Antioxidants: Carcinogenic and Chemopreventive Properties

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Chemical carcinogens, which are present widely in our environment, are considered to play an important role in the causation of most human cancers. They may be classified into genotoxic and nongenotoxic types, the former including nitrosamines, aromatic hydrocarbons, aromatic amines, and nitrofurans, and the latter being exemplified by peroxisome proliferators, antioxidants, chlorinated pesticides, uracil, and D-limonene. For primary prevention of human cancer, it is essential that we eliminate carcinogens as far as possible and ingest possible chemopreventors. One problem with eliminating carcinogens is the existence of nongenotoxic compounds which escape detection in short-term assays. The development of chemopreventors therefore assumes particular importance in approaches to the prevention of cancer. Since antioxidants possess both carcinogenic and chemopreventive properties, a detailed analysis of this group of agents should contribute to their introduction.

I. INTRODUCTION

Antioxidants have been used widely in the food industry to prevent or retard autooxidation of fats, oils, and fat-soluble compounds. Some water-soluble antioxidants prevent oxidation in aqueous material.      
Apart from foodstuffs, they are contained in various cosmetics, medicines, plastics, and rubbers. They may be synthetic or naturally occurring; the latter type of antioxidants are widely distributed in plants. Humans may thus be exposed to mixtures of synthetic and naturally occurring antioxidants orally from foodstuffs or through the skin from cosmetics. Generally, antioxidants can be classified into five types:
1. Primary antioxidants which terminate free radical chain reactions (phenolic compounds, tocopherols, tertiary amines, flavonoids)
2. Oxygen scavengers which react with oxygen and remove it (ascorbic acid and its derivatives)
3. Secondary antioxidants which decompose lipid peroxides into stable products (sulfur compounds, seleno-compounds)
4. Enzymic antioxidants which remove highly oxidative species (superoxide dismutase, catalase, glutathione peroxidase)
5. Chelating agents which chelate metallic ions such as copper and iron (citric acid, phytic acid)      
Of these antioxidants, ascorbic acid and chelating agents are able to enhance the antioxidant action of tocopherols or phenolic compounds, and are therefore called synergists. Antioxidants are generally not mutagenic as evaluated by the Ames test and may even inhibit the mutagenic activity of mutagens. Moreover, they inhibit chemical carcinogenesis in various organs of rodents when they are given prior to and/or simultaneously with certain carcinogens.       
Therefore, antioxidants may have potential applications as potent chemopreventors in man. However, some of them have been shown to enhance second stage chemical carcinogenesis in rodents when administered after exposure to carcinogens. In addition, the synthetic antioxidant BHA, which is commonly used throughout the world as a food additive, was demonstrated to induce forestomach carcinomas in F344 rats of both sexes and in male Syrian golden hamsters. Subsequently, modifying effects of antioxidants and the mechanism(s) underlying antioxidant induction of tumors have been the focus of extensive investigations.      
In addition to their antioxidant effects, many antioxidants possess various kinds of biological activities such as enzyme induction, interference with the immune response, anti-viral activity, anti-inflammatory activity, interference with prostaglandin synthesis, inhibition of platelet aggregation, and protection against reperfusion injury. Some of these properties are closely linked to chemical carcinogenesis.      
The present review article covers the latest results of research on antioxidants, especially in relation to neoplastic and chemopreventive properties.

