* Department of Environmental and Molecular Toxicology and the Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331;
College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331; and
Statistics Department, Oregon State University, Corvallis, Oregon 97331
Received January 20, 2003; accepted March 26, 2003
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ABSTRACT |
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Key Words: indole-3-carbinol; 3,3-diindolylmethane; CYP1A1/1A2; CYP 3A2; bone density; 25-OH vitamin D3; clinical chemistry; histopathology; rat.
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INTRODUCTION |
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Both I3C and DIM are marketed as dietary supplements and are under investigation as potential chemopreventive agents. I3C requires conversion to these acid condensation products in order to be chemopreventive against a wide variety of carcinogens and is especially effective when administered before or during the initiation phase of carcinogenesis (Bradlow et al., 1999; Murillo and Mehta, 2001
; Shertzer and Senft, 2000
). DIM inhibits aflatoxin B1-DNA binding in trout (Dashwood et al., 1994
) and mammary tumor growth in the rat (Chen et al., 1998
; Wattenberg and Loub, 1978
).
Among the hypothesized mechanisms of chemoprevention of I3C and DIM is their ability to modulate xenobiotic metabolizing enzymes and induce estrogen metabolism. When administered through the diet in short-term studies, I3C and DIM induce a number of phase I enzymes in liver and colon, including cytochrome P450 (CYP) 1A1, CYP1A2, and CYP 3A (Bonnesen et al., 2001; Horn et al., 2002
; Jellinck et al., 1993
; Stresser et al., 1994
; Vang et al., 1990
). Increased activity of phase I drug-metabolizing enzymes can protect against some carcinogens by increasing their rate of oxidative metabolism to less toxic metabolites (He et al., 2000
; Park and Bjeldanes, 1992
; Stresser et al., 1994
; Xu et al., 1997
); however, an increase in activity of certain CYP isozymes could enhance carcinogenecity of some chemicals by increasing their rate of bioactivation (Ioannides and Parke, 1993
).
I3C- and DIM-dependent alterations of the monooxygenase systems also raise concerns relative to their potential adverse effects on drug/xenobiotic metabolism. Studies have shown differential metabolism of tamoxifen and nicotine by liver microsomes from rats fed I3C (Katchamart et al., 2000). I3C induction of CYPs in the 1 family markedly enhances estradiol metabolism. CYP1A1 and 1A2 catalyze the 2-hydroxylation of ß-estradiol (E2), whereas CYP1B1 is an effective E2-4-hydroxylase (Hayes et al., 1996
). Both of these metabolites are catechol estrogens and represent potential toxic metabolites. Evidence suggests that 4-OH-E2 is the more reactive and toxic metabolite (Newbold and Liehr, 2000
).
Preliminary evidence to date indicates that DIM may be a safer alternative to I3C, as it is relatively stable in acid and does not undergo further condensation reactions, preventing the formations of toxic metabolites such as indolo[3,2-b]carbazole (ICZ), a potent aryl hydrocarbon receptor agonist (Bjeldanes et al., 1991) and potential promoter of hepatocarcinogenesis in the rat (Herrmann et al., 2002
). DIM induces apoptosis and inhibits growth of human cancer cells (Chen et al., 2001
; Ge et al., 1996
; Hong et al., 2002
; Leong et al., 2001
) and inhibits estrogen-dependent mammary tumorigenesis in rats (McDougal et al., 2001
). However, due to poor bioavailability of DIM, its use as a supplement has been limited. The objective of this study was to evaluate the biological effects of long-term exposure to rats of an absorbable formulation of pure DIM, [BioResponse DIM® (Indolplex®)] at the current maximal human dose (Anderton et al., in press
; U.S. Patent #6,086,915; Zeligs et al., 2002
). In addition, a pharmacological dose 10 times the current human dose was tested for toxicity and as a comparison to a similar and previously studied dose of I3C.
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MATERIALS AND METHODS |
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Animals.
