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Indole-3-carbinol modulation of hepatic monooxygenases CYP1A1, CYP1A2 and FMO1 in guinea pig, mouse and rabbit
Comparative Biochemistry and Physiology Part C 129 Ž2001. 377᎐384
Indole-3-carbinol modulation of hepatic monooxygenases CYP1A1, CYP1A2 and FMO1 in guinea pig, mouse and rabbit S. Katchamart, D.E. WilliamsU Department of En¨ ironmental and Molecular Toxicology and the Linus Pauling Institute, Oregon State Uni¨ ersity, Cor¨ allis, OR 97331-6512, USA Received 21 February 2001; received in revised form 27 May 2001; accepted 30 May 2001
Abstract Indole-3-carbinol ŽI3C., a major component of cruciferous vegetables, has been shown to be chemoprotective against cancer in a number of animal models and is being evaluated as a potential agent to prevent breast cancer in healthy women. Some concern has been raised related to the long-term use of I3C, as in some models chronic dietary post-initiation exposures promote cancers. I3C administration to rats marked induces several cytochrome P450s ŽCYPs., especially CYP1A1 Žapprox. 25-fold., while at the same time inhibiting the expression of FMO1. The consequence is a marked shift in the metabolic profile of drugs such as nicotine and tamoxifen, that are substrates for both monooxygenases. Such an effect could lead to adverse drug reactions in humans. In order to determine if the effect of I3C was manifest in species other than the rat, we fed 2000-ppm I3C to male guinea pigs, mice and rabbits for a period of 4 weeks. In each species, induction of CYP1A1r1A2 expression was observed in the liver but little or no effect on FMO1 was evident, with the possible exception of the rabbit. These data demonstrate that the ability of I3C to both induce CYP1A1 and inhibit FMO1, as observed in the rat, may not be common to other mammals for which FMO1 is the major isoform in the liver. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Cytochrome P450; FMO; Indole-3-carbinol; Phytochemicals; Drug metabolism; Monooxygenases
1. Introduction Indole-3-carbinol ŽI3C. is a breakdown product of glucobrassin, a major component of cruciferous vegetables ŽMcDannell and McLean, 1988.
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Corresponding author. Tel.: q1-541-737-3277; fax: q1541-737-5077. E-mail address: [email protected] ŽD.E. Williams..
and is also available as a dietary supplement. I3C has been documented to be chemopreventive against cancer in a number of animal models and is being investigated by NCI as a potential chemopreventive agent against breast cancer ŽBradlow et al., 1991; Grubbs et al., 1995; Guo et al., 1995; Jin et al., 1999; Manson et al., 1998; Nixon et al., 1984; Oganesian et al., 1997; Wattenberg and Loub, 1978; Wong et al., 1997; Xu and Dashwood, 1999.. Our laboratory and others
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have raised potential concerns about the longterm use of I3C based on findings showing it to be a tumor promoter in the liver of trout and rat ŽDashwood et al., 1991; Kim et al., 1994; Oganesian et al., 1999.. We have been interested in the mechanismŽs. of action of I3C as a tumor modulator in order to better predict the riskrbenefits for long-term usage in humans. Following oral administration, I3C is rapidly converted in the GI tract through a series of acid catalyzed polymerization reactions, to oligomers including dimers, trimers and tetramers ŽBjeldanes et al., 1991; DeKruif et al., 1991; Stresser et al., 1995.. One postulated mechanism for the blocking effects of I3C is through the ability of a number of these acid condensation products to induce phase I and phase II carcinogen drug metabolizing enzymes by acting as ligands for the Aryl Hydrocarbon Receptor ŽAHR. ŽBjeldanes et al., 1991.. Upon dietary administration of I3C to rats, we observed a 25fold induction of CYP1A1 in liver along with more modest Ž2᎐4-fold. induction of CYPs 1A2, 2B1r2 and 3A1r2 ŽStresser et al., 1994a. as well as induction of glutathione S-transferase ŽGST.Yc2 ŽHayes et al., 1998; Stresser et al., 1994b.. In contrast, to the induction of CYP1A1 and GST Yc2, administration of I3C to male Fischer 344 rats resulted in a dose- and time-dependent down-regulation of the major flavin-containing monooxygenase ŽFMO. in rodent liver, FMO1 ŽLarsen-Su and Williams, 1996; Katchamart et al., 2000.. FMO catalyzes the oxygenation of numerous drugs and xenobiotics containing a softnucleophile, such as N, S or Se ŽCashman, 1995.. FMO and CYP are often active towards the same xenobiotic or drug substrate but usually produce distinct metabolites Ž N-oxygenation. or exhibit distinct stereoselectivity Ž S-oxygenation.. For example, with tertiary amines, FMO only yields the N-oxide whereas CYP primarily catalyzes N-demethylation ŽZiegler, 1984.. As is the case with CYP, FMO exists as a gene superfamily, but with only six families each containing a single member ŽFMOs 1᎐6. ŽHines et al., 1994; Lawton et al., 1994.. Again, as with CYPs, isoforms of FMO are expressed in a developmental- and tissue-specific fashion Žreviewed in Cashman, 1995; Ziegler, 1993., but are not inducible by xenobiotics. FMO1 is the major FMO in the adult liver of most animals with the notable
