactivation product of aflatoxin B₁-8,9-oxide (117 , 118) . Formation of aflatoxin M₁ (a hydroxylated CYP-dependent inactivation product of aflatoxin B₁) was not affected (118) . In contrast to these observations, administration of 500 mg of oltipraz once a week for 4 weeks decreased the urinary excretion of aflatoxin M₁, presumably by inhibiting CYP-dependent hydroxylation (or by enhancing the further metabolism of aflatoxin M₁), and the urinary excretion of aflatoxin B₁-mercapturic acid was not affected (118) . The results indicated that the effects of oltipraz administration on aflatoxin B₁ metabolism in humans depended on the dosing regimen.

Potential Problems Associated with the Use of Enzyme Induction as an Approach to Cancer Chemoprevention
Although the induction of CYP enzymes that metabolize carcinogens usually inhibits carcinogenesis in experimental animals (presumably because detoxification pathways are enhanced to a greater extent than activation pathways), sometimes carcinogenesis is enhanced as occurs for the effect of phenobarbital administration on safrole carcinogenesis (63) . Some enzyme inducers that inhibit carcinogenesis when given together with a carcinogen are tumor promoters when given after the carcinogen (e.g., phenobarbital, indole 3-carbinol, and DDT; Refs. 47 , 119, 120, 121, 122, 123, 124, 125, 126 ).

Another potential problem associated with the use of enzyme induction as an approach to cancer chemoprevention is that enzyme inducers can stimulate the metabolism and alter the action of therapeutic drugs thereby leading to drug interactions in patients, and the metabolism and action of important normal body constituents (such as vitamin D, arachidonic acid, thyroid hormone, and steroid hormones) may also be modified. Although the scientific community is searching for monospecific inducers of Phase II enzymes, several inducers of Phase II enzymes (e.g., butylated hydroxyanisole, sulforaphane, oltipraz, and some related compounds) can also induce or inhibit Phase I enzymes (111, 112, 113, 114 , 127) .

The current state of our knowledge indicates that the selective induction of carcinogen-detoxifying enzymes (Phase I and/or Phase II enzymes) may be a useful approach for inhibiting carcinogenesis in individuals or populations at high unavoidable risk because of exposure to high levels of specific carcinogens, but this approach may not be useful in the general population. The possibility that enzyme inducers that increase cellular antioxidant defense systems may have a broad protective effect in the general population is a promising approach that requires additional research. Careful benefit/risk assessments need to be made before and during clinical trials with chemopreventive inducers of Phase I and/or Phase II enzymes.

Effects of Dietary Changes on the Metabolism of Foreign Compounds in Humans
Because chemical carcinogens are metabolized by the same enzyme systems that metabolize drugs, studies on the effects of environmental factors that influence drug metabolism in humans may lead to new approaches for stimulating the detoxification or inhibiting the metabolic activation of environmental carcinogens. Increasing the ratio of protein to carbohydrate in the diet or ingestion of charcoal broiled beef or cabbage and brussels sprouts increased the oxidative metabolism of several drugs (128, 129, 130, 131, 132) , and feeding cabbage and brussels sprouts also enhanced the glucuronidation of acetaminophen (a Phase II biotransformation; Ref. 133 ).

Other studies have shown that switching people from their home diet to a semisynthetic diet caused a 57% decrease in the CYP-dependent oxidative dealkylation of 7-ethoxycoumarin in the intestinal mucosa, but NADPH-cytochrome c reductase and 1-naphthol glucuronyltransferase activities were not affected (134) . These results indicate that certain CYP-dependent oxidations in the intestinal mucosa are very sensitive to dietary changes.

In an additional study, feeding a brussels sprouts and broccoli-containing diet to human subjects for 12 days enhanced the metabolism of caffeine (CYP1A2 substrate) and decreased the urinary excretion of unchanged 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) and PhIP ingested in a cooked meat meal. The results suggested that the vegetable diet may have enhanced the metabolism of these heterocyclic amines (135) .