One hundred forty Sprague-Dawley rats (70 of each sex) were purchased from Simonsens (Gilroy, CA) at 4 weeks of age. After a 1-week acclimation period, animals were randomly divided into 10 different treatment groups, each containing seven rats of each sex (Table 1). Rats were housed individually in hanging metal wire cages at the Laboratory Animal Resource Center, Oregon State University, and maintained at 22°C and 40 to 60% humidity on a 12-h light/dark cycle. Both tap water and powdered semisynthetic diet were available ad libitum throughout the study. Groups 1 and 7 received only control (AIN-76A) diet. The diet for groups 2, 5, and 8 was supplemented with I3C to levels providing a dose of 50 mg/kg/day. The diets are prepared by thoroughly mixing the indoles as a powder into the powdered AIN76A diet. The diets were made weekly and stored at 4°C in sealed containers protected from light. I3C is known to be unstable, especially under acidic conditions. Although we did not assay I3C and DIM in the diets used in this study, a previous study involving long-term feeding of mice with AIN76A diet demonstrated that under these same conditions at least 80% of the I3C was present after one week (Oganesian et al., 1997
). The stability of DIM is expected to be much greater, as we have found it to be relatively stable under mild acid conditions in air at room temperature (unpublished observations). The diets for groups 3, 4, 6, 9, and 10 provided 6.6 mg/kg/day or 66 mg/kg/day BioResponse DIMTM. On a molar basis, the daily doses of I3C and low and high DIM were 0.34, 0.008, and 0.08 mmol/kg, respectively. The approximately 2.5 and 25% molar levels of DIM (relative to I3C) span the range for the percentage of DIM found after acid condensation reaction of I3C in vitro or in vivo.
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Blood collection and analysis.
Blood was collected from the abdominal aorta while rats were anesthetized under 4% isoflurane with 02 at a flow of 2 l/min. Blood was stored at 4°C for 2 h and then spun for 20 min at 11,000 r.p.m. to isolate serum. One aliquot of serum was sent to the Texas Veterinary Medical Diagnostic Laboratory (College Station, TX) where a small animal clinical chemistry analysis was performed utilizing a Hitachi 911 Chemical Analyzer (Roche, Indianapolis, IN). A second aliquot was sent to the Animal Health Diagnostic Laboratory (Lansing, MI), where circulating 25-hydroxyvitamin D3 (25-OH-D3) levels were determined using a commercial radioimmunoassay from DiaSorin (Stillwater, MN) and testosterone levels were determined (males only) using a commercial radioimmunoassay from Diagnostic Products Corporation (Los Angeles, CA).
Bone density analysis.
Three carcasses of each sex, from groups 1, 2, and 4 were sent on ice to the University of Colorado Health Science Center (William. E Huffer, M.D., Denver, CO). The carcasses were equilibrated to 4°C, and the proximal knee joint with approximately one half of the distal femur and proximal tibia were removed and fixed for 24 h in absolute methanol, also at 4°C. The specimens were then embedded in glycol methacrylate and sectioned on a rotary microtome with a D-profile carbon-tungsten steel knife at 5 µm. The sections were stained by the von Kossa technique (Bills et al., 1971) with a hematoxylin and eosin counter-stain to demonstrate general histology and calcified bone and osteoid, and for tartrate resistant acid phosphatase to demonstrate osteoclasts on bone surfaces. Measurements of the total and cancellous bone areas, cortical bone thickness, and the number of osteoclasts in a single section of the tibial metaphysis were made by semiautomated histomorphometric methods (Huffer and Lepoff, 1992
; Huffer et al., 1994
).
Histopathology.
Tissues were fixed in 10% neutral buffered formalin and processed on a LX300 Tissue Processor (Fisher Scientific). Sections were cut at 45 microns and stained on the S/P Automatic Slide Stainer GLX with Haematoxylin (Gill-3, Shandon Inc., Pittsburgh, PA) and Eosin Y (alcoholic, Shandon Inc., Pittsburgh, PA). Slides were coverslipped with Shur/Mount (Triangle Biomedical Sciences, Durham, NC).
Microsome preparation and total CYP.
Liver samples were homogenized with a Brinkmann Polytron (Westbury, NY) into three volumes of cold homogenization buffer (10 mM potassium phosphate [pH 7.5] containing 0.15 M potassium chloride, 1 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride [PMSF]). Microsomes were prepared by ultracentrifugation according to Guengerich (1989), and protein levels were determined by the method of Lowry et al.(1951)
. The total liver microsomal CYP content was quantified by the CO versus CO-reduced difference spectra (Omura and Sato, 1964
) on a Cary 300 UV-Vis spectrophotometer (Varian, Walnut Creek, CA).
Colon lysate preparation.