exception of primates and certain mouse strains. In humans, FMO1 is the major FMO in fetal liver; shortly after birth expression of FMO1 is ameliorated in liver and replaced by FMO3. In adult human kidney, the major FMO is FMO1 ŽCashman, 2000.. Recently, we have documented that liver microsomes isolated from rats fed I3C exhibit concurrent CYP1A1 induction and FMO1 repression, resulting in markedly different metabolites with tertiary amines such as N, N-dimethylaniline, tamoxifen or nicotine ŽKatchamart et al., 2000., confirming our hypothesis concerning potential adverse drug interactions Žat least in vitro.. In order to better predict whether or not such an effect could exist in humans, we initiated a study to determine if a similar effect could be observed in other common laboratory animals. Feeding I3C to male guinea pig, mice or rabbits induced CYP1A1 expression in liver with no concurrent repression of FMO1 expression. Based on these findings we conclude that CYP1A1 induction and FMO1 repression by dietary I3C occur through independent mechanisms and that FMO1 downregulation by I3C may not be common to other mammals expressing FMO1 in the liver.
2. Material and methods 2.1. Chemicals and diet I3C was purchased from Aldrich Chemical Co. ŽMilwaukee, WI, USA. and incorporated into powdered, synthetic diets prepared without preservatives and just prior to initiation of the experiment. The diets were stored frozen in the dark and protected from air until the day of feeding. Goat anti-rabbit CYP1A1r2 was from Gentest ŽWoburn, MA, USA.. This antibody cross-reacts both with CYP1A1 and CYP1A2 and although rat CYP1A1 and CYP1A2 can be resolved by SDS-PAGE, this was not the case with guinea pig, mouse and rabbit and therefore, the results presented represent the sum of CYP1A1 and CYP1A2. Rabbit anti-porcine FMO1 was a generous gift from Dr Daniel Ziegler ŽUniversity of Texas at Austin.. Although this polyclonal antibody appears to recognize a single band on western blots, the absolute specificity with respect to possible cross-reaction with other FMO isoforms, has not been tested ŽD.M. Ziegler,
S. Katchamart, D.E. Williams r Comparati¨ e Biochemistry and Physiology Part C 129 (2001) 377᎐384
personal communication.. Rabbit anti-goat and goat anti-rabbit secondary antibody, conjugated to horseradish peroxidase, were from Bio-Rad ŽRichmond, CA, USA.. The chemiluminescence kit was purchased from Amersham Corp. ŽArlington Heights, IL, USA..
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2.4. Statistical analysis Statistical analyses of the data were performed using the Student’s t-test. All data points are the mean " S.D. for four animals per group; P-values less than 0.05 were considered significant.
2.2. Animals 3. Results and discussion Weanling male Hartley guinea pigs and C57BLr6J mice were purchased from Simonsens Laboratories, Inc. ŽGilroy, CA, USA.. Weanling male New Zealand White rabbits were purchased from Western Oregon Rabbit Co. ŽPhilomath, OR, USA.. The animals were acclimated for 7 days on powdered diet ŽAIN76A for mouse, guinea pig diet, semipurified for guinea pigs and rabbit diet, purified for rabbits, all from ICN Biomedicals, Inc., Costa Mesa, CA, USA. before being switched to diet containing 2000-ppm I3C and fed ad libitum for 4 weeks. The animals were euthanized with CO 2 and the livers immediately removed, frozen in liquid nitrogen and stored at y80⬚C until analysis. The protocols used in this study were approved by the Oregon State University Institutional Animal Care and Use Committee. 2.3. Microsome preparation and immunodetection of CYP1A1r 1A2 and FMO1 Liver microsomes were prepared by ultracentrifugation as previously described ŽGuengerich, 1989.. Protein was measured by the method of Lowry et al. Ž1951.. The liver microsomal proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ŽSDS-PAGE. ŽLaemmli, 1970. and electrophorectically transferred to nitrocellulose membranes ŽTowbin et al., 1979.. The blots were incubated in blocking buffer containing 2% bovine serum albumin in phosphate-buffered saline ŽPBS., followed by the primary antibody, goat anti-rat CYP1A1r1A2 or rabbit anti-porcine FMO1. After washing in PBS-Tween 20, the blots were probed with rabbit anti-goat IgG or goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase and then visualized using a chemiluminescence kit. Quantification was performed by densitometry, using a Hewlett-Packard ScanJet IIcx flatbed scanner employing NIH Image software version 1.54 Žpublic domain, NIH..
