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

Aram Oganesian1,3, Jerry D. Hendricks1, Cliff B. Pereira2, Gayle A. Orner1, George S. Baileyand1 and David E. Williams1,4

1 Department of Environmental and Molecular Toxicology and Marine/Freshwater Biomedical Sciences Center, Wiegand Hall and
2 Department of Statistics, Oregon State University, Corvallis, OR 97331-6602, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.

Abbreviations: AFB1, aflatoxin B1; CYP, cytochrome P450; I3C, indole-3-carbinol (3-indolemethanol); OTD, Oregon Test Diet; RP, relative potency; VG, vitellogenin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 3,3'-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 (promotion–progression 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
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 14°C (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, trout were fed ad libitum (2.8–5.6% body wt), five times per week. I3C was added to the aqueous portion of the diets during preparation. Diets were prepared biweekly and stored frozen at –20°C until 2–4 days prior to feeding, when diets were allowed to thaw at 4°C. Each treatment group contained 200–400 fish (larger numbers of fish were used for low dose initiated groups fed lower levels of I3C in the diet) housed at 79–142 per tank in 100 gallon continuous flow tanks. A maximum of 100 trout were sampled per tank. At 11 months of age trout were sampled for liver tumors while still sexually immature. Because of the large number of fish involved, the sampling was carried out over 66 days (all groups were switched to OTD diet during sampling). Blood was drawn from the caudal vein [in order to examine vitellogenin (VG) induction] and, at the start of the sampling period (while trout were still on I3C diets), 10 livers from each group were frozen and later analyzed for CYP1A induction.

Electrophoresis and immunoblotting
VG levels were determined in plasma from trout. CYP1A levels were examined in liver microsomal fractions, prepared as previously described (44). Proteins were separated by SDS–PAGE on 8% acrylamide gels (45) and electrophoretically transferred onto nitrocellulose (Trans-Blot; Bio-Rad, Richmond, CA). Blots were probed with rabbit polyclonal antibodies to salmon VG (for plasma) and trout CYP1A (liver microsomes) followed by a horseradish peroxidase-linked secondary antibody. Proteins were detected using an Amersham ECL chemiluminescence kit (Amersham, Arlington Heights, IL). Western blots were scanned on a flatbed HP Scanjet IIcx scanner. Densitometry was performed with the public domain software NIH Image v.1.57 (written by W.Rasband at the US National Institutes of Health).

Necropsy and histopathology
At termination, fish were killed by a combination of deep anesthesia, resulting from an overdose of tricaine methane sulfonate (MS-222), and bleeding, after cutting the gill arches on the left side of the fish. The fish were weighed, livers removed and weighed and the livers inspected for neoplasms under a dissecting microscope. After marking and recording the size and location of all surface tumors, the livers were fixed in Bouin's solution for 2–7 days. All livers were then cut into 1 mm slices with a razor blade to retrieve previously marked tumors and to locate any internal, previously unseen tumors. At least one piece of liver from each tumor-bearing fish was then processed by routine histological procedures and stained with hematoxylin and eosin for histological evaluation. Neoplasms were classified by the criteria of Hendricks et al. (46). The relative numbers of different tumor types were thus based on a random, non-exhaustive sample of all the tumors that occurred. When multiple tumors occurred on a tissue slide, only the largest or the most frequently occurring tumor type was recorded.

Statistical analysis
Tumor incidence and incidence of multiple tumors in tumor-bearing fish (in 78 tanks containing initiated fish) were modeled by logistic regression (Genmod procedure in SAS v.6.12). For tumor incidence data, variation between replicate tanks was larger than expected under the binomial model (overdispersion). A covariate for date sampled was included in the model because it explained a large proportion of the overdispersion (tanks sampled later tended to have higher tumor incidence). For multiple tumor incidence data, variation between tanks was only slightly larger than expected (slight overdispersion) and no covariates were included. Statistics, such as standard errors and likelihood ratio statistics were adjusted to account for overdispersion (Dscale option in Genmod procedure). Parsimonious models were chosen based on quasi-likelihood F-tests.

For parallel dose–response curves, relative potency (RP) for promotion was calculated using RP = TDx0/TDxi, where x is the tumor incidence being compared (e.g. 50%), TDx0 is the dose of AFB1 needed to yield that tumor incidence in the 0 p.p.m. I3C (initiated control) group and TDxi is the dose of AFB1 that is required to produce that tumor incidence in the group receiving i p.p.m. I3C. Estimates and confidence intervals were generated from logistic regression modeling results [log relative potency = (difference between estimated intercepts) ÷ estimated slope (47)] with the delta method used to estimate standard errors for relative potency.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A dose–response effect was observed for tumor incidence with increasing I3C levels in the diet (Table IGo and Figure 1Go). The tumor promotional effects of I3C were apparent at all levels across the entire AFB1 initiator dose range (P < 0.0001 for 750, 1000 and 1250 p.p.m. I3C; P < 0.03 for 500 p.p.m. I3C) except for 250 p.p.m. I3C (significant only for the highest AFB1 groups; Table IIGo). Based on these observations, the existence of a threshold for promotion of AFB1-induced hepatocarcinogenesis by dietary I3C could neither be demonstrated nor discounted. If a threshold exists, it must be <500 p.p.m.