Colons were rinsed with phosphate buffered saline (PBS) to remove mucus and then scraped with the back of a scalpel blade to remove cells. Cells were lysed in a modified RIPA buffer [1x PBS, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) with 1 tablet of Complete-Mini protease inhibitor cocktail (Roche, Indianapolis, IN) added for every 10 ml of buffer] and then sheared by passing through a 23-gauge needle. Sheared cells were incubated 45 min on ice after the addition of 0.574 mM PMSF (1:100 from an isopropanol stock) and then spun at 10,000 r.p.m. for 10 min at 4°C. The supernatant was removed, protein concentration determined by the method of Lowry et al. (1951), and stored at -80°C until analysis.
Electrophoresis and immunoblotting.
Microsomal and lysate proteins were resolved by SDSpolyacrylamide gel electrophoresis (SDSPAGE) (Laemmli, 1970) and electrophoretically transferred to nitrocellulose membrane (Towbin et al., 1979
). The membranes were incubated with goat antibodies recognizing both rat CYP1A1 and CYP1A2 or CYP3A2 (Gentest, Woburn, MA), and probed with rabbit anti-goat secondary antibody conjugated with horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) or rabbit antibody raised against rat CYP1B1 (Gentest, Woburn, MA), and probed with goat anti-rabbit secondary antibody also conjugated to horseradish peroxidase (Gentest, Woburn, MA). The blots were visualized by a chemiluminescence detection kit (New England Nuclear, Boston, MA) and densitometry was performed using an HP Scanjet IIcx flatbed scanner and NIH Image software version 1.61/ppc (public domain, National Institutes of Health).
Statistical analysis.
Males and females were analyzed separately. When assumptions were reasonably satisfied, treatments were compared using one-way ANOVA and all pairwise comparisons with Tukeys multiple comparison adjustment. When outliers or other non-normality were indicated, simple transformations, such as logarithmic, were examined. When the problem was nontransformable, nonparametric rank tests (Kruskal-Wallis) were used. In that case, pairwise comparisons were adjusted for multiple comparisons using the global permutation distribution, after first determining that there were no problems with extreme heteroscedasticity or badly unbalanced sample sizes (Westfall et al., 1999). When preliminary analysis indicated normality but heterogeneity of variance, a general mixed model allowing heterogeneity of variance was fit by residual maximum likelihood (REML) with Tukey adjusted pairwise comparisons. The denominator degrees of freedom for testing were adjusted by the method of Kenward and Rogers (SAS, 1999
). Statistical analyses were conducted using SAS version 8.2 (Cary, NC). Within SAS/STAT the GLM, Mixed, Npar1way, and Multtest procedures were used.
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RESULTS |
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Serum 25-OH-D3 levels were increased (p = 0.0371 and 0.0031 for males and females, respectively) about 50% by dietary I3C in both sexes after 12 months. At 3 months only the males exhibited elevated serum levels of 25-OH-D3. Again, the I3C diet increased levels by about 50%. The high dose of DIM also appeared to increase 25-OH-D3 in serum, but the change was not significant.
Serum testosterone levels in males were not altered by test diets. E2 levels were unchanged by I3C or DIM at both the 3- and 12-month time points (data not shown).
Histopathology
No significant differences between groups of either sex at 12 months was noted upon necropsy or following histopathology. Most notably, no effects were seen in hormone-responsive tissues such as prostate in male rats and ovaries in female rats, and no toxicities were indicated in treatment groups as compared to controls in the liver (Figs. 2 and 3
). There were large numerous hyaline casts found in the kidney tubules. The appearance of such casts are common in rats with age (Lord and Newberue, 1990
), and no treatment-related differences were evident (data not shown).
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DISCUSSION |
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The body weights and organ weights did not suggest any chronic treatment-related toxicity, with the possible exception of liver. A previous study showed that after a 15-day treatment of 0.5% I3C in the diet of rats, the liver somatic index was significantly increased from 4.4% in controls to 6.4% (Manson et al., 1997). The smaller but chronic doses of I3C and DIM in this study also resulted in a significant increase in the LSI. A 3-month oral administration of I3C to rats at doses of 4, 20, or 100 mg/kg/day also demonstrated an increased LSI (at the 20 and 100 mg/kg/day doses). In that same study, histopathological changes in liver were observed at the 100 mg/kg/day dose, as well as a decrease in testes weight at all doses (NCI, 1996
). These differences may be related to the method of administration (diet versus gavage). This increase in LSI observed in our study with I3C or DIM correlates with the degree of CYP induction that was also observed, as I3C was a more efficacious inducer of CYP and also had a greater effect on LSI when compared to DIM. I3C significantly enhanced LSI in males after 3 or 12 months. The high dose of DIM also increased LSI but only after 12 months. The increase was not as marked as with I3C and was seen only in males. We have no explanation for the effect of sex on LSI.