Previous results from our laboratory and others have documented that dietary exposure to I3C markedly induces a number of hepatic CYPs in the rat, especially CYP1A1 ŽBjeldanes et al., 1991; Stresser et al., 1994a.. I3C is converted into a number of acid condensation products in the GI tract that are capable of binding to the aryl hydrocarbon receptor ŽAHR. and eliciting CYP1A induction ŽBjeldanes et al., 1991.. The cancer chemopreventive properties of I3C may be mediated in part through this mechanism, although recently other potential mechanisms, including regulation of apoptosis and cell cycle control have been documented for various I3C acid condensation products ŽChang et al., 1999; Cover et al., 1998, 1999; Ge et al., 1996, 1999; Katdare et al., 1998.. In the present study we have shown that the AHR-mediated induction of CYP1A1r1A2 by I3C is not limited to the rat. CYP1A1r1A2 levels in liver microsomes from juvenile male guinea pig ŽFig. 1a. and rabbit ŽFig. 1c. were below the limits of detection in animals fed control diet, but were markedly elevated following dietary exposure to 2000-ppm I3C. CYP1A1r1A2 was expressed constitutively at low levels in the mouse and markedly induced following exposure to dietary I3C. It is of interest to note that constitutive hepatic expression of CYP1A2 in guinea pig and rabbit is below the limits of detection. This differs markedly from the situation in human liver, in which CYP1A2 is a major constitutive isoform of CYP ŽGuengerich, 1995.. Our laboratory has characterized an interesting finding with respect to the effect of dietary I3C on liver monooxygenases in the juvenile male rat. As stated above, CYP1A1r1A2 is markedly induced, whereas the other major monooxygenase in liver FMO1, is down regulated in a dose- and time-dependent fashion ŽLarsen-Su and Williams, 1996.. The net result for tertiary amine substrates such as N, N⬘-dimethylaniline, nicotine or tamox-
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Fig. 1. Western blots of hepatic microsomal proteins probed with antibody to CYP1A1r1A2. Lanes 1, 2 and 3 are 0.18, 0.45 and 1.1 pmole of purified CYP1A1 standard, respectively; lanes 4᎐7 and 8᎐11 contained 20 g of liver microsomal protein from animals fed control diet or 2000-ppm I3C containing diets, respectively. Ža. Guinea pig, Žb. mouse, and Žc. rabbit.
ifen, is a metabolic shift away from N-oxygenation and in some cases enhanced N-demethylation ŽKatchamart et al., 2000.. If a similar phenomenon occurs in humans taking I3C as a dietary supplement or as a chemopreventive agent against breast cancer, there would be the potential risk for adverse drug reactions. In order to determine how broadly this phenomenon occurs across species Žand thereby enhance predictability for humans., we assessed the levels of liver FMO1 expression following dietary administration of I3C to juvenile male guinea pigs, mice and rats at doses and for length of exposures known to inhibit rat hepatic FMO1 expression by over 75᎐90% ŽLarsen-Su and Williams, 1996; Katchamart et al., 2000.. FMO1 is expressed at fairly high levels in male guinea pig liver microsomes. Dietary administration of I3C to male guinea pigs did not significantly alter the expression of hepatic FMO1 ŽFig. 2a, and Fig. 3.. In a previous study, total liver FMO activity, as determined by N, N⬘dimethylaniline N-oxygenation, was reduced significantly in ascorbic acid-deficient andror caloric-restricted guinea pigs ŽBrodfuehrer and Zannoni, 1987.. Currently, relative to the rat and mouse,
little is known concerning the physiological and nutritional factors that determine expression of FMO in guinea pig. Compared to the rat, the mouse has previously been shown to exhibit the opposite sex-dependent expression of FMO1. In rat, FMO1 expression in liver is markedly higher in males than females ŽDannan et al., 1986; Lemoine et al., 1991.. In the mouse, FMO1 expression in liver is lower in males ŽCherrington et al., 1998; Duffel et al., 1981; Falls et al., 1995, 1997; Wirth and Thorgeirrson, 1978.. In both species, the expression of hepatic FMO1 appears to be due to repression by sex steroids, estradiol in the case of the female rat and testosterone in the case of the male mouse. Based on these previous results, it was not surprising to see that FMO1 levels in control male C57 black mice was markedly lower than male guinea pig Ž4-fold higher than mice. and rabbit Ž6-fold higher than mice. ŽFig. 2b and Fig. 3.. Administration of I3C in the diet did not significantly repress hepatic FMO1 expression in male mice. The rabbit expresses relatively high levels of FMO1 in the liver ŽFig. 2c and Fig. 3., but unlike the rat or mouse, does not exhibit a sex difference ŽShehin-Johnson et al., 1995.. Although the levels
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Fig. 2. Western blots of hepatic microsomal proteins probed with antibody to FMO1. Lanes 1, 2 and 3 correspond to 0.5, 0.9 and 1.8 pmole of purified porcine FMO1 standard; lanes 4᎐7 and 8᎐11 contained 40 g Žguinea pig and rabbit. or 48 g Žmouse. of liver microsomal protein from animals fed control diet or 2000-ppm I3C diet, respectively. Ža. Guinea pig, Žb. mouse, and Žc. rabbit.
of liver FMO1 in rabbits fed I3C declined by about 40%, this difference did not quite reach statistical significance Ž Ps 0.06., primarily due to a relatively large interindividual variability in expression in the control group.