II. ANTIOXIDANT CARCINOGENICITY

Carcinogenicity studies of antioxidants have been extensive since the demonstration of forestomach carcinogenicity for the synthetic antioxidant BHA in 1983. Before then, BHA had been used widely in the world as a safe food antioxidant and was generally regarded to be beneficial to humans due to its lack of mutagenicity and its inhibition of rodent carcinogenesis induced by several carcinogens.      
In the first examination of long-term BHA exposure, male and female F344 rats were continuously administered 2% (maximum tolerable dose) or 0.4% BHA in the diet for 2 years. Histopathological examination showed that the high dose of BHA induced forestomach squamous cell carcinomas at incidences of 34.6% in males and 29.4% in females. No neoplastic lesions which could be attributed to BHA administration were observed in any other organs. This was the first report that antioxidants could induce tumors in rodents. Subsequently, BHA was also shown to be carcinogenic to make Syrian golden hamster forestomach at doses at 1 and 2% in the diet, although the results were equivocal when male B6C3F1 mice received doses of 0.5 and 1%. In a dose-response study using F344 male rats at levels of 0.125 to 2%, only the highest dose caused squamous cell carcinomas while 1% induced only a low incidence of squamous cell papillomas; no tumors were observed at doses lower than 0.5%.      
Since the carcinogenicity of BHA appeared limited to the forestomach which humans do not possess, it was necessary to examine its effects on other animals without a forestomach for the evaluation of human hazard potential. Continuous oral treatment with BHA at doses lower than 1% of 20 months in guinea pigs, daily intragastric doses of 500 mg/kg body weight of BHA for 20 days and then 250 mg/kg body weight 5 times/week until 84 days in cynomolgus monkeys, or 0-100 mg/kg/days or dietary 0.25-1% BHA for up to 1 year in beagle dogs did not cause any histopathological changes in any tissue. Therefore it has been concluded that the carcinogenic potential of BHA is limited to forestomach epithelium.      
The carcinogenicity of butylated hydroxytoluene (BHT), which is as potent as BHA as an antioxidant and is commonly used in cosmetics or as a food additive, has also been extensively studied. Wistar rats and B6C3F1 mice of both sexes were treated with BHT at dose levels of 0.25 or 1.0%, and 0.02, 0.1, or 0.5% in the diet, respectively, for up to 104 weeks. However, no tumors were induced that could be attributed to the treatment. On the other hand, administration of 1 or 2% BHT in the diet to male B6C3F1 mice for 104 weeks was associated with an increased incidence of hepatocellular adenomas or foci of alteration in a clear dose-dependent manner.       
Although there was an inverse dose relationship, male C3H mice fed diets containing 0.5 or 0.05% of BHT for 10 months also had significantly increased incidences of liver tumors as compared to those kept on basal diet alone. Olsen et al. also reported weak tumorigenicity for BHT in the rat liver in a two generation study. However, none of the evidence to date is unequivocal and the question of BHT carcinogenicity in rodents remains open.      
Although no standard carcinogenic bioassays have been reported for tert-butylhydroquinone (TBHQ), continuous feeding of this antioxidant for <20 months at doses of 0-0.5% did not result in any compound-related gross or microscopic lesions. Propyl gallate was also studied in F344 rats and B6C3F1 mice by feeding at dietary levels of 5000-20,000 mg/kg, but no dose-related increases in incidences of tumors or differences in survival were found. The potential carcinogenicity of other gallates has not been fully evaluated.      
Since BHA was found to induce pronounced forestomach hyperplasia in a short period and forestomach squamous cell carcinoma in rats and hamsters in the long term, many other structurally related phenolic antioxidants were examined for associated proliferative activity in the forestomach epithelium as an aid to prediction of potential to induce forestomach tumors. Among these are TBHQ, 4-methoxyphenol, 1,4-dimethoxybenzene, catechol, resorcinol, hydroquinone, 3-methoxyphenol, 2-methoxyphenol, anisole, 4-cresol, phenol, 4-hydroxybenzoic acid n-alkyl esters (alkyl parabenes), 4-hydroxybenzoic acid esters, gallic acid, caffeic acid, sesamol, chlorogenic acid, syringic acid, ferulic acid, eugenol, esculin, 4-methylphenol, 4-tert-buthylphenol, pyrogallor, methylhydroquinone, 2-tert-butyl-4-methylphenol, and BHT tested in short-term feeding studies in rats or hamsters at a dose of 0.7-2% in diet; 4-methoxyphenol and sesamol were found to be as active as BHA in the induction of forestomach hyperplasia and, in addition, they caused a circular deep ulceration parallel to the limiting ridge. Caffeic acid, 2-tert-butyl-4-methylphenol, and 4-tert-butylphenol also induced pronounced hyperplasia in the forestomach epithelium. The labeling index in the glandular stomach was also significantly increased in animals fed catechol and 4-methoxyphenol. This prompted investigation in long-term experiments. Chemical structures and the results of carcinogenicity studies of antioxidants are presented in Fig. 1 and Table I, respectively.


FIGURE 1 Chemical structures of carcinogenic antioxidants.

TABLE I Incidences of Tumors in the Stomach and Kidney

Chemicals Sex No. of rats No. of rats with (%) Squamous cell carcinoma (forestomach) No. of rats with (%) Adenocarcinoma (G1 stomach) No. of rats with (%) Adenoma (kidney)
BHA M 52 18 (35)*** 0 0
  F 51 15 (29)*** 0 0
4-Methoxyphenol M 30 23 (77)*** 0
  F 30 4 (13) 0 0
Caffeic acid M 30 17 (57)*** 0 4 (13)
  F 30 15 (50)*** 0 6 (20)*
Sesamol M 29 9 (31) *** 0 0
  F 30 3 (10) 0 0
Catechol M 28 0 15 (54)*** 0
  F 28 0 12 (43)*** 0
4-Methylcatechol M 30 17 (57)*** 17 (57)*** 0
  F 30 12 (40) 14 (47)*** 0
Hydroquinone M 30 0 0 14 (47)***
  F 30 0 0 0
Control M 30 0 0 0
  F 30 0 0 0

*P < 0.02.
***P < 0.001 vs control group value.

