Inhibition of microsomal activation of aflatoxin B1 by 3-dehydroretinol and 3-dehydroretinyl palmitate.

Two vitamin A2 compounds (3-dehydroretinol and 3-dehydroretinyl palmitate) which are predominantly present in fresh water fish have been found to be very effective in inhibiting the microsome catalysed formation of DNA adduct by the carcinogen aflatoxin B1. The inhibition appears to be due to modulation of microsomal enzymes which activate the carcinogen. Such inhibition may suggest a potential chemopreventive role of these compounds against carcinogenesis induced by aflatoxin B1.

Action of curcumin on the cytochrome P450-system catalyzing the activation of aflatoxin B1.

Curcumin, in a dose-dependent manner, inhibited the formation of covalent adduct between aflatoxin B1 and DNA, as catalyzed by microsomes or a reconstituted microsomal monooxygenase system. Its effect on the cytochrome P450-system was investigated in the latter system. The inhibition (50%) of aflatoxin B1-DNA adduct formation by curcumin in this system could be reversed by increasing the amount of cytochrome P450 but not by that of NADPH-cytochrome P450 reductase. Curcumin inhibited the reductase activity when measured by the reduction of cytochrome C but not when measured by the reduction of dichlorophenolindophenol, an artificial electron acceptor. These results, as well as the reversal of curcumin-induced inhibition of P450 reductase activity by higher amounts of cytochrome C, indicated a strong affinity of curcumin towards cytochromes. This was further substantiated from the observation that curcumin-pretreated cytochrome P450 had reduced ability to catalyze aflatoxin B1-DNA adduct formation in the reconstituted system. Curcumin, thus, may inhibit chemical carcinogenesis by modulating cytochrome P450 function.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of non-nutrient phenolic compounds on the catalytic activity of liver fractions.

Young adult rats were kept on a synthetic diet containing various food associated phenolic compounds each at 0.5% level. The ability of liver microsomes to catalyze reactions of aflatoxin B1 leading to its activation and DNA adduct formation was measured after an experimental feeding period of 3 weeks. A decrease in both activities was observed with several flavonoids (fisetin, kaempferol, morin, naringin and (+/-)catechin), phenolic acids (caffeic acid and chlorogenic acid), other phenolics (eugenol, vanillin) and synthetic phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene. Certain phenolics notably naringin, (+/-) catechin, eugenol, vanillin and butylated hydroxyanisole were also found to induce cytosolic glutathione S-transferase activity that stimulated the formation of specific aflatoxin B1-glutathione conjugate. The results emphasize the role of phenolic compounds in the activation and detoxification processes, and hence in modulating the carcinogenicity of aflatoxin B1.

Modulatory effects of essential oils from spices on the formation of DNA adduct by aflatoxin B1 in vitro.

Essential oils from common spices such as nutmeg, ginger, cardamom, celery, xanthoxylum, black pepper, cumin, and coriander were tested for their ability to suppress the formation of DNA adducts by aflatoxin B1 in vitro in a microsomal enzyme-mediated reaction. All oils were found to inhibit adduct formation very significantly and in a dose-dependent manner. The adduct formation appeared to be modulated through the action on microsomal enzymes, because an effective inhibition on the formation of activated metabolite was observed with each oil. The enzymatic modulation is perhaps due to the chemical constituents of the oils, and this could form a basis for their potential anticarcinogenic roles.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of riboflavin on the catalytic activity of liver fractions.

Weanling rats were kept on a synthetic riboflavin-free diet for 4 weeks, and subsequently on the same diet but supplemented with riboflavin for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. A decrease in both activities was evident during riboflavin deficiency, and this could be restored after normal supply of the vitamin. The decrease was attributed to a fall in the endogenous flavin content, specifically the coenzyme flavin adenine dinucleotide which forms an integral part of the microsomal monooxygenase that catalyzes the activation reactions. The vitamin and its coenzymes, however, inhibit the microsomal enzyme activity when added in excess in the in vitro system. It is envisaged that riboflavin may play a role in regulating the carcinogenic activity of AFB.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of copper on the catalytic activity of liver microsome.

Wealing rats were kept on a synthetic copper-free diet for 9-13 weeks and subsequently on the same diet but supplemented with copper at different levels for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of aflatoxin B1 was evident during deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from copper deficient rats as compared to rats maintained on a normal supply of copper. These activities of microsomes returned to normal after supplementation of the diet with a small level of copper. The results emphasize the role of copper in the activation, and hence in modulating the carcinogenicity of aflatoxin B1.

Modulation of flavocoenzyme levels in rat tissues by N-nitrosodiethylamine.

