1 Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH,
2 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH,
3 Statistics Department, Oregon State University, Corvallis, OR and
4 Medical Biochemistry Department, MFBS Center, Oregon State University, Corvallis, OR, USA
Abstract
Indole-3-carbinol (I-3-C) is among the most widely and popularly known antiestrogens. Due to its putative chemopreventive action, I-3-C is being marketed to the general public in health food establishments. Although it has been demonstrated to prevent cancer in animal bioassays, I-3-C also acts as a promoter in the liver and colon. Because of this potential dual biological activity, it is important to investigate both the inhibitory and promotional activities of I-3-C in multi-organ tumorigenesis animal models. 7,12-Dimethylbenz[a]anthracene, aflatoxin B1 and azoxymethane were used to initiate mammary, liver and colon carcinogenesis, respectively in female SpragueDawley rats. The rats were fed continuously on a diet containing I-3-C for 25 weeks after initiation. I-3-C treatment was begun one week after the last carcinogen treatment had been administered. I-3-C treatment resulted in a delay in latency of mammary tumor formation, but did not alter tumor incidence or multiplicity among survivors. In the colon, the protocol produced a 40% decrease in aberrant colon crypt foci. However, in the liver, it strongly-induced GST-P foci in carcinogen-treated (a four-fold increase in volume percent foci) and in the vehicle controls (a 69-fold increase). These data support previous findings in other rodent and fish tumor models that I-3-C both inhibits and promotes carcinogenesis. The results of this study clearly demonstrate that I-3-C is not an appropriate chemoprotective agent for human use, in spite of its effects in the breast and colon in this rat animal model.
Abbreviations: I-3-C, indole-3-carbinol; AFB1, aflatoxin B1; AOM, azoxymethane; ACF, aberrant crypt foci; DMBA, 7,12-dimethylbenz[a]anthracene; DEN, N-nitrosodiethylamine
Introduction
The pioneering work of Wattenberg showed that indole-3-carbinol (I-3-C) inhibited mammary tumor formation induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague Dawley (SD) rats and neoplasia of the forestomach of female ICR/Ha mice induced by benzo[a]pyrene (BaP) (1). Interest in I-3-C has remained high because it has several characteristics that are desirable in a candidate chemopreventive agent. I-3-C, in vitro, is neither cytotoxic (2) nor mutagenic (3). When given orally, it has a relatively low acute toxicity (46) and does not appear to be teratogenic (4). It has anti-initiating activity against several classes of environmental chemical carcinogens, including nitrosamines (7), polycyclic aromatic hydrocarbons (1,8), the mycotoxin, aflatoxin B1 (AFB1) (9), and the nitroazaarene, 4-nitroquinoline-1-oxide (10). Due to the diverse etiology of human cancer, the ability to protect against many classes of carcinogens is a valuable property of a chemopreventive agent. The chemopreventive activity of I-3-C has been reported in liver, forestomach, stomach and mammary gland (1), lung (7), swim bladder (8), tongue and nasal mucosa (10), larnyx (11) and endometrium (12). Its protective effects are not limited to a particular species, since I-3-C has chemopreventive activity in mice (7), trout (8) and rats (10). Few anticarcinogens offer this degree of chemoprotection. Perhaps of greatest interest are the reports that I-3-C protects against estrogen related tumors (11,12), as well as the short-term studies in humans that show a protective effect of I-3-C against estrogen-responsive breast cancer development (13,14).
Although it has been touted as a chemopreventive agent for breast cancer, enthusiasm for its use must be tempered by the evidence indicating that I-3-C poses a considerable risk, because it can act both as an inhibitor and promoter of carcinogenesis. I-3-C or its acid condensation products can be converted to mutagenic nitrosamines by treatment with acid and nitrite under stomach conditions (15,16), suggesting possible genotoxic or promotional mechanisms in vivo. In mouse epidermis, I-3-C acts as a promoter by enhancing 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induction of ornithine decarboxylase (17). Conversely, when it was given with TPA after a subcarcinogenic dose of DMBA in the two-stage mouse skin model, I-3-C significantly inhibited tumor development (18).
When dietary I-3-C was given to trout before and during administration of DMBA (8) or AFB1 (9), the number of liver tumors was reduced. In contrast, when dietary I-3-C was administered after carcinogen exposure, there was a significant dose-dependent increase in the number of tumors (8). In the case of AFB1, both inhibitory and enhancing activities were nearly equal over a wide range of I-3-C and AFB1 concentrations (6).