When male and female F344 rats were treated with caffeic acid, sesamol, 4-methoxyphenol, or 4- methylcatechol at a dose of 2% or catechol at a dose of 0.8% in the diet for 2 years, the agents except catechol induced significantly increased incidences of forestomach squamous cell carcinomas. Females were less sensitive than males. In addition, 4-methylcatechol induced carcinomas in glandular stomach epithelium. Although catechol did not induce tumors in the forestomach, it did induce glandular stomach carcinomas. In a dose-response study, even 0.16% catechol in the diet induced adenomas at a low incidence.       
Since this demonstration of catechol (1,2-dihydroxybenzene) carcinogenicity, hydroquinone (1,4-dihydroxybenzene), one of its isomers, was further examined in male and female rats at a dose of 0.8% in diet. Whereas it was not found to be carcinogenic for either the forestomach or glandular stomach, it induced a 46.6% incidence of kidney adenomas, predominantly in male rats. 1,2,4-Benzenetriol, protocatechuic acid, protocatechualdehyde, dopamine, and DLdopa, all dihydroxybenzene derivatives like caffeic acid and catechol, were examined for their potency to induce cell proliferation in the rat forestomach and glandular stomach epithelium. However, only 1,2,4- benzenetriol and dopamine, each at 1.5% in the diet, were effective in increasing the BrdU-labeling index in the forestomach epithelium. 2-Methoxyphenol, with one hydroxy substituent replaced by a methoxy substituent, also lacked any effects on cell proliferation in either forestomach or glandular stomach epithelium. Therefore, the ortho-dihydroxy structure appears important, but substituents actually determine cell proliferation on stimulus.      
Many phenolic compounds that do not show stim- ulation activity per se on the forestomach epithelium cause very strong cell proliferation or tumorigenicity when they are combined with sodium nitrite (NaNO2). For example, continuous oral treatment with 0.8% catechol alone in the diet for 51 weeks induced mild forestomach hyperplasia. The grade of forestomach hyperplasia considerably increased and papillomas were also found in 4 of 15 rats with a simultaneous administration of catechol and NaNO2.      
When the combined effects of various phenolic compounds and NaNO2 on rat forestomach cell proliferation were further examined in a 4-week experiment, the cell proliferative response to known forestomach carcinogens such as sesamol, 4-methoxyphenol, and 4-methylcatechol was further enhanced by simultaneous treatment with NaNO2. In addition, markedly increased cell proliferation was found when 2% hydroquinone, 2% pyrogallol, 2% gallic acid, or 2% TBHQ in the diet was combined with 0.3% NaNO2 in the drinking water, although individual phenolic compounds or NaNO2 did not stimulate any proliferating activity or only mild hyperplasia. It has been known that mutagenic diazocompounds are formed by the reaction of phenols and NaNO2 under acidic conditions.
Such compounds could be responsible for the cell proliferation. Cell proliferation induced by these phenolic compounds and NaNO2 in combination was much more pronounced than with the known forestomach carcinogens caffeic acid, sesamol, 4- methoxyphenol, and 4-methycatechol. Similar effects were observed when the nonphenolic antioxidant sodium ascorbate (NaASA) or ascorbic acid (ASA) was given to rats simultaneously with NaNO2. Therefore, many phenolic compounds as well as NaASA and ASA may possess carcinogenic activity for the rat forestomach epithelium in the presence of NaNO2.

III. MECHANISTIC APPROACHES TO ANTIOXIDANT CARCINOGENICITY

Since the carcinogenicity of antioxidants was found to be mostly limited to the forestomach epithelium except in the catechol, 4-methylcatechol, and hydroquinone cases, approaches to elucidating mechanisms have been primarily directed toward this tissue. All of the carcinogenic phenolic antioxidants which target the forestomach were shown to induce cytotoxicity as well as hyperplasia. To examine whether the observed hyperplasia was due to excess regeneration associated with cytotoxicity or due to primary mitogenic effects, early forestomach lesions induced by BHA, caffeic acid, or 4-methoxyphenol in rats were investigated.


FIGURE 2 Sequential observation of labeling indices in rat forestomach epithelium treated with antioxidants. ○, 2% BHA; ●, 2% caffeic acid; ▲, 2% 4-methoxyphenol; --, basal diet.