Ingestion of watercress, which contains a high level of phenethyl isothiocyanate, increased the urinary levels of 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide [metabolically inactivated metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] in smokers (136) . This treatment also increased the glucuronidation of cotinine and trans-3'-hydroxycotinine in smokers, and the glucuronidation of trans-3'-hydroxycotinine correlated with the glucuronidation of 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (137) . In other studies, ingestion of watercress inhibited the oxidative metabolism of acetaminophen and chlorzoxazone suggesting an inhibitory effect on the activity of CYP2E1 (138 , 139) . Watercress administration, however, had no effect on acetaminophen glucuronidation (138) . These results suggest that ingestion of watercress has a selective stimulatory effect on some but not all of the UDP-glucuronosyltransferase enzymes and that ingestion of watercress also inhibits CYP2E1. It is apparent from these studies that the effects of watercress ingestion on xenobiotic metabolism are complex.

A single glass of grapefruit juice increased the oral bioavailability of felodipine, nifedipine, and several other drugs that are metabolized by CYP3A4 presumably by inhibiting the first pass metabolism of these drugs in the gastrointestinal tract and/or liver (140 , 141) . Certain furanocoumarins in grapefruit juice have been suggested as possible contributors to the inhibitory effect of grapefruit juice on drug metabolism. It was shown that administration of grapefruit juice three times a day for 6 days increased the area under the plasma concentration-time curve for felodipine and caused a 62% decrease in the concentration of CYP3A4 in the small bowel epithelium without influencing the concentration of CYP1A1 or CYP2D6 in the small bowel or the hepatic CYP3A4 activity as measured by the [¹⁴C-N-methyl] erythromycin breath test (141) .

The studies described above indicate that dietary changes can influence the metabolism of foreign compounds including environmental carcinogens in humans. It is likely that these environmental modulators of foreign compound metabolism can influence the action of environmental carcinogens, and additional research may provide more leads for dietary cancer chemopreventive regimens.

Effects of Liver Microsomal Enzyme Inducers on Steroid Metabolism and Hormone-Related Cancers
Effects of Phenobarbital and Other Drugs on Steroid Metabolism.
In studies that started 40 years ago (142) , we found that treatment of rats with phenobarbital or other drugs, or with certain halogenated hydrocarbon insecticides increased liver microsomal monooxygenase activity for the metabolism of estradiol, estrone, deoxycorticosterone, progesterone, ⁴ -androstene-3,17-dione, and testosterone (reviewed in Ref. 143 ). The stimulatory effect of treatment of rats with phenobarbital on the metabolism of these steroids to polar hydroxylated metabolites by liver microsomal enzymes is shown in Fig. 4 . The stimulatory effects of liver microsomal enzyme inducers on steroid metabolism were paralleled in vivo by a decreased action of the steroids. Pretreatment of rats with phenobarbital or several other inducers of liver microsomal enzymes decreased: (a) the uterotropic effects of estradiol, estrone, and certain oral contraceptive steroids (144, 145, 146, 147) ; (b) the anesthetic effects of progesterone and deoxycorticosterone (148 , 149) ; and (c) the growth promoting effects of testosterone on the seminal vesicles (150 , 151) .

View larger version (24K): [in this window] [in a new window]   Fig. 4. Stimulatory effect of phenobarbital treatment on steroid hydroxylation by rat liver microsomes. Immature rats were given i.p. injections of 37 mg of sodium phenobarbital per kg of body weight twice daily for 4 days. The animals were killed the following day, and liver microsomes were prepared and incubated with 14C-labeled steroid in the presence of NADPH. Formation of polar metabolites with the chromatographic mobility of hydroxylated substrate was measured. (Taken from Refs. 142 , 144 , 149 .)

