Functional Foods for Disease Prevention II - American Chemical Society

Chapter 14

Garlic and Associated Allyl Sulfur Constituents Depress Chemical Carcinogenesis

Downloaded by COLUMBIA UNIV on July 16, 2013 | http://pubs.acs.org Publication Date: August 21, 1998 | doi: 10.1021/bk-1998-0702.ch014

J. A. Milner and E. M. Schaffer Nutrition Department and Graduate Program in Nutrition, The Pennsylvania State University, University Park, PA 16802

Increased intake of garlic and related Allium foods is proposed to offer protection against cancer. The ability of water-extracts of garlic and onion, but not leeks, to block spontaneous nitrosamine formation suggests all Allium foods are not equal in their anticarcinogenic properties. Likewise, the depression in nitrosamine formation resulting from S-allyl cysteine (SAC) treatment, but not diallyl disulfide (DADS), suggests not all allyl sulfur compounds can be assumed equal. Nevertheless, SAC and DADS are both effective in blocking nitrosamine bioactivation. The ability of dietary garlic powder supplements to inhibit 7,12 dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis and DNA adducts suggest this food has widespread anticarcinogenic properties. The composition of the diet can markedly modify garlic's ability to reduce chemical carcinogenesis. Alterations in phase I and II enzymes, including prostaglandin synthase, may explain some of the anticarcinogenic properties of garlic. Collectively, the ability of garlic and associated allyl sulfur compounds to depress carcinogen formation and bioactivation is consistent with epidemiological evidence indicating that higher intake of Allium foods is accompanied by a reduction in the risks of some cancers. Cancer, once thought an inevitable consequence of aging, is now considered by many to reflect cellular reactions to a myriad of environmental factors, including dietary habits. The connection between cancer risk and dietary habits is unquestionably complex and reflects dynamic interactions occurring among essential and non-essential dietary components. Despite the plethora of both positive and negative nutrient interactions, it is estimated that approximately 30 to 60% of all cancers relate to dietary habits (7). Not surprisingly, the intake of some foods correlates with an increase in risk, while others appear to provide some degree of protection. One of the food groups whose intake is associated with reduced cancer risk comesfromthe genus Allium (2,5). While, these foods (which

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©1998 American Chemical Society In Functional Foods for Disease Prevention II; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

145 include garlic, onions, leeks, and chives) have been used for centuries for their antimicrobial properties, their ability to repel insects, and their characteristic aroma and flavor, they have only recently received significant attention as naturally cwurring sources of anticarcinogenic and antiturnorigenic agents. This review will focus on some of the more recent epidemiological and laboratoryfindingsabout the anticarcinogenic properties of garlic and related foods.


Epidemiological Evidence that Allium Foods Inhibit Cancer Epidemiological evidence, although limited, provides clues about the potential health benefits associated with increased Allium food consumption. Several studies have observed that increased garlic consumption correlates inversely with cancer mortality (4-8). Mei et al. (8) reported that individuals consuming 20 g garlic per d had about 10% of the risk of gastric cancer mortality of those consuming 2 g per d. Recent information suggests the anticancer benefits of garlic may accrue at substantially lower intakes (6). In the study of Steinmetz et al. (6) the intake of garlic, about 0.6 g per week, was the only food item among 127 evaluated that correlated with a reduction in colon cancer risk in the almost 42,000 women examined. It remains to be determine why some epidemiological studies, but not all (9-11), provide evidence that garlic and related foods have anticarcinogenic properties. Some of the discrepancy among studies may relate to the interaction of garHc with other dietary components. Amagase et al. (12) observed that the dietary intake of protein, methionine, lipid, vitamin A and selenium could significantly modify the ability of garHc to reduce the bioactivation of the mammary carcinogen 7, 12 dimethylbenz(a)anthracene (DMBA). Whether or not these are other dietary interactions account for some of the variability in cancer risk in humans remains to be detenriined. Garlic and Nitrosarnine Biosynthesis GarHc has long been touted for its antimicrobial properties. Water-extracts of garHc are recognized to possess broad-spectrum activity against bacteria and fungi (13). This antimicrobial property may account for the reduced bacterial and fungalmediated synthesis of nitrite and ultimately the formation of carcinogenic nitrosamines in studies by Mei et al (14). However, it appears that the abiHty of garHc to reduce nitrosarnine formation may occur independent of its antimicrobial properties. Dion et al. (75) found that a water-extract of several sources of garHc suppressed the spontaneous (chemical) formation of nitrosamines (Figure 1). Since most nitrosamines are recognized for their carcinogenic potential, this effect alone may significantly influence human cancer risk. While the mechanism by which a water-extract of garlic leads to this effect remains largely unexplored, it is possible as suggested by Shenoy and Choughuley (16) that it relates to the formation of Snitrosothiols. Various Allium foods have the propensity to suppress spontaneous nitrosarnine formation (75). However, not aU are equivalent since garHc and onions, but not leeks, were effective in blocking the spontaneous formation of nitrosomorpholine from its precursors (Figure 1). S-aUyl cysteine (SAC) a watersoluble sulfur compound but not diaUyl disulfide (DADS), a Hpid-soluble aUyl sulfur compound, was found to suppress spontaneous nitrosomorphoHne (NMOR) formation (75). Both SAC and DADS were effective in reducing the formation of revertants in a Hver microsomal activated Salmonella typhimurium TA100 assay to which NMOR was added. While the addition of 7 u^moles SAC per plate reduced NMOR mutagenicity by 22%, adding as Httle as 0.03 pmoles DADS was accompanied by a 69% decrease in its mutagenicity. While DADS was considerably