DNA synthesis of the forestomach epithelium, expressed as the number of BrdU-labeled cells per 100 basal cells (labeling index), increased 12 hr after treatment with caffeic acid or 4-methoxyphenol. In the case of BHA, an increase in the labeling index was apparent 3 days after treatment. After 7 days of continuous antioxidant administration, the labeling index increased or continued to be high, especially in the groups treated with 4-methoxyphenol followed by caffeic acid and BHA (Fig. 2). Hyperplasia was observed 3 days after treatment with caffeic acid, but this change first became evident only later in the cases of BHA, sesamol, and 4-methoxyphenol. Evidence of toxicity, such as erosion or ulceration, developed in animals treated with caffeic acid or 4- methoxyphenol for 7 days, but were not found in those treated with BHA. A strongly elevated expression of cell proliferation-related c-fos oncogene expression in the forestomach epithelium was demonstrated 15 min after beginning treatment with BHA, but rapidly decreased thereafter. Another cell proliferation-related c-myc expression was similarly observed after 15 min of treatment, then decreased slowly. By the electron microscopical observation, the initial changes observed in the forestomach epithelial cells are the enlargement of nucleous and an increase in free ribosomes and polysomes in the basal layer. These changes are observed 24 hr after treatment with caffeic acid and 72 hr after treatment with BHA, without any cytotoxicity. These results strongly suggest that antioxidants primarily induce cell proliferation by direct stimulation and that cell proliferation is probably further enhanced by regeneration subsequent to cytotoxicity.      
Strong cell proliferation, however, does not always correlate with occurrence of carcinomas. It takes a long time (usually more than 1 year) for the development of forestomach carcinomas, and induced hyperplasia regresses after cessation of chemical treatment. We compared reversibility of rat forestomach lesions induced by an intragastric dose of 20 mg/kg body weight N-nitroso-N'-nitro-N-nitrosoguanidine (MMNG) once a week, 20 ppm N-methylnitrosourethane (MNUR) in the drinking water as genotoxic forestomach carcinogens, 2% BHA, 2% caffeic acid, or 2% 4- methoxyphenol in the diet as nongenotoxic carcinogens for 24 weeks.      
Forestomach lesions induced by genotoxic carcinogens did not regress 24 weeks after the removal of the carcinogen stimulus. In contrast, hyperplasia induced by nongenotoxic carcinogens clearly regressed after the cessation of insult. Preneoplastic atypical hyperplasia, observed at high incidences in rats treated with genotoxic carcinogens, was also evident in animals receiving nongenotoxic agents, even after their withdrawal, albeit at low incidences. These results indicate that even with nongenotoxic carcinogens, a heritable alteration at the DNA level could occur during strong cell proliferation and result in atypical hyperplasia development. This preneoplastic lesion might then progress to produce carcinomas. Indeed, weak forestomach genotoxic carcinogens show potent carcinogenicity under cell-proliferating conditions induced by BHA. Thus, when animals were treated with 2% BHA and given sc injections of 50 mg/kg body weight of the weak forestomach carcinogen 3,2'- dimethyl-4-aminobiphenyl (DMAB) once a week or an ip injection of 15 mg/kg body weight of Nmethylnitrosourea (MNU) once every 2 weeks for 22 weeks, the carcinogenic response was amplified. At week 24 the BHA treatment was associated with significant papilloma induction in the forestomach (40%), while no lesions were observed in the group given only DMAB. MNU alone did not induce forestomach carcinomas, but carcinomas were found in 75% of rats receiving BHA in combination with either of the genetoxic agents.       
Although antioxidant forestomach carcinogens are generally negative in the Ames test and in several in vitro and in vivo mutagenesis assays, they can show weak genotoxic activity under certain conditions. Three hours after a single intragastric administration of 40 mg BHA, no detectable DNA damage was present in the forestomach epithelium, but the oxidative metabolite tert-butylquinone (TBQ) did cause DNA damage at 1/1000 of the parent concentration level. Other oxidative BHA metabolites, 3-tert-butyl- 4,5-dihydroxyanisole and 3-tert-butylanisole-4,5- quinone, also showed DNA-damaging activity but were weaker than with TBQ. In vitro incubation of BHA with calf thymus DNA in the presence of an S9 mixture under acidic conditions results in DNA adducts as evaluated by a 32P-postlabeling assay.      
Quinone metabolites of BHA form DNA adducts without the presence of an S9 mixture. Recently, low levels of DNA adducts were demonstrated in the forestomach epithelium of animals treated with 2% BHA for 2 weeks. During prostaglandin H synthasemediated oxidative metabolism of phenolic compounds, active oxygen species could be produced. This finding was supported by the clear inhibition by aspirin of BHA-induced rat forestomach hyperplasia and ESR analyses which showed that prostaglandin H synthase administration resulted in a substantially accelerated metabolism of TBHQ into TBQ, which is accompanied by the formation of superxide anion, hydroxy radical, and hydrogen peroxide. Thus, it is conceivable that active oxygen species are responsible for antioxidant-induced cytotoxicity or carcinogenesis.      
However, the results of investigation of 8-hydroxyguanosine (8-OH-dG), which is a reliable marker of oxidative DNA damage by reactive oxygen species, in the forestomach epithelium in vivo have been equivocal. Caffeic acid also causes metaldependent DNA damage through H2O2 formation in vitro. In addition, food-derived mutagenic compounds could be formed in the stomach by interaction of amines and nitrite, or nitrite and phenolic compounds. Therefore, oxidative metabolites, active oxygen species, and food-derived mutagens might contribute weak genotoxicity which could act in concert with strong cell proliferation. The putative carcinogenic process driven by phenolic compounds in the forestomach epithelium is summarized schematically in Fig. 3.


FIGURE 3 Putative pathway of rat forestomach carcinogenesis induced by phenolic antioxidants. Possible contributing factors are indicated by brackets. Observed changes in the forestomach epithelium are shown to parentheses.

IV. MODIFICATION OF CARCINOGENESIS BY ANTIOXIDANTS IN TWO-STEP CARCINOGENESIS MODELS

Many antioxidants are capable of modifying chemical or ultraviolet carcinogenesis in a broad spectrum of organs. In addition to direct effects on the initiation and/or postinitiation neoplastic process, they can also exert an influence by blocking nitrosamine formation or reducing the activity of promoters such as 12-Otetradecanoylphorbol- 13-acetate (TPA) in mouse skin carcinogenesis. The mechanisms underlying modification appear to vary with the stage of carcinogenesis and with the carcinogen.