Administration of phenobarbital or other inducers of liver microsomal monooxygenases stimulated the 6ß-hydroxylation of cortisol in guinea pig liver microsomes (153) , enhanced the urinary excretion of 6ß-hydroxycortisol in humans (154, 155, 156, 157, 158, 159) , increased the urinary excretion of polar metabolites of testosterone in humans (160) , decreased the effectiveness of a synthetic glucocorticoid in steroid-dependent asthmatics (161) , and stimulated the metabolism of synthetic estrogens and progestational steroids contained in oral contraceptives (162) . Anticonvulsants and rifampicin are examples of drugs that stimulate the metabolism of the oral contraceptive, ethinylestradiol, in humans (162, 163, 164) , and unwanted pregnancies have occurred in women taking oral contraceptives simultaneously with anticonvulsants or rifampicin (discussed in Ref. 147 ). It will be of considerable interest to determine whether people treated chronically with phenobarbital, anticonvulsant mixtures, or other inducers of the CYP enzymes have a decreased risk of hormone-dependent cancers such as cancers of the endometrium, breast, and prostate.

Effects of Indole-3-Carbinol and TCDD on Steroid Metabolism.
Chronic administration of indole-3-carbinol [a constituent of cruciferous vegetables that is converted to the Ah receptor agonist indolo(3,2-b)carbazole in the stomach] stimulates the 2-hydroxylation of estradiol and inhibits the formation of spontaneous mammary tumors in female C3H/OuJ mice (165, 166, 167, 168) . Another study indicated an inhibitory effect of administration of indole-3-carbinol on the formation of spontaneous uterine tumors in Donryu rats (169) . The development of DMBA- or N-nitrosomethylurea-induced mammary tumors (both of which are highly dependent on endogenous estrogens; Ref. 170 ) in female Sprague Dawley rats was also inhibited by chronic treatment with indole-3-carbinol (57) . Additional studies by Michnovicz, Bradlow, and their colleagues (171 , 172) showed a stimulatory effect of indole-3-carbinol administration on the 2-hydroxylation of estradiol in humans, and they suggested that indole-3-carbinol may be an effective chemopreventive agent for breast cancer in women (173 , 174) . However, the use of indole-3-carbinol as a chemopreventive agent against estrogen-dependent human cancers through induction of estrogen 2-hydroxylation should receive more careful evaluation because indole-3-carbinol is a promoter of liver tumors in initiated animals (120, 121, 122, 123, 124, 125, 126) . In addition, (a) some studies suggest that total catechol estrogen production is positively associated with an increased risk of breast cancer in women (175, 176, 177) ; (b) indole-3-carbinol [a prodrug for the formation of the potent Ah-receptor agonist, indolo(3,2-b)carbazole; Ref. 178 ], as well as other Ah-receptor agonists such as TCDD, have been reported to increase the rates of formation of both 2- and 4-hydroxyestradiol in liver and in estrogen target cells (166 , 179, 180, 181) ; and (c) 4-hydroxyestradiol has carcinogenic activity when administered to Syrian hamsters or newborn mice, whereas 2-hydroxyestradiol has very low or no carcinogenic activity (182, 183, 184, 185) . Although treatment of animals or humans with indole-3-carbinol to stimulate the 2-hydroxylation of estradiol may be an effective way of inhibiting estrogen-induced carcinogenesis, and studies in this area should be encouraged, the significance of the tumor promoting activity of indole-3-carbinol and its stimulatory effect on the formation of the potentially toxic 4-hydroxyestradiol should receive more attention before its widespread use in humans.