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Table I. Summary of liver tumor incidence in trout fed I3C (initiated with AFB1 as embryos by a 30 min immersion)
 


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Fig. 1. Promotion of AFB1-induced hepatocarcinogenesis by dietary indole-3-carbinol. Fitted lines are from logistic regression on log AFB1 dose. Parsimonious modeling resulted in parallel AFB1 dose–response curves for all doses of I3C except the highest dose (1250 p.p.m.), which had a significantly greater slope. For simplicity of presentation, the covariate adjustment for sampling date was not included in the graph. Promotion was significant starting from the 500 p.p.m. group (Table IIGo). For the 250 p.p.m. I3C dose, promotion was significant only for the higher dose AFB1-initiated groups.

 

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Table II. Odds ratio and probability values for promotion of tumor incidence and multiplicity
 
Overall, the variation between replicate tanks was 1.92 times larger than expected under the binomial error assumption, indicating significant overdispersion (P < 0.0001). The variation was greatly reduced by adding a continuous covariate for day of sampling (P < 0.0001). After adjusting for the covariate, the remaining variation between replicate tanks was only 1.24 times larger than expected under the binomial error assumption (test for overdispersion, P = 0.11). Therefore, the conclusions for the more conservative overdispersed model used here will be similar to the conclusions from a model assuming only binomial variation.

The RP of promotion of AFB1-induced hepatocarcinogenesis by dietary I3C is depicted in Figure 2Go. There is a marked increase in the RP value with dietary I3C >750 p.p.m. At levels <750 p.p.m. promotion is evident, however, with lower potency.



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Fig. 2. Relative potency of I3C (95% CI) for promotion calculated as the ratio of AFB1 doses required to achieve the same tumor incidence (control over I3C). Estimates were based on logistic regression modeling (see Materials and methods, statistical analysis). The group containing fish fed 1250 p.p.m. I3C is not included since it did not fit the parallel dose–response model (i.e. relative potency was not constant).

 
The spectrum of tumor types was the same as previously observed (48) and included malignant and benign neoplasms of hepatocellular, cholangiocellular or mixed hepatocellular/cholangiocellular origin. There was a difference in this experiment, however, since the hepatocellular carcinoma type was the predominant tumor rather than the mixed carcinoma observed in previous studies (40,49,50). Some variability in the relative percentages of the various tumor types occurred, in particular at the lower incidences seen at the low dose levels, but the trend was the same throughout (Table IIIGo). The overall averages of tumor occurrences were as follows: hepatocellular carcinoma, 56.5%; mixed hepatocellular/cholangiocellular carcinoma, 29.4%; cholangiocellular carcinoma, 1.2%; hepatocellular adenoma, 8.7%; mixed adenoma, 0.3%; cholangiocellular adenoma, 3.9%. With the exception of an approximate reversal of the percentages of the first two tumor types, these data are very consistent with what has been seen previously. No obvious reason for this change in tumor type is apparent.


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Table III. Histological classification of liver tumors in trout fed I3C (initiated as embryos with AFB1 by a 30 min immersion)
 
The potential of I3C to function as an estrogen in trout was assessed by measuring plasma VG levels (51). VG bands are barely visible in plasma from controls (0 p.p.m. I3C; Figure 3Go). For the groups fed 250 p.p.m. I3C, weak VG induction is apparent. Marked induction was observed for the groups fed diets containing >=500 p.p.m. I3C.




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Fig. 3. (Top) Induction of plasma VG (estrogen biomarker) in trout from the tumor study, fed I3C continuously at 0, 250, 500, 750, 1000 and 1250 p.p.m. as determined by a western blot of trout plasma. The bars represent arbitrary densitometry units obtained from the western blot shown in the inset. (Bottom) The numbered lanes contained vitellogenin standards (1 and 2) or plasma from trout fed 0 (3), 250 (4), 500 (5), 750 (6), 1000 (7) and 1250 (8) p.p.m. I3C. The plasma protein load in each lane was 17.5 µg.

 
Induction of CYP1A, a marker for the Ah receptormediated mechanism, was apparent only at the highest I3C dose (1250 p.p.m.; Figure 4Go).



