Arthur T. Fong

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Promotion of aflatoxin B1 hepatocarcinogenesis in rainbow trout by 17β-estradiol

Abstract Four groups of 21-day-old rainbow trout ( Oncorhynchus mykiss ) embryos were exposed to static solutions of water containing 0, 0.005, 0.025, or 0.125 ppm aflatoxin B 1 (AFB 1 ) for 30 min to initiate hepatic neoplasms. Six weeks after AFB 1 exposure, two groups at each dose level were subjected to dietary 17 β-estradiol (E 2 ) treatment, while the two remaining groups received control diet (CD) only. E 2 promoted the hepatic tumor incidence in the fish exposed to the 0.025 ppm subcarcinogenic dose of AFB 1 from 0% to 9%, and enhanced the incidence in the fish exposed to the 0.125 ppm dose of AFB 1 from 5% to 60%. E 2 alone was not carcinogenic under the present experimental conditions. This promotion of AFB 1 carcinogenesis was observed despite significant mortalities and reduction in growth in the E 2 -exposed fish. In a second experiment, 21-day-old embryos were exposed to 0.025 ppm AFB 1 or sham-treated as before. Duplicate groups were started on either CD or CD-containing 5, 10 or 15 ppm E 2 . At each level, E 2 diets were fed every other week, alternating with CD, throughout the experiment. This regimen again produced promotion of AFB 1 carcinogenesis by E 2 . The intermittent exposure to E 2 still resulted in significant growth depression, but E 2 -related mortalities were eliminated. Biochemical studies revealed that E 2 stimulated hepatic ornithine decarboxylase (ODC) activity and DNA synthesis, both indicators of cellular proliferation which may be related to promotional events.

Modulation of diethylnitrosamine-induced hepatocarcinogenesis and O6-ethylguanine formation in rainbow trout by indole-3-carbinol, β-naphthoflavone, and Aroclor 1254

Rainbow trout were fed a diet containing indole-3-carbinol (2000 ppm), beta-naphthoflavone (500 ppm), or Aroclor 1254 (100 ppm) for 6 weeks before a single 24-hr exposure to an aqueous solution of 250 ppm diethylnitrosamine (DEN). The fish were killed 42 weeks later to determine the carcinogenic response. DEN exposure produced an 80.2% incidence of liver tumors and an average of 3.47 tumors per tumor-bearing fish, whereas no tumors were detected in the sham-treated control fish. Tumor induction was inhibited by indole-3-carbinol (27.5% incidence, 1.89 tumors per tumor-bearing fish) but enhanced by beta-naphthoflavone (91.8% incidence, 3.60 tumors per tumor-bearing fish). Aroclor 1254 had no effect on DEN-induced hepatocarcinogenesis (80.0% incidence, 3.03 tumors per tumor-bearing fish). The effects of these modulators on O6-ethylguanine and 7-ethylguanine formation (measured by HPLC and fluorescence spectrophotometry) were examined. Liver DNA ethylguanine levels were reduced in indole-3-carbinol-pretreated fish and increased in beta-naphthoflavone-pretreated fish compared to untreated controls after DEN exposure. Aroclor 1254 pretreatment had no significant effect on DNA ethylguanine formation. Similar O6-ethylguanine to 7-ethylguanine ratios were found among the control and treated groups. The results of this study indicate that modulation of DEN hepatocarcinogenesis by indole-3-carbinol and beta-naphthoflavone may be mediated by their effects on O6-ethylguanine formation and, therefore, on the initiation phase of carcinogenesis.

Formation and persistence of ethylguanines in liver DNA of rainbow trout (Salmo gairdneri) treated with diethylnitrosamine by water exposure

Diethylnitrosamine exposure via the water resulted in the formation of 7-ethylguanine and O6-ethylguanine in rainbow trout liver DNA. The modified bases were quantitated by high-pressure liquid chromatography and fluorescence spectrophotometry. In vivo 7-ethylguanine and O6-ethylguanine levels were directly proportional to DEN concentrations between 62.5 and 250 ppm. 7-Ethylguanine and O6-ethylguanine levels were approximately directly proportional to duration of exposure to DEN between 0 and 6 hr under the conditions used, with less than proportionate increases thereafter. Removal of ethylguanines from liver DNA following a 24-hr exposure to 250 ppm DEN (a known carcinogenic regimen) was biphasic; 24% of the O6-ethylguanine and 32% of the 7-ethylguanine found immediately after exposure were removed in 12 hr but no significant decline was found over the period from 12 to 96 hr after exposure. Alkyl acceptor protein activity in trout liver was examined to assess the role of enzymatic repair in the observed loss of O6-ethylguanine in vivo. Although an O6-alkylguanine repair system similar to the alkyltransferase system reported in rodents was found in trout liver, only 4% of the O6-ethylguanine lost from DNA after exposure to 250 ppm DEN can be accounted for by activity of the alkyl acceptor protein. The high incidence of liver tumours observed in DEN-treated rainbow trout may be related to the rapid formation and substantial persistence of the promutagenic O6-ethylguanine in liver DNA.

Tumor Dose-Response Studies with Aflatoxin B1 and the Ambivalent Modulator Indole-3-Carbinol: Inhibitory Versus Promotional Potency

Indole-3-carbinol (I3C), a natural compound from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) carcinogenesis in trout when administered prior to and during carcinogen exposure, but also promotes it in the same species when given after AFB1 initiation. To provide quantitative potency information for these opposing activities, detailed tumor dose-response studies were performed with AFB1 (10-400 ppb) and I3C (0-4,000 ppm). In a plot of (logit) percent tumor response vs log AFB1 exposure, the results generated a series of parallel AFB1 dose-response curves. Increasing I3C doses displaced these curves, respectively, toward higher and lower AFB1 doses in the inhibition and promotion studies. Similar potencies were observed over the dose range 0-1,500 ppm I3C; the 50% promotion and inhibition (P50 and I50) values were 1,000 vs 1,400 ppm I3C, respectively. Differences in the protocols used in the two studies suggest that the inhibitory activity of I3C is more likely to supersede promotion under human exposure conditions.