Previous studies showed that treatment of animals with TCDD (a potent environmental Ah receptor agonist with strong monooxygenase-inducing activity) resulted in a marked antiestrogenic effect (186, 187, 188) , but the blood level of estradiol was not altered (188) . Additional studies revealed a marked stimulatory effect of treating cultured MCF-7 human breast cancer cells with TCDD on the 2-, 4-, 6-, and 15-hydroxylation of estradiol by microsomes from these breast cancer cells, and the 2-, 6-, and 15-hydroxylation of estradiol (estrogen inactivation pathways) were quantitatively the most important pathways that were stimulated (>90% of the total metabolites formed; Ref. 179 ). The stimulatory effect of TCDD on oxidative estradiol metabolism in cultured MCF-7 cells was associated with an inhibitory effect of TCDD on estradiol-induced formation of transformed foci and plasminogen activator activity in these cells (189 , 190) . Other studies indicated that administration of TCDD to mice injected with MCF-7 breast cancer cells inhibited the growth of these explants (190) , presumably by enhancing the metabolic inactivation of estrogen in the breast cancer cell explants and/or in host tissues. These results indicate that Ah receptor agonists such as TCDD, indolo(3,2-b)carbazole (derived from indole-3-carbinol in cruciferous vegetables), and polycyclic aromatic hydrocarbons in tobacco smoke or charcoal broiled foods may decrease estrogen action in target cells because of markedly enhanced estrogen metabolism to inactive or poorly active estrogen metabolites. It should be noted that enhanced formation of 2-hydroxyestradiol may result in increased formation of 2-methoxyestradiol, a potential methylated metabolite of 2-hydroxyestradiol that has potent antiangiogenesis and anticarcinogenic activity (reviewed in Ref. 191 ). Selective modulation of estrogen metabolism in target tissues or cells may have profound biological effects without influencing the blood or urinary levels of estrogens and their metabolites (192) .

It is of interest that although prolonged administration of TCDD to female rats enhanced the incidence of liver tumors, this treatment inhibited the formation of spontaneous tumors in the uterus, mammary gland, and pituitary (193) , which are all target tissues for estrogen action. Whether the inhibitory effect of TCDD on the formation of spontaneous tumors in estrogen target tissues is caused by its strong stimulatory effect on the metabolic inactivation of estradiol in liver or in target tissues is worthy of additional investigation. Careful dose-response studies with TCDD are needed to determine whether the beneficial or toxic effects predominate at low dose levels of TCDD. Although TCDD has numerous adverse effects in humans, epidemiology studies in a population accidentally exposed to TCDD and other chemicals in Servaso, Italy suggested a decreased risk of breast cancer in these individuals in a 10-year follow-up study but not in a 20-year follow-up study (194, 195, 196) . Additional research on the effects of exposure to TCDD on endometrial and breast cancer is needed.

Effects of Cigarette Smoking on Steroid Metabolism.
Early studies from our laboratory showed that cigarette smoking (exposure to polycyclic aromatic hydrocarbons and other inducers) stimulated the oxidative metabolism of xenobiotics (10 , 197, 198, 199) . More recent studies showed that cigarette smoking increased the level of CYP1A1 in the placenta (200) and stimulated the placental 15-hydroxylation of estradiol (201) , which is an inactivation pathway of estradiol metabolism that is stimulated in liver microsomes from rats treated with 3-methylcholanthrene (152) . Cigarette smokers also have enhanced in vivo 2-hydroxylation of estradiol (202 , 203) , and some studies indicate that smokers have lower serum or urinary levels of estradiol and estrone (204, 205, 206, 207) , as well as lower urinary excretion of 16-hydroxyestradiol (an estrogenic metabolite of estradiol) than nonsmokers (208) . Enhanced metabolic inactivation of estradiol in cigarette smokers may explain why cigarette smokers have an increased risk of osteoporosis (209, 210, 211) and a decreased risk of endometrial cancer (209 , 211, 212, 213, 214) . Although the above studies suggest that cigarette smoking may inhibit endometrial cancer by enhancing the formation of 2- and 15-hydroxyestradiol (nonestrogenic metabolites), and by decreasing the formation of 16-hydroxyestradiol (an estrogenic metabolite), several investigators have not observed decreased serum levels of estradiol in cigarette smokers (208 , 215 , 216) . The possibility that cigarette smoking enhances the metabolism of estradiol to inactive or protective metabolites in the liver or in the endometrium and breast is an area that needs additional research. Enhanced metabolism of estradiol to 2-hydroxyestradiol followed by additional metabolism of 2-hydroxyestradiol to 2-methoxyestradiol, possibly in target tissues, would be expected to inhibit estradiol-induced carcinogenesis and could occur in the absence of a decrease in the serum level of estradiol (191 , 192) .