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Fig. 4. Induction of hepatic microsomal CYP1A in trout from the tumor study fed I3C continuously at 0, 250, 500, 750, 1000 and 1250 p.p.m. as determined by a western blot of trout liver microsomes. The lane assignments were as follows: trout CYP1A standard (13) and 250 (4), 500 (5), 750 (6), 1000 (7), 1250 (8) and 0 p.p.m. (9) I3C. The microsomal protein load in each lane was 20 µg. The blot was probed with rabbit antibody to trout CYP1A as described in Materials and methods.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dietary I3C supplementation for the healthy adult human population has been advanced for the purpose of chemoprevention against estrogen-related diseases (15). However, after it was recognized that long-term dietary I3C may lead to enhancement of hepatocarcinogenesis in the rat (1) and trout (3,7) and induction of both phase I and phase II enzymes involved in possible procarcinogen activation (25,26,35,52), legitimate concerns were raised regarding possible risks associated with the above approach. Earlier studies documented tumor promotional effects of I3C at relatively high dietary doses (3,7). One of the objectives of this experiment was to address the potency of post-initiation long-term dietary I3C to act as a promoter of hepatocarcinogenesis. Thus, the inclusion of treatment groups with relatively low dietary I3C exposure (250, 500 and 750 p.p.m.) allowed determination of a possible threshold below which no significant promotion would be detected.

The results are inconclusive regarding the existence of a threshold. The data are consistent with the hypothesis of a no treatment effect at 250 p.p.m. (odds ratio 1.15) and therefore consistent with the existence of a threshold in the range studied. However, the variation in the data is such that the results are also consistent with moderate treatment effects at 250 p.p.m. based on the width of the 95% confidence interval (upper limit odds ratio 1.46; Table IIGo) and on the observed increases in pooled incidences (250 versus 0 p.p.m. level) at the three highest doses of AFB1 (Table IGo). Based on the logistic regression modeling, if a threshold exists at all, it would appear to be below 500 p.p.m. Further studies focusing on <=250 p.p.m. doses of I3C would help to clarify the threshold question. A similar trend for promotion was observed with respect to the incidence of multiple tumors among tumor-bearing fish.

Post-initiation long-term dietary I3C has ambivalent effects depending on the animal model. It promotes hepatocarcinogenesis in the trout and the rat as mentioned above; however, it acts as an inhibitor in the C57BL/6J mouse (6). A possible mechanism(s) for tumor modulation could include Ah receptor-mediated enzyme induction (25,34,35) and/or the ability of I3C derivatives to exhibit estrogenic and/or anti-estrogenic properties (29). It is known that estrogens promote chemically induced carcinogenesis in the rat (5358) and trout (40), but function as inhibitors in the mouse (6,59,60). It is unclear how estrogens inhibit liver tumors in mice. The mechanisms of estrogen-related hepatocarcinogenesis promotion in the rat are proposed to involve alterations in the cell cycle with ensuing increased cellular proliferation and/or the ability of some estrogen metabolites to cause direct and/or indirect DNA damage resulting from reactive oxygen intermediates generated by redox cycling involving catechol metabolites of estrogens (61). The ability of I3C derivatives to induce P450s through the Ah receptor and also to possess estrogenic and/or anti-estrogenic activities suggest that either of these mechanisms could be involved in promotion in the trout liver model.

VG, the precursor for egg yolk protein, is found in all oviparous vertebrates (62) and its synthesis is mediated through the estrogen receptor (63) and is required for oocyte maturation. Thus, VG synthesis is a reliable estrogen biomarker in sexually immature trout. We observed in this experiment that in the low I3C treatment groups VG synthesis was evident whereas CYP1A induction was evident only at the highest dose (1250 p.p.m.). Previous studies showed that Ah agonists promote chemical hepatocarcinogenesis in trout (64). The present study suggests that estrogenicity of I3C may play a more important role in promotion of chemically induced hepatic tumors in trout, especially at lower dietary levels. In fact, the slope for the relative potency for promotion increases markedly above 750 p.p.m. (Figure 2Go), a possible indication of more than one mechanism being involved in the promotion. Perhaps, at higher I3C levels both estrogenic and Ah receptor-mediated mechanisms are responsible for the observed promotion, but in the lower dose range just the estrogenic pathway is active.

When estrogenic activities of a compound of interest are examined, trout are a very sensitive model for tumor studies. Estrogens promote liver tumors in trout. This is particularly relevant considering that human and trout estrogen receptors have similar specificities for binding estrogens (65).

If I3C indeed promotes hepatocarcinogenesis in trout by acting as an estrogen, its proposed supplementation for chemoprevention purposes should be approached with caution, until its mechanism(s) of promotion and the relation to human cancer risk are thoroughly understood.


    Acknowledgments
 
The authors thank Dan Arbogast and other members of the OSU Food Toxicology and Nutrition Laboratory for care and feeding of the fish and especially their help in sampling. This work was supported by US PHS grants ES04766, ES03850 and CA34732.


    Notes
 
3 Present address: Wyeth-Ayerst Research, Princeton Corporation Plaza, CN-8000, Monmouth Junction, NJ 08852, USA Back

4 To whom correspondence should be addressed Email: david.williams{at}orst.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 16, 1998; accepted October 9, 1998.