A. Modification of Carcinogenesis by Antioxidants in the Initiation Stage
In this stage, antioxidants could modify carcinogenesis by (1) altering the metabolic activation of procarcinogens, (2) altering detoxifying enzymes, (3) direct interaction with the proximate carcinogenic species, (4) trapping active oxygen species, or (5) influencing absorption of carcinogens from the gastrointestinal tract. BHA inhibits benzo[a]pyrene (BP)- or its proximate carcinogen (±)-trans-7,8-dihydrobenzo[a]pyreneinduced mouse forestomach and lung carcinogenesis in the initiation stage. In this case, inhibition of the cytochrome P450-dependent monooxygenase, which metabolizes BP via 7,8-dihydrodiol to the ultimate carcinogen 7,8-diol-9,10-epoxide, and induction of phase II enzymes, such as glutathione S-transferase, which detoxify the proximate carcinogen, eventually resulted in the decreased formation of diol epoxide-DNA adducts. The plant flavonoid ellagic acid inhibits BPinduced mouse lung carcinogenesis by intraperitoneal and/or oral administration. It also reduces the mutagenic activity of BP 7,8-diol-9,10-epoxide and 7,8-diol- 9,10-epoxide-induced mouse pulmonary tumor formation when administered prior to carcinogen. The observed inhibition may be due to ellagic acid decreasing hepatic and pulmonary cytochrome P450 levels, increasing hepatic glutathione S-transferase activity, and directly interacting with ultimate carcinogen. Many phenolic antioxidants, flavonoids, and seleno-compounds are thought to inhibit carcinogenesis by modifying metabolic pathways. On the other hand, BHA was found to enhance dibutylnitrosamine (DBN)-induced hepatocarcinogenesis, possibly due to enhancing cytochrome P450-mediated oxidation of DBN to proximate carcinogenic studies.      
In cases of active oxygen-mediated carcinogenesis, some antioxidants lower the tumor yield or cytotoxicity induced by carcinogens, but protective effects are not general. Continuous oral treatment with 500 ppm potassium bromate (KBrO3) induces renal cell tumors in 90% of rats when given for up to 2 years. Intraperitoneal or intragastric administration of KBrO3 induces cytotoxicity, lipid peroxidation, and increases in 8-hydroxydeoxyguanosine (8-OH-dG) formation at the target site of carcinogenesis, and therefore active oxygen might be involved in its carcinogenic action.      
Combined treatment with glutathione, cystein, or ascorbic acid, but not superoxide dismutase or vitamin E, protected against its associated oxidative DNA damage and nephrotoxicity. On the other hand, ferric nitrilotriacetate-induced nephrotoxicity and lipid peroxidation were protected against by vitamin E. It is known that this renal carcinogen generates hydroxy radicals in the presence of H2O2 in vitro, which cause DNA cleavage and base damage. It produces 8-OH-dG in the kidney DNA and causes lipid peroxidation and nephrotoxicity in the proximal tubules. Hepatocarcinogenesis induced by peroxisomal proliferators, in which excess production of H2O2 may be responsible, is also blocked by BHA and ethoxyquine, and the ascorbic acid derivative (CV 3611), N,N'-diphenyl-p-phenylenediamine, or BHT protect against liver tumor induction by a cholinedeficient diet in which lipid peroxidation and formation of 8-OH-dG are thought to be involved.      
Chlorophyllin inhibits mutagenesis by several carcinogens, such as the heterocyclic amine Trp-P-2, in the Ames test by absorbing carcinogens to form carcinogen-chlorophyllin complexes. In the rat, chlorophyllin accelerates the excretion of Trp-P-2 into feces. Although an inhibition action for chlorophillin in in vivo carcinogenesis has not been demonstrated to date, this chemical might be expected to exert beneficial effects.

B. Modification of Carcinogenesis by Antioxidants on the Promotion/Progression Stage
The modifying effects of antioxidants on carcinogenesis when administered after carcinogen treatment have been examined in various organs by a large number of investigators. Both promoting and inhibitory effects in various organs have been documented, dependent on the organ site and agent, as shown in Table II. Carcinogenic antioxidants usually strongly enhance carcinogenesis in their target organs. Thus BHA, caffeic acid, and 4-methylcatechol promote forestomach carcinogenesis, and catechol and 4- methylcatechol enhance both forestomach and glandular stomach carcinogenesis after pretreatment with MNNG. Both BHA and BHT promote rat urinary bladder carcinogenesis initiated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN), and BHT enhances rat urinary bladder, esophagus, and thyroid carcinogenesis in animals pretreated with BBN, DBN, and DHPN, respectively. Without carcinogen pretreatment, they induce hyperplasia or increase the BrdUlabeling index in their target organs. Therefore, promotion effects appear closely related to their potency in inducing cell proliferation.      
On the positive side, BHA was found to inhibit DHPN-initiated lung carcinogenesis, 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated mammary carcinogenesis, and diethylnitrosamine (DEN)-induced hepatocarcinogenesis. BHT also inhibits colon, kidney, and mammary carcinogenesis, and similar findings have been widely gained for synthetic as well as naturally occurring antioxidants. Modulation of ornithine decarboxylase (ODC) activity, generation of active oxygen species, interaction with calcium- and phospholipid-dependent protein kinase C, altered prostaglandin synthesis, and influence on intercellular communication are all factors that might play a role in antioxidant effects on cell proliferation and/or modulation of carcinogenesis.

TABLE II Modifying Effects of Antioxidants on Rat Carcinogenesis after Carcinogen Exposure

Target organ Antioxidant Synthetic BHA Synthetic BHT Synthetic TBHQ Synthetic PG Antioxidant Naturally occurring SA Naturally occurring α-TOC Naturally occurring PA Naturally occurring DDS
Esophagus
Forestomach
Glandular stomach
Colon
Liver
Lung
Kidney
Urinary bladder
Mammary gland NE NE
Thyroid

Note: TBHQ, t-butylhydroquinone; PG, propyl gallate; SA, sodium ascorbate; α-TOC, α-tocopherol; PA, phytic acid; DDS, diallyldisulfide. ↑, enhancement; →, no effect; ↓, inhibition, NE, not examined.