In Functional Foods for Disease Prevention II; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


146 more effective than SAC in blocking the mutagenicity of NMOR, both were more effective than isomolar ascorbic acid (75). Collectively, these investigations demonstrate that garlic and related sulfur compounds can inhibit carcinogenesis by at least two mechanisms, namely suppressing carcinogen formation and inhibiting carcinogen bioactivation. The abilty of a water extract of garlic to suppress the mutagenicity of nitrosamines is consistent with previous observations that dietary garlic supplementation retards liver DNA adducts caused by treatment with preformed nitrosamines (77). This suppression in adducts likely reflects a suppression in carcinogen bioactivation possibly attributable to altered cytochrome p450 activity. Brady et al. (18) found that diallyl sulfide (DAS), another oil-soluble allyl compound from processed garlic, could inactivate cytochrome p450 2E1 (CYP2E1). The metabolism of chlorzoxazone has been used as an in vivo biomarker for CYP2E1 activity (79). Recent studiesfromour laboratory reveal that dietary intake of garHc (2%), SAC (57 mmol/kg) and DADS (57 mmol/kg) decrease the hydroxylation of chlorzoxazone (20). A depression in cytochrome p450 may explain the abiHty of DAS to completely inhibit N nitrosomethylbenzylamine-induced esophageal tumors (27). Increased glutathione and glutathione-S-transferases (factors involved in phase II detoxification) also has been observed foUowing the consumption of processed garHc powder in experimental animals (22). While the response in phase II enzymes and substrates likely is highly dependent on the quantity of garHc or associated constituents consumed, these changes may contribute significantly to the anticarcinogenic properties of garHc. Garlic Blocks Action of Other Carcinogens The protection offered by garHc against chemicaUy induced tumors is not limited to nitrosamines. Several studies have shown that garHc and its associated sulfur compounds are effective in blocking the incidence of tumors in experimental animals treated with polycycHc hydrocarbons, such as 7,12 dimemylbenz(a)anthracene (DMBA) (23), and alkylating compounds, such as 1,2 dimethylhydrazine (24). Topical appHcation of garHc o5 was shown several years ago to substantiaUy reduce skin cancers DMBA treated mice (25). Studies from our laboratory show that the occurrence of DMBA induced DNA adducts in mammary tissue 24 h after carcinogen treatment is highly correlated with ultimate tumor incidence (23). Thus, alterations in these DNA adducts can serve as a sensitive biomarker of the impact of dietary constituents on DMBA carcinogenesis. Studies from our laboratory reveal that both SAC and DADS markedly inhibit the occurrence of DNA adducts and the carcinogenicity of DMBA (23, 26, 27). This protection results from biological effects occurring during both the initiation and promotion phases of carcinogenesis (23). GarHc preparations likely vary in the amount of specific organosulfur compounds. Part of this variation may be attributable to the growing conditions. Thus, not aU garHc sources can be assumed to be equaUy effective blockers of chemicaUy induced tumors in experimental animals, or presumably in humans. The quantity of SAC occurring in various garHc preparations was found to correlate with their abiHty to inhibit the in vivo formation of DMBA-DNA adducts (26). Nevetheless, in addition to aUyl sulfur compounds constituents such as arginine, polysaccharides, saponins and/or selenium likely influence the anticarcinogenic potential of garlic. Studies by Ip et al. (28) reveal that variation in the content of selenium in garHc can markedly influence its abiHty to influence DMBA carcinogenesis. While additional attention is needed to evaluate the influence of processing on the abiHty of garHc and related constituents to inhibit the cancer