In summary, weanling males from all three species resembled the juvenile male rat in responding to dietary administration of I3C with a marked induction of CYP1A1r1A2. This is not surprising given that induction is AHR-dependent
Fig. 3. Densitometry of FMO1 expression in animals fed control diet or I3C. The bands on the western blots shown in Fig. 2 were scanned and quantified by densitometry. The data are reported as pmol FMO1 Žporcine FMO1 equivalents.rmg microsomal protein. There was no statistical Ž P- 0.05. difference between control and I3C treatment groups for any of the three species.
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and all these mammals possess a functional AHR in liver beginning very early in development. In contrast, the down-regulation of FMO1 expression in the liver was not evident, with the possible exception of the rabbit. Even in the latter case, the degree of repression Ž40%. was markedly less than in the rat Ž75᎐90%. under this exposure protocol. The mechanism by which I3C down-regulates FMO1 expression in male rat liver and intestine is currently unknown, but may involve indirect effects mediated through perturbations in hormone homeostasis. Such an effect may only be evident in sexually immature animals, as we have not observed repression of FMO1 in longterm feeding trials of mature males. FMO1 expression is regulated by growth hormone and glucocorticoids in addition to, or in combination with, sex steroids ŽCoecke et al., 1998; Lee et al., 1993; Lemoine et al., 1991.. The down regulation of FMO1 in weanling male rats takes 4 weeks to plateau and cannot be reproduced in vitro with rat liver slices Žin which CYP1A1 induction with I3C is evident., again supporting the involvement of an indirect mechanism. The evidence to date would indicate that humans might not be at risk for this potential adverse supplement-drug interaction, as the effect is not evident in all mammals. We, as yet, do not know if the effect is also FMO isoform-selective or only observed in sexually immature males. Humans express predominantly FMO1 in fetal and neonatal liver, and shortly after birth repress FMO1 and express FMO3. Female mice resemble adult humans in expressing FMO3 in the liver. Preliminary studies with dietary administration of I3C does not show a repression of FMO3 expression, supporting the hypothesis that the modulation by I3C may be isoform selective ŽKatchamart et al., unpublished.. Dietary exposure of I3C to pregnant women could alter the expression of CYPs and FMO1 in the liver of the fetus. We have found that I3C administered to pregnant rats through the diet is bioavailable to the fetus and induces CYP1A1r2 and CYP1B1; however, we find no evidence of down-regulation of FMO1 ŽLarsen-Su and Williams, submitted to Toxicol. Sci... Future studies need to address the potential for hormonal or dietary modulators of FMO Žsuch as I3C. to alter FMO expression in humans at various stages of development in order to predict potential adverse drug interactions with FMO substrates.
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T.M., Bailey, G.S., 1994b. Indole-3-carbinol induces a rat liver glutathione transferase subunit ŽYc2. with high activity toward aflatoxin B 1 exo-epoxide: association with reduced levels of hepatic aflatoxin-DNA adducts in vivo. Drug Metabol. Dispos. 22, 392᎐399. Stresser, D.M., Williams, D.E., Griffin, D.A., Bailey, G.S., 1995. Mechanisms of tumor modulation by indole-3-carbinol. Disposition and excretion in male Fischer 344 rats. Drug Metab. Dispos. 23, 965᎐974. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350᎐4354. Wattenberg, L.W., Loub, W.D., 1978. Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 38, 1410᎐1413. Wirth, P.J., Thorgeirrson, S.S., 1978. Amine oxidase in mice-sex differences and developmental aspects. Biochem. Pharmacol. 27, 601᎐603. Wong, G.Y., Bradlow, L., Depkovic, D., Mehl, S., Mailman, J., Osborne, M.P., 1997. Dose-ranging study of indole-3-carbinol for breast cancer prevention. J. Cell. Biochem. Suppl. 28, 111᎐116. Xu, M., Dashwood, R.H., 1999. Chemoprevention studies of heterocyclic amine-induced colon carcinogenesis. Cancer Lett. 143, 179᎐183. Ziegler, D.M., 1984. Metabolic oxygenation of organic nitrogen and sulfur compounds. In: Mitchell, J.R., Horning, M.G. ŽEds.., Drug Metabolism and Drug Toxicity. Raven Press, New York, pp. 33᎐53. Ziegler, D.M., 1993. Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Annu. Rev. Pharmacol. Toxicol. 33, 179᎐199.