Administration of I-3-C (combined with wheat bran and cholesterol) to rats before, during, and after treatment with the colon-specific carcinogen dimethylhydrazine (DMH), enhanced tumor incidence in the colon (19). The main effect was attributed to I-3-C, possibly due to induction of AHH activity and enhancement of DNA alkylation by DMH. When male F344 rats were given I-3-C before and during treatment with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) the number of aberrant crypt foci (ACF) was significantly reduced (20). I-3-C also inhibited the formation of ACF induced by the heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), in fried ground beef when it was given before and during PhIP treatment or in the post-initiation phase (21).
In addition to the trout studies (8), two other attempts have been made to weigh the chemoprotective benefits of I-3-C against its promotional risk. In a multi-organ rat model, the chemopreventive potential of allyl sulfide (AS), I-3-C, and germanium (GE) were evaluated for their anti-tumor activity in tumors induced by sequential administration of N-diethylnitrosamine (DEN), N-methylnitrosourea (MNU), and N,N-dibutylnitrosamine (DBN) in the liver, lung, thyroid, and urinary bladder (22). When AS and GE were given after initiation, all three agents inhibited the formation of GST-P foci in liver. In addition, AS and GE also inhibited lung and thyroid adenomas, whereas only AS inhibited urinary bladder carcinogenesis (22). In another multi-organ rat model, Kim et al. (23) found that I-3-C enhanced liver and thyroid neoplasia when given 1 week after a 3-week initiation period with DEN, NMU, and dihydroxy-di-N-propyl-nitrosamine (DHPN).
It was hoped that I-3-C might offer a non-genotoxic alternative to tamoxifen or synergin for adjuvant therapy of breast cancer. Unfortunately, the possibility of cancer promotion by I-3-C in other organs, especially if taken as a `health food' by the disease-free general public is unknown. The protocol reported here was designed to measure inhibition and promotion by I-3-C in a multi-organ system in which mammary, liver and colon carcinogenesis were initiated sequentially in each target organ. This model was used to quantify opposing I-3-C post-initiation effects on mammary adenocarcinomas, GST-P foci in the liver, and aberrant crypt foci and carcinomas in the colon.
Materials and methods
Chemicals and diet
DMBA, AFB1, and I-3-C were purchased from Aldrich Chemical Company (Milwaukee, WI). AOM was purchased from Ash Stevens (Detroit, MI). The vehicle, tricaprylin, was obtained from Sigma Chemical (St Louis, MO). Powdered AIN-93G diet was purchased from Dyets (Bethlehem, PA).
Animals
Female 40-day-old SpragueDawley rats were obtained from Harlan SpragueDawley (Indianapolis, IN) and quarantined for 2 weeks before use in an experiment. Rats were randomly assigned to nine treatment groups (n = 20, Table I) and housed two per cage in animal rooms maintained at 20 ± 2°C with a 50 ± 10% relative humidity and a 12 h light/dark cycle. All animals were fed newly formulated AIN-93G rodent base diet (24) without antioxidants throughout the study. Food and water were available ad libitum with food consumption recorded at each scheduled diet renewal. Fresh diet was prepared bi-weekly and stored at 4°C. Animals were weighed weekly and the size and location of new (or regressing) palpable mammary tumors (
3 mm3) recorded for each animal. All animal protocols were in accordance with National Institutes of Health guidelines and the Institutional Animal Care and Use Committee of The Ohio State University.
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Statistical analysis
All analyses comparing groups with and without 1-3-C were performed using the SAS System for Windows release 8.1 (SAS Institute, Cary, NC). Two-sample t-tests (SAS Test procedure) or Exact Wilcoxon tests (SAS Npar1way procedure) were used to detect shift alternatives. t-tests were used when approximate normality was a reasonable assumption on either the original (aberrant crypts/foci counts for groups 6 and 8) or log scale (all liver foci measurements for groups 6 and 8 and per foci bearing animal measurements for groups 7 and 9). Exact Wilcoxon tests were used whenever a normality assumption was not reasonable (other liver foci measurements for groups 7 and 9, all aberrant crypt foci counts for groups 7 and 9, mammary tumor multiplicity, time until first mammary tumor, and colon tumors per animal). To detect more general alternatives, the Kuiper two-sample test (SAS Npar1way procedure) was used for time until first mammary tumor data. Fisher's Exact test (SAS Freq procedure) for r by c tables was used to detect general differences in mammary tumor multiplicity and colon tumors per animal. Fisher's Exact test was also used for comparing colon tumor incidence, liver foci incidence, and proportion killed.