Although epidemiology studies consistently indicate that cigarette smokers have a decreased risk of endometrial cancer (209 , 211, 212, 213, 214) , studies to determine whether cigarette smoking alters the risk of breast cancer have been inconclusive (217) . A recent study suggests a dual/competing effect of cigarette smoking on breast cancer risk (218) . It was observed that the risk of breast cancer was increased in parous women who started smoking within 5 years of menarche or in nulliparous women who smoked 20 cigarettes daily for 20 pack-years (218) . In contrast with these observations, postmenopausal women whose body-mass index increased from age 18 and who started to smoke after a full-term pregnancy had a decreased risk of breast cancer (218) . These observations suggested that in older women, the antiestrogen effects of cigarette smoking may modulate the increased risk seen with an increase in body weight (218) . These possible competing effects of cigarette smoking on breast cancer risk are discussed additionally in a commentary by Russo (219) .

In a recent study, it was found that cigarette smoking was associated with a decreased risk of breast cancer in women with a BRCA1 or BRCA2 mutation (220) , and this report was discussed additionally in a commentary by Baron and Haile (221) . In contrast to these observations in women with a BRCA1 or BRCA2 mutation, in a cohort study of high-risk breast cancer families in the general population it was found that cigarette smoking was associated with an increased risk of breast cancer (222) . Although cigarette smoking is clearly associated with substantial increases in the risk for lung cancer, bladder cancer, oral cancer, osteoporosis, and cardiovascular disease, the available evidence suggests that cigarette smoking increases the risk for breast cancer in some women and decreases the risk for breast cancer in other women. Overall, the risks of cigarette smoking in the general population greatly exceed potential benefits, and all efforts to discourage people from smoking should continue.

In early studies, exposure of dogs to cigarette smoke was reported to stimulate the hepatic 6ß-hydroxylation of testosterone, decrease the serum level of testosterone, and to decrease the size of the prostate gland (223) . It was also reported that human cigarette smokers had a decreased risk of benign prostate hypertrophy (224) . In more recent studies, cigarette smoking was not found to be associated with an altered risk of benign prostate hypertrophy (225) or prostate cancer (226, 227, 228) , but cigarette smoking was reported to be associated with an increased risk of fatal prostate cancer (227 , 229) . Another report indicated that cigarette smoking was positively related to benign prostate hypertrophy but only in men who smoke 35 cigarettes per day (230) . More research is needed to resolve the conflicting reports on the effects of cigarette smoking on benign prostate hypertrophy and prostate cancer.

Metabolism of Xenobiotics by Intestinal Microorganisms
It is well known that many foreign chemicals can be metabolized by microorganisms in the gastrointestinal tract. The kinds of microorganisms differ in different individuals and can also differ in the same individual depending on the diet. Induction of enzymes that inactivate potentially toxic chemicals in the intestinal microflora, and the development and use of stable microorganisms that detoxify foreign chemicals in the gastrointestinal tract are additional approaches for enhancing the metabolic inactivation of carcinogens and other toxic chemicals.

