Plant Phenolic Compounds as Inhibitors of Mutagenesis and

Chapter 4

Plant Phenolic Compounds as Inhibitors of Mutagenesis and Carcinogenesis

Downloaded by GEORGE MASON UNIV on June 24, 2014 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch004

Harold L. Newmark Laboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0789 Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 It has been known for decades that at least a few types of human cancer are related to substances in our environment, i.e. the chemical composition of our food, drink, atmosphere, as demonstrated by defined tests for mutagenicity and carcinogenicity. Attention has recently focussed on substances in the environment that act as antimutagens (desmutagens), or protective against carcinogenesis. Plant phenolics, originally hypothesized to inhibit mutagenesis and/or carcinogenesis by virtue of antioxidant or electrophile trapping mechanisms, can also act as modulators of arachidonic metabolism cascade pathways. Certain plant phenols can be effective inhibitors of chemical mutagens, in vitro, and/or carcinogenesis in vivo. The historical origins, hypothesis of actions, current status and potential adverse effects of the utility of plant phenolics to reduce risk of cancer are discussed, as well as future possibilities and needs and objectives for future research. It has been known for several decades that there are substances in commonly consumed foods that reduce the incidence of chemically induced carcinogenesis in laboratory rodents. In early pioneering studies, Wattenberg found that rodents on a purified or semi-synthetic diet developed more chemically-induced lesions than on a mixed "natural food" diet. Further studies elucidated many active "chemopreventative" substances in foods, including terpenes, aromatic isothiocyanates, organosulfur compounds, protease inhibitors, dithiolthiones and indoles (1-3). Of particular interest as chemopreventative agents were the monophenols, polyphenols, flavones, flavonoids and tannins in foods, which may be consumed in large quantities (up to 1-2 grams per day) in some human diets. The Antioxidant Hypothesis. Wattenberg found several food antioxidants, such as butylated hydroxyanisole (BHA), reduced the incidence of neoplasia induced by some carcinogens in laboratory animals, and expanded the studies to show similar effects for the plant phenolics caffeic and ferulic acids (4). The mechanism for B H A

0097-6156/92/0507-0035$06.00/0 © 1992 American Chemical Society In Phenolic Compounds in Food and Their Effects on Health II; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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inhibition of neoplasia was ascribed to alteration of carcinogen metabolism towards inactive products, but no explanation was offered specifically for the tumor inhibitory activities of caffeic and ferulic acids. Inhibition of Nitrosation. In the mid 1970's Dr. W J . Mergens and I found that caffeic and ferulic acids were highly effective consumers of nitrite ion, particularly in acid pH (5). This results in strong activity of these plant phenols, commonly present in many human foods, in preventing nitrosation of susceptible secondary amines and amides to form highly potent carcinogenic nitrosamines and nitrosamides in vitro, in our foods, and in vivo (6). Sources of nitrites in foods are almost ubiquitous, particularly in fermented or smoked foods, or added as aids to preservation, as in processed meats. In addition, nitrates naturally in our foods are readily recycled to the saliva after ingestion and absorption, and then reduced to nitrite by buccal flora, resulting in gastric nitrosation of susceptible amines. The function of dietary plant phenolics in blocking these reactions in foods in food processing and cooking and in vivo has probably been underrated as a major cancer prevention process. Electrophile Radical T r a p Hypothesis. The current axiom of chemical carcinogenesis is that many, perhaps most, carcinogens are converted by either nonenzymatic (in the case of direct acting carcinogens) or metabolic activation to highly reactive species that can attack cellular components. The best known form of the reaction species is the electrophilic reactant, possessing a positively charged group such as a carbonium ion, and which reacts with electron-rich moieties chemically termed nucleophiles. Many cellular components can be targets for such electrophilic attack, but the (probably minor) attack and resultant chemical and structurally alteration of D N A is believed to be a key step in carcinogenic initiation in the cells. Protection of the D N A in the cells is largely achieved by competitive efficient chemical nucleophiles in the cell such as glutathione, however, this protection can be overwhelmed. Prodded by Dr. Allan Conney to consider additional methods of increasing protection of cellular D N A from activated carcinogen electrophilic attack, we realized that some plant phenolics, such as caffeic and ferulic acids could act as potent chemical nucleophiles, based on our previous studies of their reaction with nitrite (5). On testing as inhibitors of mutagenesis in vitro induced by benzo[a]pyrene diol epoxide, these plant phenolics were indeed found to be potent, particularly the related ellagic acid. The mechanisms of reaction, involving π bond interactions between the planar molecules involved, and capacity to act as electron-rich donors (i.e. electrophilic trap for electron-poor carcinogenic electrophiles) was reported in a series of papers by Wood, Huang, Chang, Sayer, Jerina, Conney, Newmark, and others as reviewed by Newmark (7,8). Thus, plant phenolics may be inhibitors of initiation processes in carcinogenesis. Arachidonic Metabolism Modulation. It has long been known that several plant phenolics such as salicylic acid, quercetin and others can inhibit the cyclooxygenase pathway of arachidonic acid metabolism to prostaglandins. Some plant phenolics also inhibit lipoxygenase pathways to other prostanoids (9,10). Kato et al., in their demonstration of inhibition of phorbol ester promotion of mouse skin tumors by quercetin suggested the possible involvement of lipoxygenase inhibition (77). Indeed, several inhibitors of lipoxygenase pathways of arachidonic acid metabolism in mammalian cells, as well as cyclo-oxygenase inhibitors have demonstrated anti-tumor activity (72). Arachidonic acid metabolism modulation appears to affect promotion