Results
Establishment of treatment schedule
Two preliminary experiments were carried out to determine a satisfactory carcinogen treatment schedule. In the first trial, two groups of rats (n = 8 for both high and low dose experiments) were given a single subcutaneous (s.c.) dose of AOM at either 15 or 30 mg/kg body weight on day 1, followed by administration of a single gavage of DMBA at 25 or 50 mg/kg body weight on day 8, and multiple gavages of AFB1 (62.5 and 125 µg/kg body weight) beginning at day 15 for five days per week over a 2-week period. The rats were killed 21 weeks after initiation and analyzed for mammary tumors, GST-P foci in the liver, and colonic ACF. Although there was some variation in body weight and food consumption between the high and low dose groups over a period of 23 weeks, there were no significant differences in either parameter for most of the experimental period. Mammary tumor incidence reached 100% by week 16 in both high and low dose groups. The frequency of induced liver foci was sufficient for future chemopreventive studies (data not shown). However, the rapid development of mammary tumors did not allow sufficient time for development of either ACF or colon tumors. This treatment schedule was therefore deemed inappropriate for additional studies.
In the second trial, the treatment schedule shown in Figure 1 and in Table I
was evaluated (four rats per group). In this modified dosing schedule animals received two rather than one AOM treatment, and the post-initiation period was lengthened from 26 weeks to 30 weeks. In addition, DMBA was applied as the final carcinogen in order to avoid undue delay between mammary initiation and onset of I-3-C treatment (C.Ip and H.Thompson, personal communication). This protocol allowed adequate time for development of mammary adenocarcinoma, liver GST-P foci and colon ACF as quantifiable endpoints. Though the AFB1 protocol used is sufficient to produce hepatic tumors after 12 months, mammary tumor development limits the total post-initiation period to
6 months. In the liver, the volume % GST-P foci ranged from 3.3% to 16%, the number of foci per cm3 from 2465 to 5040 and the mean diameter/focus from 137 to 362 µm. For colon ACF, total numbers per rat ranged from 100 to 225 with 50 to 140 ACF showing
4 aberrant crypts per focus.
Based upon the data from these studies, a final study was performed using the scheme outlined in Figure 1. The study design used 20 animals per variable to establish and validate the multi-organ initiation protocol (Table I
, groups 17), and to examine the effects of I-3-C on initiated and control animals (group 6 versus group 8 and group 7 versus group 9, respectively).
Effect of I-3-C on mammary tumor response
In the final protocol, mammary tumors were induced by the administration of DMBA alone (group 1), in combination with AOM (group 4), or with AOM and AFB1 (groups 5 and 6) (Table II). Neither AOM nor AFB1 alone induced formation of mammary tumors (Table II
, groups 2 and 3) when compared with the vehicle-treated controls (group 7). At terminal sacrifice (week 30, Figure 1
), tumor incidence in the DMBA alone, DMBA + AOM, and DMBA + AOM + high AFB1 groups (groups 1, 4 and 6, respectively) was
100%, with no significant differences in mean yield as tumors/rat, or multiplicity (mean tumors/tumor bearing rat) between carcinogen treatments. The effects of post-initiation I-3-C treatment on mammary tumor response are revealed by comparing groups 6 and 8. As seen in Table II
, 2000 p.p.m. dietary I-3-C treatment of initiated animals for 25 weeks post-initiation did not result in a reduction of cumulative mammary tumor incidence during the study. Moreover, I-3-C did not reduce tumor multiplicity among rats surviving at week 30, nor did it produce any evident reduction in mean tumor size. We note that animals in any treatment group were killed if and when mammary tumor burden exceeded six tumors per animal or individual tumor size exceeded 1.5 cm in diameter. Thus, information from these animals was included in cumulative mammary tumor incidence and in time-to-first-tumor data, but not in final tumor multiplicity at week 30. We note, however, that the distribution of survivors was very similar between groups 6 and 8, and statistical tests did not detect any evidence for differences for surviving tumor bearing animals (t-test, Wilcoxon test, Fisher's Exact test, all P > 0.5). Although the percent killed prematurely due to excessive tumor burden is higher in group 6 and in group 8, the difference is not statistically significant (Fisher's Exact test, P = 0.30).