	INHIBITORY EFFECTS OF DIETARY CHEMICALS ON CARCINOGENESIS: STUDIES WITH CURCUMIN, CAFFEINE, EGCG, AND TEA

Top ABSTRACT INDUCTION OF CARCINOGEN... INHIBITORY EFFECTS OF DIETARY... INHIBITORS OF CARCINOGENESIS IN... TAILORING CANCER CHEMOPREVENTIVE... REFERENCES

 
Epidemiology studies indicate that a substantial portion of human cancer in the United States is related to dietary factors (231 , 232) . Although it has been difficult from the epidemiology studies to identify specific dietary components that are inhibitory, the available evidence suggests that people who eat large amounts of fruit and vegetables have a lower risk of several kinds of cancer (233, 234, 235, 236, 237) . These observations in human populations pointed out the importance of identifying dietary substances that modulate carcinogenesis. The pioneering research of Wattenberg et al. (reviewed in Refs. 86 , 238 , 239 ) that demonstrated cancer chemopreventive activity for a large number of dietary chemicals in animals and the work by Sporn and his colleagues (reviewed in Ref. 240 ) on the chemopreventive effects of retinoids fostered the field of cancer chemoprevention. In recent years, there has been an increased emphasis on dietary modulators of carcinogenesis, and work in this field is being pursued in many laboratories. Our early research on diet and cancer focused on dietary inhibitors of nitrosamine formation (241, 242, 243, 244) , dietary inhibitors and activators of carcinogen-metabolizing enzymes (245) , dietary antagonists of ultimate carcinogens (246, 247, 248) , and effects of dietary factors on the in vivo metabolism of foreign compounds in humans by Phase I and Phase II reactions (128, 129, 130, 131, 132, 133) . More recently, we have studied the effects of curcumin, caffeine, EGCG, and tea on carcinogenesis in experimental animals, and these studies are described below.

Studies with Curcumin
Inhibitory Effect of Curcumin and Some of Its Analogues on Tumor Promotion by TPA.
Curcumin is the major yellow pigment in turmeric, curry, and mustard, and it is obtained from the rhizome of the plant Curcuma longa Linn. The ground dried rhizome of this plant (turmeric) has been used for centuries for the treatment of inflammatory diseases (249 , 250) , and curcumin is currently in wide use as a spice, food preservative, and yellow coloring agent for foods, drugs, and cosmetics.

Several studies indicated that compounds that possess antioxidant or anti-inflammatory activity inhibit TPA-induced tumor promotion in mouse skin. Because curcumin was reported to possess both antioxidant and anti-inflammatory activity (251, 252, 253, 254, 255, 256) , we studied the effect of topical applications of curcumin on TPA-induced tumor promotion on mouse skin, and we also studied the effect of curcumin as a potential inhibitor of tumor initiation by polycyclic aromatic hydrocarbons. Studies in our laboratory showed that topical application of curcumin inhibited TPA-induced tumor promotion as well as BP- and DMBA-induced tumor initiation on mouse skin (257 , 258) . These were the first studies to demonstrate an inhibitory effect of curcumin on carcinogenesis. In additional studies, we also found that topical application of curcumin inhibited TPA- or arachidonic acid-induced inflammation (mouse ear edema), as well as TPA-induced increases in hydrogen peroxide formation, ornithine decarboxylase activity, the synthesis of mRNA for ornithine decarboxylase, DNA synthesis, and hyperplasia in mouse skin (257 , 259, 260, 261) .

We examined the potential inhibitory effects of topical applications of several structural analogues of curcumin on TPA-induced tumor promotion in mouse skin. The structures of the compounds tested and the results obtained are shown in Fig. 5 . Among the compounds tested, demethoxycurcumin (17% of commercial grade curcumin) and bisdemethoxycurcumin (3% of commercial grade curcumin) are naturally occurring substances present in the rhizome of Curcuma longa Linn and in turmeric. Caffeic acid phenethyl ester is a constituent of the propolis of honeybee hives, and caffeic acid, ferulic acid, and chlorogenic acid are widespread constituents of fruits and vegetables. Chlorogenic acid accounts for 6–8% of the dry weight of the coffee bean (262) . The results of our studies indicate that curcumin, demethoxycurcumin, and caffeic acid phenethyl ester are highly active inhibitors of TPA-induced tumor promotion on mouse skin (Fig. 5 ; Refs. 257 , 261 , 263 , 264 ). Bisdemethoxycurcumin, tetrahydrocurcumin, caffeic acid, ferulic acid, and chlorogenic acid were less active than curcumin (257 , 263) , and bisdemethylcurcumin (a dicatechol synthesized by Dr. Toshihiko Osawa at Nagoya University, Nagoya, Japan) was inactive (Fig. 5) . Commercial grade curcumin had the same inhibitory effect as pure curcumin on TPA-induced tumor promotion (263) .