In Phenolic Compounds in Food and Their Effects on Health II; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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rather than initiation processes in carcinogenesis. Plant phenolics as modulator of arachidonic metabolism (e.g. as lipoxygenase inhibitors) can act as inhibitors of carcinogenic promotion processes.


Alteration of Carcinogen Metabolism. The mechanism of inhibition of chemically induced carcinogenesis by B H A , and a few other food phenolic antioxidants has been related to altered metabolism of certain carcinogens, including: diminished microsomal metabolism to DNA-binding metabolites, decreased epoxidation, increased formation of readily conjugated and excreted metabolites, and enhanced activity and level of G S H and glucuronide conjugating enzymes (4). However, the plant food phenolics have not been extensively studied for activity in altering metabolism of carcinogens, except to suggest a wide range of potency (4). C u r r e n t Status. The hypotheses discussed above gave rational cause for experimentatial investigation of inhibitory effects of plant phenolics as inhibitors of mutagenesis (anti-mutagens or desmutagens) and carcinogenesis. With the information currently available, some of the original hypotheses seem less plausible and useful for further studies. The electrophile trap hypothesis is less attractive. The originally promising studies of anti-mutagenic activity were later shown to be partly dependent on reactions in vitro of the phenolics with the tested mutagen (often benzo[a]pyrene) outside the cell, before cell entry (Chang, R., Rutgers University, personal communication.) Also the phenolics appear highly reactive within the cells in a variety of functional systems and determination of specificity of effective phenolic amounts in reaching a tissue, entering the cells, and performing a useful tumor inhibitory function, with adequate safety to normal cell function, will probably require much further study. Most of the hypotheses above have some current applicability, but a uniform single mechanism is unlikely, since the plant phenolics appear to have a range of biochemical activities in the cells. Although each plant phenolic component will be somewhat specific in actions, tissue localization, and effects on stresses induced by initiation (e.g. genotoxic) as well as promotion (non-genotoxic) carcinogens, it seems more likely that plant phenolics, being multifunctional, can inhibit carcinogenesis by several activities simultaneously. Many plant phenolics have been shown to be effective as antimutagens, particularly against activated aromatic carcinogens, somewhat less active against nonaromatic carcinogens (7,8). Several have shown moderate to strong activity as inhibitors of neoplasia development by chemical carcinogens in laboratory rodents. Problems and Adverse Effects. In a series of studies, Ito and co-workers have shown that caffeic acid (2% of diet), sesamol (2% of diet) and catechol (0.8% of diet) could induce stomach cancer in rodents. However, there is a difference in species sensitivity, rats being more sensitive than mice (13). These effects seem to derive from a gastric mucosal hyperplasia stemming from irritation by the chronically ingested dietary phenolics in the studies. For decades it has been known that phenols, especially ortho dihydroxy phenols (catechols), can readily oxidize in highly aerobic exposure. Trace metal ions such as copper, or iron present act as potent catalysts for oxidation of phenols in vitro where rate increases as the pH rises. The oxidation reaction produces hydrogen peroxide via an intermediate superoxide. Thus, when phenolic substances are tested for "mutagenicity" in vitro in an Ames-type assay, in highly aerobic conditions, and appreciable trace metals are present in the media, it is no surprise that the phenols appear to test positive as mutagens. This is probably largely the "mutagenicity" of the