Clinical chemistry panels failed to uncover any significant differences between control, I3C, and DIM treated rats that would indicate toxicity. Conversely, the significant reduction in creatine kinase (CK) in male rats fed DIM, and the significant reduction in alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in rats fed I3C or DIM could indicate possible protective effects against age-related tissue damage. This may be explained by the antioxidant and electrophilic scavenging properties described for I3C and DIM (Arnao et al., 1996; Fong et al., 1990
; Shertzer and Senft, 2000
; Shertzer et al., 1988
). Again, this significant reduction in the serum enzyme markers was only evident in male rats after 12 months of dietary exposures. With one exception, no other significant alterations in serum chemistry following 12 months of exposure to I3C or DIM was evident. In previous work some acid condensation products of I3C have been shown to lower serum LDL/VLDL cholesterol levels in mice (Dunn and LeBlanc, 1994
), resulting from the inhibition of acyl-CoA:cholesterol acyltransferase (ACAT). Treatment with I3C or DIM failed to provide cholesterol-lowering effects in this study. The dietary indoles did not alter serum levels of testosterone in males or E2 in females. The fact that there were no significant changes in testosterone levels in this study may not be surprising, considering the individual variation normally observed in testosterone levels in rats (Overpeck et al., 1978
) and that 750 mg/kg of I3C were needed to cause a significant effect in shorter-term studies (Wilson et al., 1999
). Clinical trials with I3C have documented reductions in urinary E2 levels in both men and women concurrent with an increase in the 2-OH-E2/16a-OH-E2 ratio (Michnovicz et al., 1997
) and absorption-enhanced DIM also increased this ratio in a pilot clinical study (Zeligs et al., 2002
).
The basis for investigation of both the 25-OH-D3 levels and bone density stem from reports of individuals with low 25-OH-D3 and a decrease in bone density while on I3C. The concern regarding vitamin D3 is that enzyme induction in the colon or liver could influence levels of this vitamin/hormone, which in turn could effect bone density. It has also been shown that bone density can be influenced by estrogen metabolism, especially CYP1A-dependent 2-hydroxylation (Leelawattana et al., 2000). There appears to be greater bone density with lower ratios of 2-OH/ 16a-OH estrogen levels in postmenopausal women, implying that increasing this ratio with I3C or DIM could result in lower bone density. No effect on bone density was observed in this study following I3C or DIM exposure. DIM had no significant effects on 25-OH-D3 levels; however, significant increases were observed in males fed I3C for 3 or 12 months and in females fed I3C for 12 months.
The absence of data indicating toxicity in the chemistry panel and other blood work was confirmed by the histopathological examination. Other than the increase in hyaline casts in the kidney, no apparent lesions were observed in any of the tissues examined. The appearance and severity of this kidney pathology increased with age but was not treatment related.
The induction of CYP isoforms observed in this study are mostly consistent with data from previous acute or subchronic studies. The 24-, 3-, and 4- fold induction of CYPs 1A1, 1A2, and 3A1/2, respectively, in male Fischer 344 rats consuming 0.2% I3C in their diet for 7 days (Stresser et al., 1994) can be compared to the 82-, 40-, and 2- fold inductions observed in male Sprague-Dawley rats in this study after administration of a similar dose of I3C over a much longer time.
Whereas direct toxicity by long-term exposure to I3C and DIM is not evident in this study, the induction of CYPs, especially those of the 1A subfamily could be a cause for concern, given the role of these enzymes in activation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene and aromatic amines such as 4-aminobiphenyl or PhIP. The induction of CYP 3A may also be significant, as this subfamily contributes to the metabolism of 60% of all clinically relevant drugs (Guengrich, 1999). The dampened induction of CYPs seen with DIM exposure may result in fewer drug interactions with DIM supplementation, when compared to I3C. When all endpoints in this study are considered in the comparison between I3C and DIM, the differences can be contributed to a magnified effect in the increased liver somatic index, total CYP, and induction of specific CYPs in the I3C treated group. The higher efficacy/potency of I3C is expected and related to the fact that in the acidic conditions of the stomach after oral exposure, I3C becomes a complex mixture of not only DIM but more than 20 different I3C-derived compounds, all having pharmacological/toxicological effects, such as possessing different affinities for the Ah receptor. One of these compounds, ICZ, binds to the Ah receptor with an affinity similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). DIM has been shown to be relatively more stable in acid and does not robustly undergo further condensation reactions.