Indole-3-carbinol modulation of hepatic monooxygenases CYP1A1, CYP1A2 and FMO1 in guinea pig, mouse and rabbit S. Katchamart, D.E. WilliamsU Department of En¨ ironmental and Molecular Toxicology and the Linus Pauling Institute, Oregon State Uni¨ ersity, Cor¨ allis, OR 97331-6512, USA Received 21 February 2001; received in revised form 27 May 2001; accepted 30 May 2001
Abstract Indole-3-carbinol ŽI3C., a major component of cruciferous vegetables, has been shown to be chemoprotective against cancer in a number of animal models and is being evaluated as a potential agent to prevent breast cancer in healthy women. Some concern has been raised related to the long-term use of I3C, as in some models chronic dietary post-initiation exposures promote cancers. I3C administration to rats marked induces several cytochrome P450s ŽCYPs., especially CYP1A1 Žapprox. 25-fold., while at the same time inhibiting the expression of FMO1. The consequence is a marked shift in the metabolic profile of drugs such as nicotine and tamoxifen, that are substrates for both monooxygenases. Such an effect could lead to adverse drug reactions in humans. In order to determine if the effect of I3C was manifest in species other than the rat, we fed 2000-ppm I3C to male guinea pigs, mice and rabbits for a period of 4 weeks. In each species, induction of CYP1A1r1A2 expression was observed in the liver but little or no effect on FMO1 was evident, with the possible exception of the rabbit. These data demonstrate that the ability of I3C to both induce CYP1A1 and inhibit FMO1, as observed in the rat, may not be common to other mammals for which FMO1 is the major isoform in the liver. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Cytochrome P450; FMO; Indole-3-carbinol; Phytochemicals; Drug metabolism; Monooxygenases
1. Introduction Indole-3-carbinol ŽI3C. is a breakdown product of glucobrassin, a major component of cruciferous vegetables ŽMcDannell and McLean, 1988.
U
Corresponding author. Tel.: q1-541-737-3277; fax: q1541-737-5077. E-mail address: [email protected] ŽD.E. Williams..
and is also available as a dietary supplement. I3C has been documented to be chemopreventive against cancer in a number of animal models and is being investigated by NCI as a potential chemopreventive agent against breast cancer ŽBradlow et al., 1991; Grubbs et al., 1995; Guo et al., 1995; Jin et al., 1999; Manson et al., 1998; Nixon et al., 1984; Oganesian et al., 1997; Wattenberg and Loub, 1978; Wong et al., 1997; Xu and Dashwood, 1999.. Our laboratory and others
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have raised potential concerns about the longterm use of I3C based on findings showing it to be a tumor promoter in the liver of trout and rat ŽDashwood et al., 1991; Kim et al., 1994; Oganesian et al., 1999.. We have been interested in the mechanismŽs. of action of I3C as a tumor modulator in order to better predict the riskrbenefits for long-term usage in humans. Following oral administration, I3C is rapidly converted in the GI tract through a series of acid catalyzed polymerization reactions, to oligomers including dimers, trimers and tetramers ŽBjeldanes et al., 1991; DeKruif et al., 1991; Stresser et al., 1995.. One postulated mechanism for the blocking effects of I3C is through the ability of a number of these acid condensation products to induce phase I and phase II carcinogen drug metabolizing enzymes by acting as ligands for the Aryl Hydrocarbon Receptor ŽAHR. ŽBjeldanes et al., 1991.. Upon dietary administration of I3C to rats, we observed a 25fold induction of CYP1A1 in liver along with more modest Ž2᎐4-fold. induction of CYPs 1A2, 2B1r2 and 3A1r2 ŽStresser et al., 1994a. as well as induction of glutathione S-transferase ŽGST.Yc2 ŽHayes et al., 1998; Stresser et al., 1994b.. In contrast, to the induction of CYP1A1 and GST Yc2, administration of I3C to male Fischer 344 rats resulted in a dose- and time-dependent down-regulation of the major flavin-containing monooxygenase ŽFMO. in rodent liver, FMO1 ŽLarsen-Su and Williams, 1996; Katchamart et al., 2000.. FMO catalyzes the oxygenation of numerous drugs and xenobiotics containing a softnucleophile, such as N, S or Se ŽCashman, 1995.. FMO and CYP are often active towards the same xenobiotic or drug substrate but usually produce distinct metabolites Ž N-oxygenation. or exhibit distinct stereoselectivity Ž S-oxygenation.. For example, with tertiary amines, FMO only yields the N-oxide whereas CYP primarily catalyzes N-demethylation ŽZiegler, 1984.. As is the case with CYP, FMO exists as a gene superfamily, but with only six families each containing a single member ŽFMOs 1᎐6. ŽHines et al., 1994; Lawton et al., 1994.. Again, as with CYPs, isoforms of FMO are expressed in a developmental- and tissue-specific fashion Žreviewed in Cashman, 1995; Ziegler, 1993., but are not inducible by xenobiotics. FMO1 is the major FMO in the adult liver of most animals with the notable
exception of primates and certain mouse strains. In humans, FMO1 is the major FMO in fetal liver; shortly after birth expression of FMO1 is ameliorated in liver and replaced by FMO3. In adult human kidney, the major FMO is FMO1 ŽCashman, 2000.. Recently, we have documented that liver microsomes isolated from rats fed I3C exhibit concurrent CYP1A1 induction and FMO1 repression, resulting in markedly different metabolites with tertiary amines such as N, N-dimethylaniline, tamoxifen or nicotine ŽKatchamart et al., 2000., confirming our hypothesis concerning potential adverse drug interactions Žat least in vitro.. In order to better predict whether or not such an effect could exist in humans, we initiated a study to determine if a similar effect could be observed in other common laboratory animals. Feeding I3C to male guinea pig, mice or rabbits induced CYP1A1 expression in liver with no concurrent repression of FMO1 expression. Based on these findings we conclude that CYP1A1 induction and FMO1 repression by dietary I3C occur through independent mechanisms and that FMO1 downregulation by I3C may not be common to other mammals expressing FMO1 in the liver.