The hepatocarcinogen N-nitrosodiethylamine (NDEA) was administered continuously in drinking water (200 mg/l) to male Wistar rats, and at intervals tissues were examined for flavocoenzyme levels. Flavin adenine dinucleotide (FAD) and non-FAD riboflavin were determined fluorimetrically. Results revealed significant depletion in hepatic levels of these compounds at the end of 60 days, a period of exposure considered adequate for the development of hepatoma. Considerable reduction was also noticed after 15 days, and also when lower dose of NDEA was administered (50 mg/l). The flavin compounds in liver after short exposure to NDEA remained at depleted levels even after withdrawal of the carcinogen. Changes in the kidney were found to be less significant. The selective depletion of flavin compounds in the target tissue following NDEA exposure is of significance, and may be related to biochemical mechanisms involved in malignant transformation.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of vitamin A on the catalytic activity of liver fractions.

Weanling rats were kept on a synthetic vitamin A free diet for 9 weeks, and subsequently on the same diet but with oral supplementation of vitamin A palmitate at different doses for 2-3 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of AFB1 was evident during vitamin A deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from vitamin A-deficient rats as compared to rats maintained on normal supply of vitamin A. These activities of microsomes returned to normal after the rats had adequate supplements of vitamin A. The vitamin A status was also found to play a role in regulating glutathione S-transferase activity of the liver cytosol fraction, the activity being low in deficiency but increased progressively with increasing supplementation of vitamin A. Results suggest that vitamin A can afford protection against adverse effects of the potent hepatocarcinogen AFB.

Action of vitamin A on DNA adduct formation by aflatoxin B1 in a microsome catalyzed reaction.

The effect of vitamin A and some derivatives has been studied on the formation of DNA adduct by aflatoxin B1 (AFB1) in an in vitro reaction catalyzed by rat liver microsomes. Retinol, retinal, all trans retinoic acid and two retinyl esters were found to inhibit the adduct formation in a dose-dependent manner. The inhibition by retinol showed that it was prompt and could be reversed by increasing microsome concentration. Retinol also inhibited the formation of AFB1 adduct with microsomal protein. The inhibition of adduct formation with both DNA and protein was still apparent when repurified microsomes were used after pretreatment with retinol. Retinol inhibited adduct formation by virtue of its ability to interact with microsomal enzyme component(s), thereby interfering with the bioactivation of AFB1. The data are suggestive of a potential anticarcinogenic role for vitamin A against AFB1.

Modulation by certain factors of metabolic activation of aflatoxin B1 as detected in vitro in a simple fluorimetric assay.

Forty compounds belonging to various chemical groups have been tested for their ability to suppress metabolic activation of aflatoxin B1 (AFB1) mediated by rat liver microsome. Microsomal activation has been carried out in an in vitro system containing Tris-buffer at pH 7.2. Production of the reactive metabolite, AFB1 8,9-epoxide, has been measured by separation and detection of its hydrolysis product AFB1 8,9-dihydrodiol as the Tris-diol complex. The complex is separated using simple procedures of biphasic extraction and deproteinization, and detected by its characteristic fluorescence. Quantitation is made by direct comparison of its fluorescence with that of an authentic Tris-diol prepared synthetically. The method is rapid and proved to be highly sensitive and reproducible. A large number of compounds have been observed to modulate at varying degrees the activation of AFB1 in this in vitro system. Many compounds have been tested at several concentration ranges and inhibition curve is constructed in each case from which ID50 values, i.e., the dose needed to bring about 50% inhibition can be obtained. These values expressed as nmol afford a direct and realistic comparison of the inhibitory potential of various modulators. Factors having great inhibitory potential have been identified as retinoids (retinol, retinal, retinoic acid, retinyl acetate), beta-carotene, riboflavin, ascorbic acid, copper, zinc, linoleic acid, p-hydroxy benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, disulfiram, and phenothiazine. Several other compounds have shown moderate inhibitory potential. The strong inhibition on Tris-diol formation by several vitamins, antioxidants and trace metals shows similarity with their effect on AFB1-DNA adduct formation. It is suggested that these agents may have potential anticarcinogenic activity against AFB1.

Factors modulating the formation of DNA adduct by aflatoxin B1 in vitro.

Forty-two compounds belonging to various chemical groups have been tested for their ability to suppress formation of aflatoxin B1--DNA adduct mediated by microsome in vitro. While many of these compounds have either marginal or no modulating effect, some have been identified as effective inhibitors. The strong inhibition of DNA adduct formation by retinoids (retinol, retinal, retinoic acid and retinyl acetate), riboflavin, riboflavin 5'-phosphate, flavin adenine dinucleotide, Cu2+, 7,8-benzoflavone, disulfiram, butylated hydroxyanisole, butylated hydroxytoluene and phenothiazine suggests that these agents may have potential anticarcinogenic activity against aflatoxin B1.