147 process, studies from our laboratory suggest that crushed and dehydrated garlic, commercially processed high sulfur garlic and commercially processed deodorized garlic are all effective in reducing the binding of DMBA to mammary cell DNA (26). Clearly, our studies demonstrate that odor is not a necessary prerequisite for the ability of garlic preparations to inhibit chemically induced DNA adducts or tumors (23, 26, 27). As indicated previously several components of the diet can influence the protection provided by garlic against the cancer process. Schaffer et al. (27) and Ip et al. (28) have shown that providing selenium either as a constituent of the garlic or as a separate supplement can enhance the protection provided by garlic. It remains to be determine what metabolic changes account for this combined benefit. Studies from our laboratory show that glutathione S-transferase and UDPglucuronosyltransferase activities were enhanced by dietary selenite supplementation, while the garlic, SAC or DADS do not significantly affect activities of these enzymes (27). SAC and DADS may be modifying phase I and II enzymes other than those influenced by selenium intake. Our laboratory has also shown that the quantity and type of lipid consumed by rats can also influence the ability of garlic to depress DNA adducts in mammary tissue resulting from treatment with DMBA (Figure 2) (12). Inclusion of 2% deodorized garHc in the diet inhibited DMBA-induced mammary DNA adducts by approximately 50% when the diet contains 20% com oil, but has virtuaUy no effect when the diet contains 5% com oil (12). The impact of Hpid intake likely relates to the intake of specific fatty acids. Recent studies from our laboratory have shown that while dietary linoleic acid and oleic acid supplementation can markedly enhance the presence of mammary ceU DNA adducts following treatment with DMBA, a similar effect is not seen when palmitic acid is provided (29). Inclusion of garHc in the diet has the greatest inhibitory effect on DMBA carcinogenesis when the diet contains high Hnoleic acid than high amounts of oleic acid (30) (Figure 3). Bioactivation of benzo[a]pyrene, another polycycHc aromatic hydrocarbon, is known to be mediated by prostaglandin synthase (PHS) (31). We undertook studies to determine if PHS completes DMBA bioactivation and if the garHc constituents SAC and/or DADS might alter this reaction (Figure 4). Indeed, PHS was found to mediate the formation of DNA adducts when 3,4-DMBA-cHhya^cdiol was incubated with PHS, arachidonic acid and calf-thymus DNA (Figure 5). Indomethacin is a recognized inhibitor of PHS activity (32). Interestingly, the addition of DADS or SAC to the incubation media in the present studies was also found to significantly inhibit the PHS mediated formation of DNA adducts in vitro (Figure 5). DADS and SAC were approximately 20 and 45% less effective on a molar basis than was indomethacin in suppressing the bioactivation of 3,4-DMBAdihydrodiol. The present studies suggest PHS activity may represent a key site of action of garlic and related sulfur compounds in blocking DMBA carcinogenesis. At least one other mechanism may account for the anticarcinogenic properties of garlic and related allyl sulfur compounds. Supplemental dietary garHc has been found to significantly reduce adducts caused by the treatment with methylnitrosurea, a carcinogen that does not require metaboHc activation. These results raise the possibility that enhanced DNA repair may also be occurring following increased garHc consumption (17). More recent studies have demonstrated that feeding garHc powder (2%), SAC (57 nimol/kg) or DADS (57 mmol/kg) are aU effective in decreasing the incidence of mammary tumors resulting from treatment with this carcinogen (76, 41, and 53% inhibition, respectively) in a 23 week feeding study (33).


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Allium Extract (mg/mL) Figure 1. Effect of water extracts of garlic, elephant garlic, leeks, onions and deodorized garlic on the spontaneous formation of N-nitrosomorpholine. Control incubations resulted in the formation of 7.4 mM NMOR after 1 h. Values are means ± SEM of 3 observations per treatment. Values occurring following addition of garlic, elephant garlic or onion extracts differ signficantly from control values (P

Functional Foods for Disease Prevention II - American Chemical Society

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