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Effect of I-3-C on GST-P foci in liver
Treatment of rats with DMBA plus AOM plus low or high doses of AFB1 produced strong induction of GST-P foci in the liver (e.g. focal volume percent = 3.05 and 6.96 for groups 5 and 6, respectively) (Table III). Responses in groups receiving one or two carcinogens (groups 14) were much lower, but still 35400 fold greater than that seen in the group 7 vehicle control. Protocol treatment for group 6 (or 5) provided adequate dynamic range for detection of promotional or suppressive effects of I-3-C treatment in this model. Comparison of groups 8 and 6 shows that 2000 p.p.m. I-3-C had a significant promotional effect (P < 0.0001, t-test) in the development of hepatic GST-P foci when evaluated as mean focal area (55.1 versus 14.7), mean volume (43.9 versus 5.7), or by the focal volume percent (26.8 versus 6.9). The focal volume percent is considered to be the most robust measure of foci formation and is analogous to tumor burden (26). Analysis of the focal volume percent response revealed that I-3-C induced
4.1-fold increase over that observed in the carcinogen combination treatment (group 6 versus group 8; 95% CI, 2.5- to 6.8-fold).
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Effect of I-3-C on colon tumors and aberrant crypt foci (ACF)
The relatively short duration carcinogenesis protocol was aimed at colonic ACF rather than tumors as the endpoint. Nonetheless, treatment of rats with 30 mg/kg body weight of AOM alone and in combination with DMBA was able to elicit colon tumors (tubular adenoma and adenocarcinomas) in 26% (group 2) and 22% (group 4) of the animals, respectively (Table IV). When AOM treatment was combined with AFB1 and DMBA, tumor incidence was similar, at 45% (AFB1 low dose) and 27% (AFB1 high dose) (groups 5 and 6). Post-treatment of animals for 25 weeks with I-3-C (Table IV
, groups 6 versus 8) did not appear to reduce tumor incidence or tumor multiplicity, but the tumor response in this protocol was minimal (only three and two animals had tumors in groups 6 and 8, respectively).
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Effect of I-3-C on mean body weight
Figure 3 shows the cumulative weight data in all nine groups during the 30-week bioassay. In most of the weeks 17 to 29, the mean weight of rats treated with vehicle + I-3-C (group 9) was significantly (P < 0.05) lower than that in rats treated with vehicle only (group 7). Similarly, beginning in week 18, rats treated with the three carcinogens + I-3-C (group 8) weighed less than those treated with the three carcinogens only (group 6; P < 0.05). These data indicate that the addition of the dietary I-3-C led to a potential toxic effect in both control and carcinogen-treated animals.
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Development of the three-organ rat model
A major feature of the multi-organ rat model developed in this study is that it permitted an assessment of the activity of I-3-C against mammary, liver and colon cancer in the same animal. These three cancer types were selected because mammary and colon cancer are two of the leading causes of cancer death in the United States, whereas liver cancer is one of the top three causes of cancer mortality world-wide. Previous studies in multi-organ models (22,23,29) either did not permit or did not report simultaneous comparison of I-3-C post-initiation effects in these three tissues. I-3-C is currently being proposed for suppression of mammary cancer in women, yet most publications indicate that post-initiation treatment with I-3-C promotes liver cancer (8,23), and one report indicates possible promotion of colon cancer (19). A limitation in the usefulness of this three-organ model became apparent as this study progressed. While tumors can be generated in the mammary gland and in the colon within the span of the 6-month bioassay, liver tumor development requires at least 12 months using this protocol. Due to this, it was necessary to use a well-established marker for liver tumor risk, GST-P foci. This marker would appear to be predictive of I-3-C effects since, in other model systems such as trout, I-3-C has been shown to promote the development of hepatocellular carcinomas (8).
Post-initiation effects of indole-3-carbinol
The results of the present study indicate that post-initiation dietary I-3-C at 2000 p.p.m. provided some degree of protection against mammary tumors and colon ACF, but greatly promoted hepatic GST-P foci in animals jointly initiated for all three endpoints. I-3-C increased the mean focal area and the volume % of GST-P foci in the liver of rats treated with all three carcinogens 4-fold. I-3-C also increased the volume % GST-P foci in vehicle-treated control animals as well as the number of foci per cm3 >100-fold. These data are consistent with the observations of Kim et al. (30), who found that I-3-C inhibited DEN-induced hepatic GST-P foci if given prior to DEN injection, but significantly increased GST-P foci if given in the diet for 6 weeks beginning 4 weeks after initiation. Both the number (no./cm2) and area (mm2) of GST-P foci were increased as a result of I-3-C post-treatment.