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hydrogen peroxide produced in the media by the conditions of testing. In several instances, such as the apparent mutagenicity of caffeic acid, addition of catalase enzyme to the in vitro system virtually eliminated clastogenic activity, emphasizing the role of artifactual generation of hydrogen peroxide in laboratory tests for mutagenicity of plant phenolics (14). A re-evaluation of the literature of apparent mutagenicity of plant phenolics to eliminate artifactual errors due to hydrogen peroxide formation would probably find most of these phenolics free of mutagenic activity. Hydrogen peroxide formation in the dietary systems used in testing may also be partly responsible for promotion of stomach cancer in rats (13). Plant phenols, particularly high molecular weight polyphenols such as the gallotannins, can precipitate proteins by physico-chemical interactions. In higher concentrations in food products, such as strong black coffee without added milk as a neutralizing protein source, this can be a source of chronic gastric irritation. Future Possibilities. A newly emerging area is the endogenous production of phenolic lignans (diphenolic compounds). These are produced from plant precursors (probably plant phenolics) through modification by the colon microflora, possibly the Clostridia group (75). The two most common mammalian lignans are enterolactone and enterdiol. In limited studies, they appear to have tumor inhibitory properties, particularly as anti-estrogens. These lactones were quantified in human urine in subjects with a varied large range of high fiber cereal diets. Linseed (flaxseed) in the diet gave particularly high levels of urinary lignans (76). Recent studies seem to confirm these early reports (Thompson, D . , University of Toronto, private communication.) This approach may be of practical use in reducing mammary and possibly colon cancer risk in human studies. Tyrosine kinase and other protein kinases are enzymes involved in cell proliferation. Plant phenolics could be useful dietary inhibitors of these kinases, and act to reduce hyperproliferation of epithelial cells as a means of reducing cancer risk. Quercetin is an inhibitor of protein kinase C, tyrosine protein kinase and a specific protein kinase in rat colonic epithelium (77). Needs and Objectives for Future Research. I wish to emphasize that plant phenols have multifunctional biochemical activities, as illustrated by quercetin in Table I. Most of these involve modulation of one or more processes thought to be involved in carcinogenesis development. While substances may be chosen as candidates for chemoprevention of carcinogenesis based on a single hypothesized mechanism, probably several activities are involved, adding to the total anticarcinogenic potential. Table I. Multifunctional Activity of Phenolics: Quercetin Antioxidant:

Lipids

Antimutagen:

P A H electrophiles

Anti-prostanoid:

Lipoxygenase inhibitor

Anti-kinases:

Inhibitor of tyrosine protein and other kinases

Plant phenolics, components of human foods, have shown interesting activities as inhibitors of mutagenic and carcinogenic processes. In order to utilize these properties for chemoprevention for reduction of risk for human cancer, much further