2. Material and methods 2.1. Chemicals and diet I3C was purchased from Aldrich Chemical Co. ŽMilwaukee, WI, USA. and incorporated into powdered, synthetic diets prepared without preservatives and just prior to initiation of the experiment. The diets were stored frozen in the dark and protected from air until the day of feeding. Goat anti-rabbit CYP1A1r2 was from Gentest ŽWoburn, MA, USA.. This antibody cross-reacts both with CYP1A1 and CYP1A2 and although rat CYP1A1 and CYP1A2 can be resolved by SDS-PAGE, this was not the case with guinea pig, mouse and rabbit and therefore, the results presented represent the sum of CYP1A1 and CYP1A2. Rabbit anti-porcine FMO1 was a generous gift from Dr Daniel Ziegler ŽUniversity of Texas at Austin.. Although this polyclonal antibody appears to recognize a single band on western blots, the absolute specificity with respect to possible cross-reaction with other FMO isoforms, has not been tested ŽD.M. Ziegler,
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personal communication.. Rabbit anti-goat and goat anti-rabbit secondary antibody, conjugated to horseradish peroxidase, were from Bio-Rad ŽRichmond, CA, USA.. The chemiluminescence kit was purchased from Amersham Corp. ŽArlington Heights, IL, USA..
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2.4. Statistical analysis Statistical analyses of the data were performed using the Student’s t-test. All data points are the mean " S.D. for four animals per group; P-values less than 0.05 were considered significant.
2.2. Animals 3. Results and discussion Weanling male Hartley guinea pigs and C57BLr6J mice were purchased from Simonsens Laboratories, Inc. ŽGilroy, CA, USA.. Weanling male New Zealand White rabbits were purchased from Western Oregon Rabbit Co. ŽPhilomath, OR, USA.. The animals were acclimated for 7 days on powdered diet ŽAIN76A for mouse, guinea pig diet, semipurified for guinea pigs and rabbit diet, purified for rabbits, all from ICN Biomedicals, Inc., Costa Mesa, CA, USA. before being switched to diet containing 2000-ppm I3C and fed ad libitum for 4 weeks. The animals were euthanized with CO 2 and the livers immediately removed, frozen in liquid nitrogen and stored at y80⬚C until analysis. The protocols used in this study were approved by the Oregon State University Institutional Animal Care and Use Committee. 2.3. Microsome preparation and immunodetection of CYP1A1r 1A2 and FMO1 Liver microsomes were prepared by ultracentrifugation as previously described ŽGuengerich, 1989.. Protein was measured by the method of Lowry et al. Ž1951.. The liver microsomal proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ŽSDS-PAGE. ŽLaemmli, 1970. and electrophorectically transferred to nitrocellulose membranes ŽTowbin et al., 1979.. The blots were incubated in blocking buffer containing 2% bovine serum albumin in phosphate-buffered saline ŽPBS., followed by the primary antibody, goat anti-rat CYP1A1r1A2 or rabbit anti-porcine FMO1. After washing in PBS-Tween 20, the blots were probed with rabbit anti-goat IgG or goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase and then visualized using a chemiluminescence kit. Quantification was performed by densitometry, using a Hewlett-Packard ScanJet IIcx flatbed scanner employing NIH Image software version 1.54 Žpublic domain, NIH..