Beginning at 17 weeks, animals treated with I-3-C had lower body weights than vehicle controls indicating that the compound itself elicited some toxicity in the animals. In addition, animals treated with the three carcinogens plus I-3-C had lower body weights than those treated with the three carcinogens alone. Thus, it is possible that the mild reduction in mammary tumors and colon ACF in I-3-C treated rats was a result of the reduction in body weight.
There is evidence for two basic mechanisms for I-3-C post-initiation suppression of mammary tumorigenesis alterations in hepatic estrogen metabolism leading to reduced systemic estrogens, and receptor-mediated suppressive mechanisms within the mammary epithelial cell. The systemic anti-estrogenic effect is widely ascribed to induction of hepatic cytochrome P450 (CYP) 1A1 by dietary I-3-C (3133). At the cellular level, treatment of mouse mammary epithelial cells (34) and human MCF-7 cells with I-3-C (35) induced CYP1A1 and produced anti-estrogenic responses including a reduced C16- to C2 hydroxylation ratio, decreased nuclear estrogen receptor (ER) and progesterone receptor binding, and decreased estrogen-induced cellular proliferation. Bradlow et al. (36) have suggested that an increased mammary cancer risk is associated with a decrease in the C2-hydroxylation: C16-
hydroxylation ratio, a mechanism that might be overcome by I-3-C mediated induction of C2-hydroxylation.
The inhibition of hepatocarcinogenesis by I-3-C when given before carcinogen treatment is thought to be attributed to the induction of metabolizing and detoxification enzymes (33,37,38), a decrease in covalent binding of carcinogens (3941), or the scavenging of free radicals (42). However, the means by which I-3-C promotes liver tumors is less well defined. One report (43) attributed post-initiation I-3-C promotion of hepatocarcinogenesis in trout to both estrogenic and Ah receptor pathways. Protection was biphasic, with I-3-C levels as low as 250 p.p.m. giving evidence for induction of estrogen biomarkers, whereas induction of P450 CYP1A1 was observed at dietary levels of 1000 p.p.m. and higher. Both Ah receptor and estrogen pathways were significantly elevated at I-3-C doses above 1000 p.p.m.
As with I-3-C inhibition of liver carcinogenesis, mechanisms for blocking the initiation of colon carcinogenesis by pretreatment with I-3-C have been described by several investigators. These include induction of metabolizing and detoxification enzymes (23,44,45) and a reduction of DNA adducts (20). The present study provided evidence that post-initiation I-3-C suppresses development of AOM-initiated ACF in the colon. This finding is consistent with previous evidence (21) for I-3-C suppression of PhIP-induced ACF. However, mechanisms for I-3-C suppression of the ACF biomarker, and the relationship to final carcinoma development, have not been fully elucidated. Recent results from our laboratory suggest that post-initiation effects of I-3-C or chlorophyllin in the rat colon is dependent upon the initiator used (46,47). Post-initiation treatment was either inhibitory or enhancing, apparently depending on the particular spectrum of ß-catenin mutations elicited by the carcinogen chosen (46). Until these opposing activities are better understood, caution should be applied in the proposed use of I-3-C for the suppression of human colon cancer.
With the exception of the studies cited in this report (6,22,23), no other attempts have been made to weigh chemoprotective benefits against promotional risk for I-3-C. In spite of the hope that I-3-C might be a non-genotoxic alternative for tamoxifen or synergin for adjuvant therapy, the risk of promoting colon and liver cancer, especially if I-3-C is used as a `health food' by the presumably disease-free general public is unwise and potentially dangerous.
The results of this study and other published reports clearly show that I-3-C is not an appropriate chemopreventive agent for human use, despite the protective effects in the breast and colon in the rat multi-organ model.
Notes
5 To whom reprint requests are to be sent: Division of Environmental Health Sciences, School of Public Health, 1148 CHRI, 300 W. 10th Avenue, Email: stoner.21{at}osu.edu
Acknowledgments
The authors wish to acknowledge the technical assistance of Charlotte Morgan, Balvinder Sidhu, and Laura Kresty in conduct of animal experiments and Denise MacMillan and Karen Baumgartner for assistance in evaluating the hepatic foci. This work was supported in part by NIH/NCI Grants CA-34732 (GSB)and CA-39416 (BDR).
References