A range of potential chemoprotective agents, most of them natural dietary constituents, has been examined for ability to modulate both phase I (cytochrome P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A, 4A) and phase II drug metabolizing enzymes (glutathione S-transferases, in particular subunits Yc2 and P, aflatoxin B1-aldehyde reductase and quinone reductase) in rat liver. In addition to assays of total enzyme activity and Western blots for individual isozymes, the ability of microsomes to metabolize aflatoxin B1, and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to GSH and to produce AFB1-dialcohol, were measured. Induction of gamma-glutamyl transpeptidase activity was examined by histochemistry. Differing patterns of induction were observed, reflecting differences in the control of expression of the individual enzymes studied. Of the compounds examined, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol and phenethyl isothiocyanate were the most potent bifunctional agents (inducing both phase I and II activities). Oltipraz, while only weakly inducing CYP1A2 and 2B1/2, was a potent inducer of phase II enzymes. Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce phase II enzymes. 4-Methyl catechol, alpha-tocopherol and red wine decreased certain phase I enzyme activities, while inducing total GST activity. Butylated hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz, phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if administered prior to AFB1. While these studies indicate the relative contributions of phase I and II metabolism in the overall protective effect in rat, care should be taken that a similar balance is achieved in man, and that relevant enzymes or iso forms are induced.

Certain dietary constituents can protect against chemically induced carcinogenesis in rodents. A principal mechanism by which these chemopreventive compounds exert their protective effects is likely to be via induction of carcinogen detoxification. This can be mediated by conjugation with glutathione, which is synthesized by the sequential actions of glutamate-cysteine ligase (GLCL) and glutathione synthetase. We have demonstrated that dietary administration of the naturally occurring chemopreventive agents, ellagic acid, coumarin or alpha-angelicalactone caused an increase in GLCL activity of between approximately 3- and 5-fold in rat liver. Treatment with the synthetic antioxidant ethoxyquin or the classic inducer phenobarbital caused < 2-fold induction of GLCL activity in rat liver, which was not found to be significant. The increases in GLCL activity were accompanied by increases (between 2- and 4-fold) in levels of both the catalytic heavy subunit (GLCLC) and regulatory light subunit (GLCLR). No substantial induction of GLCL was observed in rat kidney. The glutathione S-transferase (GST) subunits A1, A3, A4, A5, P1 and M1 were all found to be inducible in rat liver by most of the agents. The greatest levels of induction were observed for GST P1, following treatment with coumarin (20-fold), alpha-angelicalactone (10-fold) or ellagic acid (6-fold), and GST A5, following treatment with coumarin (7-fold), alpha-angelicalactone (6-fold) and ethoxyquin (6-fold). Glutathione synthetase was induced approximately 1.5-fold by coumarin, alpha-angelicalactone, ellagic acid and ethoxyquin. The expression of glutathione-related enzymes was also examined in preneoplastic lesions induced in rat liver by aflatoxin B(1). The majority of gamma-glutamyltranspeptidase (GGT)-positive preneoplastic foci contained increased levels of GLCLC relative to the surrounding tissue. This was usually found to be accompanied by an increase in GLCLR. Cells in the inner cortex of rat kidney were found to contain the highest levels of both GLCLC and GLCLR. The same cells showed the strongest staining for GGT activity.

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