Previous results from our laboratory and others have documented that dietary exposure to I3C markedly induces a number of hepatic CYPs in the rat, especially CYP1A1 ŽBjeldanes et al., 1991; Stresser et al., 1994a.. I3C is converted into a number of acid condensation products in the GI tract that are capable of binding to the aryl hydrocarbon receptor ŽAHR. and eliciting CYP1A induction ŽBjeldanes et al., 1991.. The cancer chemopreventive properties of I3C may be mediated in part through this mechanism, although recently other potential mechanisms, including regulation of apoptosis and cell cycle control have been documented for various I3C acid condensation products ŽChang et al., 1999; Cover et al., 1998, 1999; Ge et al., 1996, 1999; Katdare et al., 1998.. In the present study we have shown that the AHR-mediated induction of CYP1A1r1A2 by I3C is not limited to the rat. CYP1A1r1A2 levels in liver microsomes from juvenile male guinea pig ŽFig. 1a. and rabbit ŽFig. 1c. were below the limits of detection in animals fed control diet, but were markedly elevated following dietary exposure to 2000-ppm I3C. CYP1A1r1A2 was expressed constitutively at low levels in the mouse and markedly induced following exposure to dietary I3C. It is of interest to note that constitutive hepatic expression of CYP1A2 in guinea pig and rabbit is below the limits of detection. This differs markedly from the situation in human liver, in which CYP1A2 is a major constitutive isoform of CYP ŽGuengerich, 1995.. Our laboratory has characterized an interesting finding with respect to the effect of dietary I3C on liver monooxygenases in the juvenile male rat. As stated above, CYP1A1r1A2 is markedly induced, whereas the other major monooxygenase in liver FMO1, is down regulated in a dose- and time-dependent fashion ŽLarsen-Su and Williams, 1996.. The net result for tertiary amine substrates such as N, N⬘-dimethylaniline, nicotine or tamox-
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Fig. 1. Western blots of hepatic microsomal proteins probed with antibody to CYP1A1r1A2. Lanes 1, 2 and 3 are 0.18, 0.45 and 1.1 pmole of purified CYP1A1 standard, respectively; lanes 4᎐7 and 8᎐11 contained 20 g of liver microsomal protein from animals fed control diet or 2000-ppm I3C containing diets, respectively. Ža. Guinea pig, Žb. mouse, and Žc. rabbit.
ifen, is a metabolic shift away from N-oxygenation and in some cases enhanced N-demethylation ŽKatchamart et al., 2000.. If a similar phenomenon occurs in humans taking I3C as a dietary supplement or as a chemopreventive agent against breast cancer, there would be the potential risk for adverse drug reactions. In order to determine how broadly this phenomenon occurs across species Žand thereby enhance predictability for humans., we assessed the levels of liver FMO1 expression following dietary administration of I3C to juvenile male guinea pigs, mice and rats at doses and for length of exposures known to inhibit rat hepatic FMO1 expression by over 75᎐90% ŽLarsen-Su and Williams, 1996; Katchamart et al., 2000.. FMO1 is expressed at fairly high levels in male guinea pig liver microsomes. Dietary administration of I3C to male guinea pigs did not significantly alter the expression of hepatic FMO1 ŽFig. 2a, and Fig. 3.. In a previous study, total liver FMO activity, as determined by N, N⬘dimethylaniline N-oxygenation, was reduced significantly in ascorbic acid-deficient andror caloric-restricted guinea pigs ŽBrodfuehrer and Zannoni, 1987.. Currently, relative to the rat and mouse,
little is known concerning the physiological and nutritional factors that determine expression of FMO in guinea pig. Compared to the rat, the mouse has previously been shown to exhibit the opposite sex-dependent expression of FMO1. In rat, FMO1 expression in liver is markedly higher in males than females ŽDannan et al., 1986; Lemoine et al., 1991.. In the mouse, FMO1 expression in liver is lower in males ŽCherrington et al., 1998; Duffel et al., 1981; Falls et al., 1995, 1997; Wirth and Thorgeirrson, 1978.. In both species, the expression of hepatic FMO1 appears to be due to repression by sex steroids, estradiol in the case of the female rat and testosterone in the case of the male mouse. Based on these previous results, it was not surprising to see that FMO1 levels in control male C57 black mice was markedly lower than male guinea pig Ž4-fold higher than mice. and rabbit Ž6-fold higher than mice. ŽFig. 2b and Fig. 3.. Administration of I3C in the diet did not significantly repress hepatic FMO1 expression in male mice. The rabbit expresses relatively high levels of FMO1 in the liver ŽFig. 2c and Fig. 3., but unlike the rat or mouse, does not exhibit a sex difference ŽShehin-Johnson et al., 1995.. Although the levels
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Fig. 2. Western blots of hepatic microsomal proteins probed with antibody to FMO1. Lanes 1, 2 and 3 correspond to 0.5, 0.9 and 1.8 pmole of purified porcine FMO1 standard; lanes 4᎐7 and 8᎐11 contained 40 g Žguinea pig and rabbit. or 48 g Žmouse. of liver microsomal protein from animals fed control diet or 2000-ppm I3C diet, respectively. Ža. Guinea pig, Žb. mouse, and Žc. rabbit.
of liver FMO1 in rabbits fed I3C declined by about 40%, this difference did not quite reach statistical significance Ž Ps 0.06., primarily due to a relatively large interindividual variability in expression in the control group.