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work is needed. This includes further extension of anti-cancer studies, but also fundamental studies in allied areas, including: 1. Reliable food composition data of amounts of specific phenolics in fresh foods, and losses in processing, storage, etc. to get realistic estimates of dietary intake. 2. Absorption and metabolism. Little is known about the fate of most plant phenolics after ingestion. Rutin and quercetin are poorly absorbed (18) while caffeic acid appears well absorbed, but only one-fifth identified as urinary metabolites (79) in human studies. However, these studies were performed with pure crystalline substances, while in foods the substances are usually present as glycosides or esters, or in solution in the terpene-lipid components of the foods. 3. Cellular reactions of the plant phenolics with mammalian tissues, including mode and chemical form of delivery to target tissues, effects on cell membranes, attention of cytosolic enzymes and activation systems. Of theoretical interest would be information on the effects of individual plant phenolics on specific cytochrome P450 systems, and resultant effects on detoxification activities towards endogenous and xenobiotic substances.

Literature Cited. 1. Wattenberg, L.W. Cancer Res. 1985, 45, 1-8. 2. Wattenberg, L.W. Proc. of the Nutrition Soc. 1990, 49, 173-183. 3. Hartman, P.E.; Shanekl, D.M. Env. and Mol. Mutagenesis, 1990, 15, 145-182. 4. Wattenberg, L.W.; Coccia, J.B.; Lam, L.K.T. Cancer Res. 1990, 40, 28202823. 5. Newmark, H.L.; Mergens, W.J. In: Inhibition of Tumor Induction and Development; Zedeck, M.S.; Lipkin, M., Ed.; Plenum Press: New York, NY, 1981; pp 127-168. 6. Kuenzig, W.; Chan, J.; Norkus, E.; Holowaschenko, H.; Newmark, H., Mergens, W.; Conney, A.H. Carcinogenesis 1984, 5, 309-313. 7. Newmark, H.L. Nutr. Cancer 1984, 6, 58-70. 8. Newmark, H.L. Can. J. Physio. Pharmacol. 1987, 65, 461-466. 9. Dehirst, F.E. Prostaglandin 1980, 20, 209-214. 10. Baumann, J.; Wuma, G.; Bruchhausen, F. Arch. Pharm(Weinheim). 1980, 313, 330-337. 11. Kato, R.; Makadate, S.; Yamamoto, S.; Sugimura, T. Carcinogenesis 1983, 5, 1301-1305. 12. Karmali, R.A. In: Biochemistry of Arachidonic Acid Metabolism; Lands, W.E.M., Ed.; Martinas Nijhoff Publishing: Boston, MA; pp 203-212. 13. Hirosi, M.; Fukushima, S.; Shirai, T.; Hasegawa, R.; Kato, T.; Tanaka, M.; Asakawa, E.; Ito, M. Jpn. J. Cancer Res. 1990, 81, 202-212. 14. Hanham, A.F.; Dunn, B.P.; Stich, H.F. Mutation Res. 1982, 116, 333-339. 15. Adlercreutz, M . Scand. J. Lab. Invest. 1990, 50, Suppl 201, 3-23. 16. Horwitz, C.; Walker, A.P.R. Nutr. Cancer 1984, 6, 73-76. 17. Schwartz, B.; Fraser, G.M.; Levy, J.; Sharoni, Y.; Guberman, R.; Krawiec, J.; Lamprecht, S.A. Gut 1988, 29, 1213-1221. 18. Gugler, R.; Leschick, M., Dengler Europ. J. Clin. Pharmacol. 1975, 9, 229234. 19. Jacobson, A.E.; Newmark, H.; Baptista, J.; Bruce W.R. Nutri. Reports Int. 1983, 28, 1409-1417. RECEIVED

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Plant Phenolic Compounds as Inhibitors of Mutagenesis and

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