Potency of dietary indole-3-carbinol as a promoter of aflatoxin B1-initiated hepatocarcinogenesis: results from a 9000 animal tumor study

Aram Oganesian 0 2 Jerry D.Hendricks 2 Cliff B.Pereira 1 Gayle A.Orner 2 George S.Bailey 2 David E.Williams 2 0 Present address: Wyeth-Ayerst Research , Princeton Corporation Plaza, CN-8000, Monmouth Junction, NJ 08852 , USA 1 Department of Statistics, Oregon State University , Corvallis, OR 97331-6602 , USA 2 ; CYP , cytochrome P450; I3C, indole-3- carbinol (3-indolemethanol); OTD, Oregon Test Diet; RP, relative potency; VG, vitellogenin 4To whom correspondence should be addressed Email: Indole-3-carbinol (I3C), a metabolite of glucobrassicin found in cruciferous vegetables, is documented as acting as a modulator of carcinogenesis and, depending on timing and dose of administration, it may promote hepatocarcinogenesis in some animal models. In this study we demonstrate that, when given post-initiation, dietary I3C promotes aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rainbow trout model at levels as low as 500 p.p.m. Trout embryos (~9000) were initiated with 0, 25, 50, 100, 175 or 250 p.p.b. AFB1 by a 30 min immersion. Experimental diets containing 0, 250, 500, 750, 1000 or 1250 p.p.m. I3C were administered starting at 3 months and fish were sampled for liver tumors at 11-13 months. Promotion at the level of tumor incidence was statistically significant for all dietary levels, except 250 p.p.m. Relative potency for promotion markedly increased at dietary levels >750 p.p.m. We propose that more than one mechanism could be involved in promotion and that both estrogenic and Ah receptor-mediated pathways could be active. The estrogenicity of I3C, measured as its ability to induce vitellogenin (an estrogen biomarker in oviparous vertebrates) was evident at the lowest dietary level (250 p.p.m.), whereas CYP1A (a P450 isozyme induced through the Ah receptor pathway) was not induced until dietary levels of 1000 p.p.m. Therefore, at lower dietary levels, promotion by I3C in this model could be explained by estrogenic activities of I3C acid derivatives, as it is known that estrogens promote hepatocarcinogenesis in trout. Much stronger promotion was observed at high dietary I3C levels (1000 and 1250 p.p.m.), at which levels both CYP1A and vitellogenin were induced. Introduction Indole-3-carbinol (I3C), a plant secondary metabolite found in cruciferous vegetables such as broccoli, cauliflower, Brussels sprouts, etc., is available to the general public for purchase as a dietary supplement in various forms over the Internet and through health food stores and distribution networks. Dietary I3C has been documented as inhibiting tumorigenesis (14) in various target organs, including mammary tissue (5), liver (6,7), endometrium (8), lung (912) and other target organs (13,14) in various animal models, and is currently being evaluated in human clinical trials as a potential chemopreventive agent against breast and ovarian cancers (15). The chemopreventive properties of I3C are proposed to occur through several possible mechanisms, including the alteration of estrogen metabolism (1621). Furthermore, I3C is reported to inhibit glutathione S-transferase-mediated steroid binding activity (22), act as a scavenger of free radicals (23), modulate the activity of multidrug resistance (24) and alter the expression of various phase I and phase II drug metabolizing enzymes (20,2528) contributing to detoxification of carcinogenic compounds. On dietary intake, I3C undergoes acidic condensation reactions in the stomach, yielding various derivatives believed to be responsible for its biological effects (2933). Some of the condensation products of I3C have anti-estrogenic as well as estrogenic activities (29) and also possess relatively high affinity for binding to the Ah receptor (34,35). A major condensation product, the dimer 3,39-diindolylmethane, is capable of inducing apoptosis in human cancer cells (36) and is an effective inhibitor in vitro of cytochromes P450 (CYP) (35,37,38). Carcinogenesis chemoprevention properties of dietary I3C in most of the models are evident when it is administered concurrently with the carcinogen or prior to initiation. Yet there are reports that, when given after initiation (promotionprogression stage), I3C can enhance carcinogenesis (1,3,7). There is also some evidence that I3C may be mutagenic when co-administered in the diet along with nitrites (39). Earlier studies (3,7) documented the ability of I3C to promote aflatoxin B1 (AFB1)-initiated hepatocarcinogenesis at relatively high dietary levels (1000 p.p.m.). The objective of this study was to evaluate the tumor promoting properties of I3C at relatively low dietary levels, across a wide range of initiator doses, and to investigate the possibility of a threshold for promotion within these range of doses. We report that I3C significantly promoted hepatocarcinogenesis across the entire AFB1 dose range except at 250 p.p.m. I3C, in which case promotion was only evident at the highest AFB1 doses. We further demonstrate that the estrogenicity of I3C is evident at the lowest dietary levels, at which point CYP1A was not induced. Perhaps, with the lower dietary I3C treatment the mechanism of hepatocarcinogenesis promotion involves estrogenic pathways, as it is known that estrogens promote chemically induced hepatocarcinogenesis in the trout (40). The Ah receptor-mediated pathway could play a role in promotion with higher dose treatment (1000 p.p.m.), where the ability of I3C to induce CYP1A was evident. It is documented that certain estrogens (oral contraceptives) are implicated in promotion of hepatic adenomas and carcinomas in humans (41,42). Based on observations from this study and the relevance of estrogens in human cancer risk, we suggest that dietary I3C supplementation be approached with caution until the mechanism(s) of hepatocarcinogenesis promotion in the trout and rat and the implications for human cancer risk are fully understood. Materials and methods AFB1 was purchased from Sigma (St Louis, MO). I3C was purchased from Aldrich (Milwaukee, WI). Rabbit polyclonal antibody against Coho salmon vitellogenin was kindly provided by Dr A.Hara (Hokkaido University, Japan). Rabbit polyclonal antibody to CYP1A was generously provided by Dr Donald Buhler (Oregon State University, Corvallis, OR). Animals and treatments Rainbow trout (Oncorhynchus mykiss) were hatched and reared at the Oregon State University Food Toxicology and Nutrition Laboratory in 14C (average temperature) flowing well water. Approximately 9000 embryos were initiated with 25, 50, 100, 175 or 250 p.p.b. AFB1 for 30 min. Sham-exposed embryos were exposed to vehicle alone (0.01% ethanol) and served as non-initiated controls. After hatching, fry were fed Oregon Test Diet (OTD), a semi-purified casein-based diet (43), for 3 months, after which trout were randomly (within initiator treatment groups) divided into experimental treatment groups and fed OTD diets containing 0, 250, 500, 750, 1000 or 1250 p.p.m. I3C. Once on experimental diets (...truncated)