C. Antipromoting Activity of Antioxidants
Antipromotion effects have been primarily demonstrated in the two-stage mouse skin carcinogenesis model. In this system, mice are given a single topical application of DMBA, 3-methylcholanthrene, BP, or BP-7,8-diol-9,10-epoxide as an initiator, then receive a continuous topical treatment with TPA or teleocidin together with synthetic antioxidants such as BHA, BHT, α-tocophenol, or other naturally occurring antioxidants such as green tea polyphenols, curcumin, chlorogenic acid, caffeic acid, and ferulic acid. The observed inhibition of TPA-induced skin promotion may be partly due to a scavenging action of these antioxidants against TPA-induced active oxygens.      
TPA induces superoxide anion radicals and H2O2 release in human peripheral leukocytes in vitro and H2O2 and 8-OH-dG in mouse skin in vivo. These phenomena can be strongly inhibited by copper(II)- (3,5-disopropylsalicylate)2 (CuDIPS) and by nordihydroguaiaretic acid (NDGA), which are superoxide anion radical scavengers and detoxifiers. In an in vivo experiment, CuDIPS and NDGA significantly inhibited TPA-induced skin tumor promotion in mice initiated with DMBA. Induction of ODC in mouse epidermis also appears to be an important factor for TPA-induced skin tumor promotion. This TPAinduced ODC increase could be inhibited by lipoxygenase inhibitors NDGA, morin, fisetin, kaempferol, propyl gallate, esculetin, and BHA. In addition, morin or esculetin treatment was associated with significant inhibition of skin tumor promotion. Thus, the inhibitory effects of flavonoids on TPA-induced ODC induction and tumor promotion roughly paralleled their lipoxygenase inhibition. These results therefore suggest that antioxidants act by scavenging the superoxide anion radicals that are responsible for tumor promotion or by interfering with lipoxygenase in the epidermis induced by TPA.

D. Modification by Blocking Nitrosamine Formation
From the epidemiological viewpoint, a number of studies have suggested that an intake of nitrite and nitrate correlates with a high incidence of human gastric cancer and that this was due to formation of carcinogenic N-nitroso compounds in the stomach by the reaction of nitrite with amines present in foods and certain drugs. Some antioxidants have been shown to prevent nitrosation in vitro and tumor formation in animals by preventing this reaction between nitrite and amines to form N-nitroso compounds. Ascorbic acid and α-tocophenol are well-known inhibitors of nitrosation. For example, they significantly inhibit in vitro nitrosation of secondary amines such as morpholine, piperazine, diethylamine, and N-methylurea. The reaction of ascorbic acid with nitrite proceeds with the reduction of 2 mol of nitrite to nonnitrosating nitric oxide per mole of ascorbic acid, which is oxidized to dehydroascorbic acid. However, the nonnitrosating nitric oxide can, in the presence of oxygen, give rise to higher oxides of nitrogen, which are themselves powerful nitrosating species. Therefore, under certain conditions ascorbic acid can catalyze nitrosation. Nevertheless, sodium ascorbate at 11.5 or 23 g/kg in the diet gave 89-98% inhibition of lung adenoma induction when NaNO2 was applied with a piperazine, morpholine, or methylurea system. Inhibition of nitrosation by ascorbic acid can be observed by examination of human urine following sequential oral doses of nitrite and proline.      
However, the dose of antioxidant required to return the N-nitrosoproline excretion to basal levels was far in excess of the proline administered. Phenolic antioxidants and flavonoids are also capable of blocking nitrosamine formation. In one carcinogenesis study, gallic acid strongly inhibited adenoma induction in mouse lung by morpholine plus NaNO2. The mechanisms by which phenolic compounds inhibit nitrosation involve reduction of nitrite to nitric oxide, or formation of C-nitroso compounds and mutagenic diazoquinone. Therefore, the possibility remains that the C-nitroso compounds or diazoquinone formed could exert carcinogenic activity. Recently, continuous oral administration of ascorbic acid or some phenolic compounds, including catechol, hydroquinone, and gallic acid, and NaNO2 in combination induced strong cell proliferation or papillomas in rat forestomach epithelium. Therefore, much care and attention should be given to this type of cancer prevention.