The data from this study confirms results from short-term studies indicating that both I3C and DIM are relatively non-toxic compounds. Furthermore, these results confirm earlier long-term feeding studies in other models, including the rainbow trout and the same strain of rat used in the present study, that I3C is not a complete carcinogen (Dashwood et al., 1991; Oganesian et al., 1999
; Stoner et al., 2002
). A concern, however, with the prolonged use of I3C for cancer chemoprevention is its potential for promotion of liver neoplasms. I3C is an effective promoter of liver cancer in the trout (Dashwood et al., 1991
; Oganesian et al., 1999
) and recent studies in a multi-organ model (female Sprague-Dawley rats initiated with 7,12-dimethylbenz[a]anthracene for breast, aflatoxin B1 for liver and azoxymethane for colon) demonstrate that long-term post-initiation with dietary I3C could provide some chemoprotection in breast and colon, but not without a significant increased risk for liver neoplasms (Stoner et al., 2002
). The long-term post-initiation effects of I3C in hepatocarcinogenesis are not consistent across species, as this treatment with C57 black mice, initiated at 15 days of age with diethylnitrosamine, provided significant protection (Oganesian et al., 1997
). The mechanism(s) of I3C tumor modulation need to be established in these models in order to assess the risk to human health.
Previous work from our laboratory utilizing the rainbow trout has shown both I3C and DIM to be estrogenic (Shilling and Williams, 2000; Shilling et al., 2001
). The estrogenicity of I3C is a likely mechanism by which I3C promotes hepatocarcinogenesis in trout (Oganesian et al., 1999
). We have not yet tested DIM in trout as a tumor promoter. DIM is primarily an anti-estrogen in mammalian systems (Chen et al., 1998
; McDougal et al., 2001
). We hypothesize that this difference may be a function of species-specific DIM metabolism. We have preliminary evidence that CYP-dependent hydroxylation of DIM is required in trout to elicit estrogenicity (Shilling et al., 2001
)
Also confirmed were the alterations observed in the monooxygenase system that could be important in carcinogen bioactivaion/detoxication and potential adverse effects on drug metabolism. The results from this study suggest that DIM is a markedly less efficacious inducer of CYP in the rat, but further studies are required to investigate the effects of both I3C and DIM on carcinogenesis, metabolism, and human health.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Arnao, M. B., Sanchez-Bravo, J., and Acosta, M. (1996). Indole-3-carbinol as a scavenger of free radicals. Biochem. Mol. Biol. Int. 39, 11251134.[ISI][Medline]
Arneson, D. W., Hurwitz, A., Crowell, J. A., and Mayo, M. S. (2001). Pharmacokinetics of 3,3'-diindolylmethane following oral administration of indole-3-carbinol to human subjects. Proc. Am. Assoc. Cancer Res. 42, 4658 (Abstract).
Bills, C. E., Eisenber, H., and Pallante, S. L. (1971). Complexes of organic acids with calcium phosphate: The von Kossa stain as a clue to the composition of bone mineral. Johns Hopkins Med. J. 128, 194207.[ISI][Medline]
Bjeldanes, L. F., Kim, J., Grose, K. R., Bartholmoew, J. C., and Bradfield, C. A. (1991). Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: Comparison with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. U.S.A. 88, 95439547.[Abstract]
Bonnesen, C., Eggleston, I. M., and Hayes, J. D. (2001). Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res. 61, 61206130.
Bradfield, C. A., and Bjeldanes, J. F. (1987). Structure-activity relationships of dietary indoles: A proposed mechanism of action as modifiers of xenobiotic metabolism. J. Toxicol. Environ. Health 21, 311322.[ISI][Medline]
Bradlow, H. L., Sepkovic, D. W., Telang, N. T., and Osborne, M. P. (1999). Multifunctional aspects of the action of indole-3-carbinol as an antitumor agent. Ann. N.Y. Acad. Sci. 889, 204213.
Chen, D. Z., Qi, M., Auborn, K. J., and Carter, T. H. (2001). Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic preneoplastic cervical epithelium. J. Nutr. 131, 32943302.