View larger version (22K): [in this window] [in a new window]   Fig. 5. Structures and inhibitory effects of curcumin and some analogues on tumor promotion by TPA. (Taken from data described in the text and from Refs. 257 , 261 , 263 , 264 .)

Studies with potential inhibitors of carcinogenesis in the TPA-induced tumor promotion model on mouse skin may be useful in predicting the cancer chemopreventive potential of agents in a variety of epithelial tissues such as the gastrointestinal tract, esophagus, oral mucosa, lung, breast, prostate, and bladder. Absorption, distribution, and pharmacokinetic studies after oral administration will help in the selection of compounds for additional study.

Inhibitory Effects of Dietary Curcumin on Colon, Duodenal, Stomach, Esophageal, and Oral Carcinogenesis.
In 1992–1994, we reported that 0.5–2% dietary curcumin inhibited the formation of azoxymethane-induced focal areas of colonic dysplasia and colon tumors, N-ethyl-N'-nitrosoguanidine-induced duodenal tumors and BP-induced forestomach tumors in mice (269 , 270) , and Tanaka et al. (271) reported an inhibitory effect of 0.05% dietary curcumin on 4-nitroquinoline-1-oxide-induced carcinogenesis of the tongue in rats. Dietary curcumin was also shown to inhibit chemically induced colon cancer in rats (272 , 273) , esophageal carcinogenesis in rats (274) , glandular stomach carcinogenesis in rats (275) , and spontaneous intestinal tumors in the Min/+ mouse (276 , 277) . The inhibitory effect of dietary curcumin on the formation of azoxymethane-induced colon tumors was associated with enhanced apoptosis in the tumors (278) . Application of curcumin three times a week to the buccal pouch of DMBA-initiated Syrian golden hamsters inhibited oral carcinogenesis, and greater effects were observed when topical applications of curcumin were combined with p.o. administered green tea (279) . In an additional study, p.o. administered or topically applied curcumin or turmeric together with DMBA inhibited DMBA-induced oral cancer in Syrian hamsters (280) .

In our studies, feeding 0.5% or 2% curcumin in the diet to CF-1 mice inhibited the number of azoxymethane-induced colon adenomas per mouse by 50% and 56%, respectively, and the number of colon adenocarcinomas per mouse was inhibited by 57% and 100%, respectively (Table 5) . Feeding curcumin also had a marked inhibitory effect on the size of the tumors (Table 5) . An inhibitory effect of curcumin on colon carcinogenesis was observed when the compound was fed either during the initiation or postinitiation phase of azoxymethane-induced colon carcinogenesis (270) .