V. CHEMOPREVENTION OF CARCINOGENESIS BY ANTIOXIDANTS

Antioxidants that have demonstrated an inhibitory effect in experimental chemical carcinogenesis have been proposed as possible chemopreventors in man. However, this has been suggested primarily on the basis of epidemiological findings, and the existence of adverse effects in different experimental models has indicated that care must be taken in application of antioxidants as chemopreventors.      
Necessary characteristics for an ideal chemopreventor include (1) ability to inhibit initiation activity, (2) ability to inhibit promotion or progression activity, (3) ability to block nitrosamine formation, (4) lack of genotoxicity, (5) lack of carcinogenicity, (6) not a carcinogen precursor, (7) lack of enhancing activity at any stage of carcinogenesis, (8) lack of toxicity, and (9) commercially available. Factors (5) to (8) might, however, be ignored if hazardous effects are only evident at doses much higher than the chemopreventive dose.      
Nevertheless, it may still be difficult to find chemopreventors which satisfy all these requirements. For example, sodium ascorbate can satisfy requirements 1, 2, 3, 5, 8, and 9, but not 4, 6, and 7, whereas α- tocopherol satisfies 1-6, 8, and 9 but not 7. The fact that antioxidants may show opposite effects in different organs, particularly in the promotion stage, means, furthermore, that a total body approach using different carcinogenic initiators is necessary for the reliable assessment of second-stage effects.      
Recently we established a multiorgan carcinogenesis model in which five different carcinogens were used as initiators. In this model, not only the enhancing but also the inhibitory effects of chemicals on carcinogenesis either in the initiating or in the promoting stage on major organs (liver, forestomach, small intestine, large intestine, lung, kidney, urinary bladder, and thyroid gland) can be examined in a single experiment, and its application for examining chemopreventive effects of several naturally occurring antioxidants suggested that green tea catechins (GTC) may be in fact possible chemopreventors. Significant decreases in the incidences of small intestinal tumors (adenomas and adenocarcinomas) were evident in the group treated with 1% GTC (content of catechins >91%, of these ( - )-epigallocatechin gallate >54%) during carcinogen exposure (13%) as compared with the carcinogen alone control value (57%). Multiplicities (average no. per rat) were also lower in groups treated with GTC both during (0.13 ± 0.35) and after (0.13 ± 0.48) carcinogen exposure than in the carcinogen alone case (1.07 ± 1.21). No significant differences in esophagus, forestomach, colon, liver, kidney, urinary bladder, lung, and thyroid gland lesion induction were observed.      
Subsequent treatment with 1.0% GTC also inhibited rat mammary tumor development and consequently increased the survival rate (93% vs. 33.3% in DMBA alone) after a single intragastric administration of 50 mg/kg body weight of DMBA. GTC potently lowers the hepatocarcinogenicity of glutamic acid pyrolisate 2-amino-6-methyldipyridol[1,2- a:3',2'-d] imidazole (Glu-P-1) as assessed in terms of number and areas of preneoplastic glutathione Stransferase placental form (GTC-P) positive foci in our medium term liver bioassay. Green tea extracts or green tea polyphenols also inhibit benzo[a]pyrene (BP)-, DMBA, 3-methylcholanthrene-, or ultraviolet light-induced tumor initiation and complete carcinogenesis in mouse skin, 12-O-tetradecanoylphobol-13- acetate caused tumor promotion in mouse skin, Nethyl- N'-nitro-N-nitrosoguanidine (ENNG)-induced mouse duodenal carcinogenesis, azoxymethaneinduced rat colon carcinogenesis, BP- and diethylnitrosamine- induced mouse lung and forestomach carcinogenesis, and 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone-induced mouse lung carcinogenesis.      
These inhibitory effects were observed not only in the initiation stage but also in the promotion or progression stage in some cases. The lowest effective dose for protection to occur was 0.005% ( - )-epigallocatechin gallate in the ENNG case and this dose is almost comparable with the daily intake of GTC in green tea drinkers. The mechanism(s) underlying how GTC inhibits carcinogenesis is not fully understood, but inhibition of ornithine decarboxylase and lipoxygenese activities, enhancement of Phase II enzymes, inhibition of oxidative stress induced by promoters or carcinogens, direct interaction between GTC and ultimate carcinogens, inhibition of promoter-induced protein kinase C, reduction of activating enzymes, and stimulation of immunity in the target organs may all play roles. In addition, some epidemiological data indicate a reduced risk of colon tumors and gastric cancers among populations with high levels of green tea consumption.      
1-O-2,3,5-Trimethylhydroquinone (HTHQ) is a strong phenolic antioxidant. In the medium term liver bioassay for the detection of hepatocarcinogens or hepatopromoters in F344 male rats, treatment with Glu- P-1 alone was associated with a significant increase in the number (per cm2 liver) and area (mm2 per cm2 liver) of preneoplastic GST-P-positive foci (47.5 ± 8.9 and 11.1 ± 4.7, respectively). Combined treatment with 1.0% HTHQ significantly reduced the number and area of GST-P-positive foci (to 8.1 ± 2.1 and 0.6 ± 0.2), almost to control level values without chemicals (3.6 ± 1.6 and 0.3 ± 0.1). HTHQ is therefore expected to be a selective potent chemopreventor which could reduce the carcinogenicity of heterocyclic amines such as Glu-P-1 (Fig. 4).


FIGURE 4 Quantitative analyses of the effects of antioxidants on Glu-P-1-induced GST-P positive foci in the medium term liver bioassay. HTHQ, 1-O-2,3,5-trimethylhydroquinone; GTC, green tea catechin. Significantly different from the Glu-P-1 group at ***P < 0.001.

Since chemopreventors exert their actions in different organs, in different stages of carcinogenesis, and dependent on the carcinogen, intake of different chemopreventors in combination may prove to be important for the prevention of human cancer.