Chen, I., McDougal, A., Wang, F., and Safe, S. (1998). Aryl hydrocarbon receptor mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19, 16311639.[Abstract]
Dashwood, R. H., Fong, A. T., Arbogast, D. N., Bjeldanes, L. F., Hendricks, J. D., and Bailey, G. S. (1994). Anticarcinogenic activity of indole-3-carbinol acid products: Ultrasensitive bioassay by trout embryo microinjection. Cancer Res. 54, 36173619.[Abstract]
Dashwood, R. H., Fong, A. T., Williams, D. E., Hendricks, J. D., and Bailey, G. S. (1991). Promotion of aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: Influence of dose, duration and intermittent exposure on indole-3-carbinol promotional potency. Cancer Res. 51, 23622365.[Abstract]
Dashwood, R. H., Uyetake, L., Fong, A. T., Hendricks, J. D., and Bailey, G. S. (1989). In vivo disposition of the natural anti-carcinogen indole-3-carbinol after PO administration to rainbow trout. Food Chem. Toxicol. 27, 385392.[CrossRef][ISI][Medline]
Dunn, S. E., and LeBlanc, G. A. (1994). Hypocholesterolemic properties of plant indoles. Inhibition of acyl-CoA:cholesterol acyltransferase activity and reduction of serum LDL/VLDL cholesterol levels by glucobrassicin derivatives. Biochem. Pharmacol. 47, 359364.[CrossRef][ISI][Medline]
Fong, A. T., Swanson, H. I., Dashwood, R. D., Williams, D. E., Hendricks, J. D., and Bailey, G. S. (1990). Mechanism of anti-carcinogenesis by indole-3-carbinol: Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation. Biochem. Pharmacol. 39, 1926.[CrossRef][ISI][Medline]
Ge, X., Yanni, S., Rennert, G., Gruener, N., and Fares, F. A. (1996). 3,3'-Diindolylmethane induces apoptosis in human cancer cells. Biochem. Biophys. Res. Commun. 228, 153158.[CrossRef][ISI][Medline]
Guengerich, F. P. (1989). Analysis and characterization of enzymes. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 777814. Raven Press, New York.
Guengrich, F. P. (1999). Cytochrome P450 3A4: Regulation and role in drug metabolism. Annu. Rev. Pharmacol. Toxicol. 39, 117.[CrossRef][ISI][Medline]
Hayes, C. L, Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996). 17ß-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. U.S.A. 93, 97769781.
He, Y. H., Friesen, M. D., Ruch, R. J., and Schut, H. A. (2000). Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: Inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats. Food Chem. Toxicol. 38, 1523.[CrossRef][ISI][Medline]
Herrmann, S., Seidelin, M., Bisgaard, H. C., and Vang, O. (2002). Indolo[3,2-b]carbazole inhibits gap junctional intercellular communication in rat primary hepatocytes and acts as a potential tumor promoter. Carcinogenesis 23, 18611868.
Hong, C., Firestone, G. L., and Bjeldanes, L. F. (2002). Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human breast cancer cells. Biochem. Pharmacol. 63, 10851097.[CrossRef][ISI][Medline]
Horn, T. L., Reichert, M. A., Bliss, R. L., and Malejka-Giganti, D. (2002) Modulations of P450 mRNA in liver and mammary gland and P450 activities and metabolism of estrogen in liver by treatment of rats with indole-3-carbinol. Biochem. Pharmacol. 64, 393404.[CrossRef][ISI][Medline]
Huffer, W. E., and Lepoff, R. B. (1992). An indirect method of measuring widths suitable for automated bone histomorphometry. J. Bone Miner. Res. 7, 14171427.[ISI][Medline]
Huffer, W. E., Ruegg, P., Zhu, J. M., and Lepoff, R. B. (1994). Semiautomated methods for cancellous bone histomorphometry using a general-purpose video image analysis system. J. Microsc. 173, 5366.[ISI][Medline]
Ioannides, C., and Parke, D. V. (1993). Induction of cytochrome P4501 as an indicator of potential chemical carcinogenesis. Drug Metab. Rev. 25, 485501.[ISI][Medline]
Jellinck, P. H., Forkert, P. G., Riddick, D. S., Okey, A. B., Michnovicz, J. J., and Bradlow, H. L. (1993) Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation. Biochem. Pharmacol. 45, 11291136.[CrossRef][ISI][Medline]
Katchamart, S., Stresser, D. M., Dehal, S. S., Kupfer, D., and Williams, D. E. (2000). Concurrent flavin-containing monooxygenase down-regulation and cytochrome P-450 induction by dietary indoles in rat: Implications for drug-drug interaction. Drug Metab. Dispos. 28, 930936.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[ISI][Medline]
Larsen-Su, S., and Williams, D. E. (1996). Dietary indole-3-carbinol inhibits FMO activity and the expression of flavin-containing monooxygenase form 1 in rat liver and intestine. Drug Metab. Dispos. 24, 927931.[Abstract]
Leelawattana, R., Ziambaras, K., Roodman-Weiss, J., Lyss, C., Wagner, D., Klug, T., Armamento-Villareal, R., and Civitelli, R. (2000). The oxidative metabolism of estradiol conditions postmenopausal bone density and bone loss. J. Bone Miner. Res. 15, 25132520.[ISI][Medline]
Leete, E., and Marion, L. (1953). The hydrogenolysis of 3-hydroxymethylindole and other indole derivatives with lithium aluminum hydride. Can. J. Chem. 31, 775784.[ISI]
Leong, H., Firestone, G. L., and Bjeldanes, L. F. (2001). Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression. Carcinogenesis 22, 18091817.