Inhibitory Effect of Curcumin on Arachidonic Acid Metabolism.
Several inhibitors of arachidonic acid metabolism inhibit TPA-induced inflammation and tumor promotion, and it is thought that arachidonic acid metabolites are important for TPA-induced inflammation and tumor promotion in mouse skin. Because of the possibility that curcumin might inhibit carcinogenesis in mouse skin by inhibiting arachidonic acid metabolism, we evaluated the effect of curcumin on the metabolism and action of arachidonic acid in the epidermis. We found that topical application of curcumin inhibited arachidonic acid-induced ear inflammation in mice (257 , 287) . Studies on the metabolism of arachidonic acid by mouse epidermis revealed that curcumin (10 µM) inhibited the epidermal metabolism of arachidonic acid to 5- and 8-hydroxyeicosatetraenoic acid by 60% and 51%, respectively (lipoxygenase pathway), and the metabolism of arachidonic acid to PGE₂, PGF₂, and PGD₂ was inhibited 70%, 64%, and 73%, respectively (cyclooxygenase pathway) (Ref. 287 ). In another study, dietary administration of 0.2% curcumin to rats inhibited azoxymethane-induced colon carcinogenesis, and decreased colonic and tumor phospholipase A₂, phospholipase C1, and PGE₂ levels (272) . In this study, dietary curcumin also decreased enzyme activity in colonic mucosa and tumors for the formation of PGE₂, PGF₂, PGD₂, 6-keto PGF₁, and thromboxane B₂ via the cyclooxygenase system, and production of 5(S)-, 8(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acid via the lipoxygenase pathway of arachidonic acid metabolism was also inhibited. More recent studies have shown that curcumin is a strong inhibitor of cyclooxygenase 2 activity as well as an inhibitor of cyclooxygenase 2 mRNA formation (288 , 289) .

Synergistic Effects of Curcumin on All-trans Retinoic Acid- and 1,25-Dihydroxyvitamin D₃-Induced Differentiation.
Suh et al. (290) suggested the use of HL-60 cell differentiation as a bioassay that is useful for the discovery of novel cancer chemopreventive agents in natural products. We considered the possibility that some of the cancer chemopreventive effects of curcumin may be related to an effect of this compound on cellular differentiation, and we investigated the effect of curcumin on differentiation in the human promyelocytic HL-60 leukemia cell model system. We also investigated the effects of combinations of curcumin together with 1,25-dihydroxyvitamin D₃ or all-trans retinoic acid on differentiation in HL-60 cells. Treatment of HL-60 cells with 10 µM curcumin for 48 h resulted in small increases in differentiation as measured by the proportion of cells that reduced nitroblue tetrazolium and expressed Mac 1 (Tables 6 and 7 ; Ref. 291 ). Synergistic induction of differentiation as measured by the above markers was observed when 1–10 µM curcumin was combined with 10–100 nM all-trans retinoic acid or with 100 nM 1,25-dihydroxyvitamin D₃ (Tables 6 and 7 ; Ref. 291 ). Combinations of all-trans retinoic acid and curcumin stimulated differentiation predominantly to granulocytes, whereas combinations of 1,25-dihydroxyvitamin D₃ and curcumin stimulated differentiation predominantly to monocytes (291) . Independent studies by Sokoloski et al. (292) have also shown a synergistic effect of curcumin and 1,25-dihydroxyvitamin D₃ on differentiation in HL-60 cells, and they suggest the importance of NF-B inhibition for this effect. In additional studies, Amir et al. (293) have shown a synergistic effect of lycopene in combination with 1,25-dihydroxyvitamin D₃ on differentiation in HL-60 cells. It is possible that many dietary chemicals such as curcuminoids, carotenoids, and other substances in fruits, vegetables, and other edible plants prevent human cancer in part by synergizing with endogenously produced stimulators of differentiation such as all-trans retinoic acid, 1,25-dihydroxyvitamin D₃, and butyrate. More research is needed to test this hypothesis.

View this table: [in this window] [in a new window]   Table 7 Effect of curcumin and 1,25-dihydroxyvitamin D3 on the differentiation of HL-60 cells as measured by nitroblue tetrazolium reduction and Mac-1 expression HL-60 cells were treated with curcumin (CUR) and 1,25-dihydroxyvitamin D3 (VD3) for 48 h, and nitroblue tetrazolium (NBT)-positive or Mac-1-positive cells were measured. The numbers in parentheses were corrected for background values from control samples. Each value represents the average of three experiments. (Taken from Ref. 291 .)