VI. EVALUATION OF ANTIOXIDANTS AS HUMAN HAZARDS

There are many synthetic and naturally occurring antioxidants in our environment. Humans may ingest considerable amounts of such compounds in foodstuffs, medicines such as vitamins C and E, and γ- oryzanol, or by absorption through the skin of antioxidant additives in cosmetics, antiseptics, disinfectants, and industrial chemicals. It is therefore possible that these antioxidants may indeed play a role in human carcinogenesis. Although there are some epidemiological and case control studies suggesting that high intake of antioxidants such as ascorbic acid, α-tocopherol, selenium, -carotene, and vegetables that contain vitamins A, C, and E may lower the mortality rate for certain cancer types in humans, no such studies have been performed for phenolic antioxidants.     
For human risk assessment of phenolic antioxidant exposure, and extrapolation from experimental data, it is of importance to take into account the target organ, dose level, and route of administration. BHA is carcinogenic for the rat, hamster, and possibly mouse forestomach epithelium, but this activity is strictly limited to this tissue, and no carcinogenic potential for other squamous epithelia, such as those lining the esophagus and oral cavity, or for glandular stomach has been found. Since humans do not have a forestomach, it appears most likely that such limited forestomach carcinogens would lack effects on human gastric epithelium. Moreover, the threshold carcinogenic dose of BHA in rats is 2% in the diet (only a small incidence of benign papillomas was induced at lower dose levels), a level that is exceedingly high as compared with the possible human exposure. The estimated daily dietary intake of BHA was reported to be less than 7 mg/person in a Canadian study and therefore the carcinogenic dose in animals is nearly 10,000 times higher than the likely human exposure level.      
On the other hand, catechol, which is present in certain foods (e.g., fruits, vegetables, coffee), in tobacco, in cosmetics such as hair dye, in film developers, and in wood smoke, promotes glandular stomach carcinogenesis and induces adenocarcinomas in the rat glandular stomach, which is anatomically and biologically similar to human gastric epithelium at a dose of 0.8% in diet. Catechol at a dose of 0.16% for 2 years also caused glandular stomach adenomas to develop at low incidence. A 0.16% dose level is equivalent to 5-7.5 g catechol per person per day. The amount of catechol and its conjugates actually excreted in urine in humans was reported to be 1.1-30 mg/day, but although the carcinogenic dose in rats is 250-6250 times higher than the estimated human exposure, this chemical might still be a factor for enhancing human gastric cancer.      
Thus, the promotion potential of antioxidants may be far more important for human environmental carcinogenesis than any complete carcinogen action. Experiments have shown that effective enhancement can be achieved at much lower levels than the carcinogenic dose, and since antioxidants can exert promoting potential in various organs that are not necessarily targets for carcinogenicity, as shown in Table II, this must be taken into account. Moreover, clear synergistic effects regarding promotion have been reported; that is, combined treatment with 0.5% caffeic acid, 0.16% catechol, 0.5% BHA, and 0.25% 2-tert-butyl-4-methylphenol in rats for 51 weeks in rats pretreated with MNNG induced an 80% incidence of forestomach squamous cell carcinomas, whereas the individual treatments resulted in only 13-27% incidences. In a long-term carcinogenicity study, rats were treated with 0.4% caffeic acid, 0.4% sesamol, 0.16% catechol, 0.4% BHA, and 0.4% 4- methoxyphenol either alone or in combination for 104 weeks. Although papillomas were found in 0-15.8% of the individual treatment groups, the incidence increased to 42.9% with the combined treatment.      
Such synergistic or additive effects in carcinogenicity or promotion of carcinogenesis have been observed not only in the forestomach. Moreover, the carcinogenic or hyperplasiagenic activity of BHA was enhanced by concurrent treatment with sodium ascorbate or vitamin A, but was inhibited by concomitant treatment with diethylmaleate, a glutathionedepleting agent, or aspirin, and some antioxidants potentiate carcinogenicity of genotoxic carcinogens possibly through metabolic activation. NaNO2 also is a factor that can greatly modify carcinogenicity of phenolic compounds.       
Therefore antioxidant effects may be considerably altered by changes in environmental or physiological conditions. Endogenous factors such as age, immunological condition, and other diseases in target organs will also influence the effective dose for promotion of carcinogenesis or carcinogenicity. Available data thus indicate that low concentrations of carcinogens or promoters, even if they do not show activity per se, may indeed be important for human environmental carcinogenesis.

Nobuyoki Ito
Masao Hirose
Katsumi Imaida
Nagoya City University Medical School, Nagoya, Japan

See Also
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION ; CANCER RISK REDUCTION (DIET/SMOKING CESSATION/ LIFESTYLE CHANGES) ; CHEMOPREVENTION, PHARMACOLOGY OF ; MOLECULAR EPIDEMIOLOGY AND CANCER RISK ; NUTRITIONAL SUPPLEMENTS AND DIET AS CHEMOPREVENTIVE AGENTS

GLOSSARY
chemoprevention The primary prevention of carcinogenesis by chemicals. Any chemical agent that inhibits initiation, promotion, or progression, or more than one of these steps, is considered to be a chemopreventor.

forestomach The proximal part of the rodent stomach, situated between the esophagus and glandular part of the stomach and lined by squamous epithelium. Occupying about half of the stomach and separated from the glandular stomach by a limiting ridge, the forestomach is found in rats, mice, and hamsters.

initiation An irreversible alteration in the heritable material of target cells caused by carcinogens; this is considered the first step of the carcinogenesis process.

progression The process by which benign tumors or premalignant lesions progress to increasing malignant behavior.

promotion The process by which initiated cells grow to form benign tumors or precancerous lesions.

squamous cell carcinoma A malignant tumor originating from squamous epithelium and demonstrating squamous differentiation such as cornification and intercellular bridges. Characteristics include frequent invasion of adjacent tissues.

squamous cell papilloma A benign papillary tumor originating from squamous epithelium with fine or abundant connective tissue stroma. Structural and cellular atypia, or invasive growth, is absent.

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