Lord, G. H., and Newberue, P. M. (1990). Renal mineralizationala ubiquitous lesion in chronic rat studies. Food Chem. Toxicol. 28, 449455.[CrossRef][ISI][Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
Manson, M. M., Ball, H. W. L., Barrett, M. C., Clark, H. L., Judah, D. J., Williamson, G., and Neal, G. E. (1997). Mechanism of action of dietary chemoprotective agents in rat liver: Induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism. Carcinogenesis 18, 17291738.[Abstract]
McDanel, R., McLean, A. E., Hanley, A. B., Heaney, R. K., and Fenwick, G. R. (1988). Chemical and biological properties of indole glucosinolates (glucobrassicin): A review. Food Chem. Toxicol. 26, 5970.[CrossRef][ISI][Medline]
McDougal, A., Gupta, M. S., Morrow, D., Ramamoorthy, K., Lee, J. E., and Safe, S. H. (2001). Methyl-substituted diindolylmethanes as inhibitors of estrogen-induced growth of T47D cells and mammary tumors in rats. Breast Cancer Res. Treat. 66, 147157.[CrossRef][ISI][Medline]
Michnovicz, J. J., Adlercreutz, H., and Bradlow, H. L. (1997). Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans. J. Natl. Cancer Inst. 89, 718723.
Murillo, G., and Mehta, R. G. (2001). Cruciferous vegetables and cancer prevention. Nutr. Cancer 41, 1728.[CrossRef][ISI][Medline]
NCI, DCPC, Chemoprevention Branch and Agent Development Committee (1996). Clinical development plan: Indole-3-carbinol. J. Cellul. Biochem. 26S, 127136.[CrossRef]
Newbold, R. R., and Liehr, J. G. (2000). Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. Cancer Res. 60, 235237.
Oganesian, A., Hendricks, J. D., Pereira, C. B., Orner, G. A., Bailey, G. S., and Williams, D. E. (1999). Potency of dietary indole-3-carbinol as a promoter of alfatoxin B1-initiated hepatocarcinogenesis: Results from a 9000 animal tumor study. Carcinogenesis 20, 453458.
Oganesian, A., Hendricks, J. D., and Williams, D. E. (1997) Long tern dietary indole-3-carbinol inhibits diethylnitrosamine-initiated hepatocarcinogenesis in the infant mouse model. Cancer Lett. 118, 8794.[CrossRef][ISI][Medline]
Omura R., and Sato, T. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemeprotein nature. J. Biol. Chem. 234, 23702378.
Overpeck, J. G., Colson, S. H., Hohmann, J. R., Applestine, M. S., and Reilly, J. F. (1978). Concentrations of circulating steroids in normal prepubertal and adult male and female humans, chimpanzees, rhesus monkeys, rats, mice and hamsters: A literature survey. J. Toxicol. Environ. Health 4, 785803.[ISI][Medline]
Park, J. Y., and Bjeldanes, L. F. (1992). Organ-selective induction of cytochrome P-450-dependent activities by indole-3-carbinol-derived products: Influence on covalent binding of benzo[a]pyrene to hepatic and pulmonary DNA in the rat. Chem. Biol. Interact. 83, 235247.[CrossRef][ISI][Medline]
Preobrazhenskaya, M. N., Bukhman, V. M., Korolev, A. M., and Efimov, S. A. (1993). Ascorbigen and other indole-derived compounds from Brassica vegetables and their analogs as anticarcinogenic and immunomodulating agents. Pharmacol. Ther. 60, 301313.[CrossRef][ISI][Medline]
SAS (1999). SAS online doc., version 8. SAS Institute, Inc., Cary, NC.
Shertzer, H. G., Berger, M. L., and Tabor, M. W. (1988). Intervention in free radical mediated hepatotoxicity and lipid peroxidation by indole-3-carbinol. Biochem. Pharmacol. 37, 333338.[CrossRef][ISI][Medline]
Shertzer, H. G., and Senft, A. P. (2000). The micronutrient indole-3-carbinol: Implications for disease and chemoprevention. Drug Metab. Drug Interact. 17, 159188.[Medline]
Shilling, A. D., Carlson, D. B., Katchamart, S., and Williams, D. E. (2001) 3,3'-Diindolylmethane, a major condensation product of indole-3-carbinol, is a potent estrogen in the rainbow trout. Toxicol. Appl. Pharmacol. 170, 191200.[CrossRef][ISI][Medline]
Shilling, A. D., and Williams, D. E. (2000) Determining relative estrogenicity by quantifying vitellogenin induction in rainbow trout liver slices. Toxicol. Appl. Pharmacol. 164, 330335.[CrossRef][ISI][Medline]
Slominski, B. A., and Campbell, L. D. (1987). Formation of indole glucosinolate breakdown products in autolyzed, steamed and cooked Brassica vegetables. J. Agric. Food. Chem. 37, 12971302.
Spande, T. F. (1979). Hydroxyindoles, indoles, alcohols and indolethiols. In Indoles, Part 3 (W. J. Houlihan, Ed.), pp. 1355. John Wiley & Sons, New York.
Stoner, G., Casto, B., Ralston, S., Roebuck, B., Pereira, C., and Bailey, G. (2002). Development of a multi-organ rat model for evaluating chemopreventive agents: Efficacy of indole-3-carbinol. Carcinogenesis 23, 265272.
Stresser, D. M., Bailey, G. S., and Williams, D. E. (1994). Indole-3-carbinol and ß-naphthoflavone induction of aflatoxin B1 metabolism and cytochrome P-450 associated with bioactivation and detoxification of aflatoxin B1 in the rat. Drug Metab. Dispos. 22, 383391.[Abstract]
Stresser, D. M., Williams, D. E., Griffen, D. A., and Bailey, G. S. (1995). Mechanism of tumor modulation by indole-3-carbinol: Disposition and excretion in male Fischer 344 rats. Drug Metab. Dispos. 23, 965975.[Abstract]
Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 43504354.[Abstract]
Vang, O., Jensen, M. B., and Autrup, H. (1990). Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone. Carcinogenesis 11, 12591263.[Abstract]
Wattenberg, L. W., and Loub, W. D. (1978). Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 38, 14101413.[Abstract]
Westfall, P., Tobias, R. D., Rom, D., Wolfinger, R. D., and Hochberg, Y. (1999). Multiple Comparisons and Multiple Tests Using the SAS System, pp. 231235. SAS Institute, Inc., Cary, NC.
Wilson, V. S., McLachlan, J. B., Falls, J. G., and LeBlanc, G. A. (1999). Alteration in sexually dimorphic testosterone biotransformation profiles as a biomarker of chemically induced androgen disruption in mice. Environ. Health Perspect. 107, 377384.[ISI][Medline]
Wortelboer, H. M., de Druif, C. A., van Iersel, A. A. J., Falke, H. E., Noordhoek, J., and Blaauboer, B. J. (1992). Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and Phase II enzymes in rat and monkey hepatocytes. Biochem. Pharmacol. 43, 14391447.[CrossRef][ISI][Medline]
Xu, M., Schut, H. A., Bjeldanes, L. F., Williams, D. E., Bailey, G. S., and Dashwood, R. H. (1997). Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: Dose-response studies in the rat colon. Carcinogenesis 18, 21492153.[Abstract]
Zeligs, M. A., Sepkovic, D. W., Manrique, C., Macsalka, M., Williams, D. E., Liebelt, D. A., and Bradlow, H. L. (2002). Absorption-enhanced 3,3'diindolylmethane: Human use in HPV-related, benign and pre-cancerous conditions. Proc. Amer. Assoc. Cancer Res. 43, 3198 (Abstract).