In summary, weanling males from all three species resembled the juvenile male rat in responding to dietary administration of I3C with a marked induction of CYP1A1r1A2. This is not surprising given that induction is AHR-dependent
Fig. 3. Densitometry of FMO1 expression in animals fed control diet or I3C. The bands on the western blots shown in Fig. 2 were scanned and quantified by densitometry. The data are reported as pmol FMO1 Žporcine FMO1 equivalents.rmg microsomal protein. There was no statistical Ž P- 0.05. difference between control and I3C treatment groups for any of the three species.
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and all these mammals possess a functional AHR in liver beginning very early in development. In contrast, the down-regulation of FMO1 expression in the liver was not evident, with the possible exception of the rabbit. Even in the latter case, the degree of repression Ž40%. was markedly less than in the rat Ž75᎐90%. under this exposure protocol. The mechanism by which I3C down-regulates FMO1 expression in male rat liver and intestine is currently unknown, but may involve indirect effects mediated through perturbations in hormone homeostasis. Such an effect may only be evident in sexually immature animals, as we have not observed repression of FMO1 in longterm feeding trials of mature males. FMO1 expression is regulated by growth hormone and glucocorticoids in addition to, or in combination with, sex steroids ŽCoecke et al., 1998; Lee et al., 1993; Lemoine et al., 1991.. The down regulation of FMO1 in weanling male rats takes 4 weeks to plateau and cannot be reproduced in vitro with rat liver slices Žin which CYP1A1 induction with I3C is evident., again supporting the involvement of an indirect mechanism. The evidence to date would indicate that humans might not be at risk for this potential adverse supplement-drug interaction, as the effect is not evident in all mammals. We, as yet, do not know if the effect is also FMO isoform-selective or only observed in sexually immature males. Humans express predominantly FMO1 in fetal and neonatal liver, and shortly after birth repress FMO1 and express FMO3. Female mice resemble adult humans in expressing FMO3 in the liver. Preliminary studies with dietary administration of I3C does not show a repression of FMO3 expression, supporting the hypothesis that the modulation by I3C may be isoform selective ŽKatchamart et al., unpublished.. Dietary exposure of I3C to pregnant women could alter the expression of CYPs and FMO1 in the liver of the fetus. We have found that I3C administered to pregnant rats through the diet is bioavailable to the fetus and induces CYP1A1r2 and CYP1B1; however, we find no evidence of down-regulation of FMO1 ŽLarsen-Su and Williams, submitted to Toxicol. Sci... Future studies need to address the potential for hormonal or dietary modulators of FMO Žsuch as I3C. to alter FMO expression in humans at various stages of development in order to predict potential adverse drug interactions with FMO substrates.
Acknowledgements We thank Dr Daniel Ziegler for his gift of purified porcine FMO1 and rabbit anti-FMO1 antibody. We also thank Dr Mei-Fei Yueh for her assistance in performing the animal work and Ms Sandy Segna for editorial assistance. This work was supported by PHS NIH grant HL38650. SK was supported by a grant from the Government of Thailand. References Bradlow, H.L., Michnovicz, J.J., Telang, N.T., Osborne, M.P., 1991. Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12, 1571᎐1574. Bjeldanes, L.F., Kim, J.-Y., Grose, K.R., Bartholomew, J.C., 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-tetrachloro-dibenzo-p-dioxin. Proc. Natl. Acad. Sci. USA 88, 9543᎐9547. Brodfuehrer, J.I., Zannoni, V.G., 1987. Flavin-containing monooxygenase and ascorbic acid deficiency. Biochem. Pharmacol. 36, 3161᎐3167. Cashman, J.R., 1995. Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem. Res. Toxicol. 8, 165᎐181. Cashman, J.R., 2000. Human flavin-containing monooxygenase: substrate specificity and role in drug metabolism. Curr. Drug Metabol. 1, 181᎐191. Chang, Y.C., Riby, J., Chang, G.H., Peng, B.C., Firestone, G., Bjeldanes, L.F., 1999. Cytostatic and antiestrogenic effects of 2-Žindol-3-ylmethyl.-3,3⬘-diindolylmethane, a major in vivo product of dietary indole-3-carbinol. Biochem. Pharmacol. 58, 825᎐834. Cherrington, J.J., Cao, Y., Cherrington, J.W., Rose, R.L., Hodgson, E., 1998. Physiological factors affecting protein expression of flavin-containing monooxygenases 1, 3 and 5. Xenobiotica 28, 673᎐682. Coecke, S., Debast, G., Phillips, I.R., Vercruysse, A., Shepard, E.A., Rogiers, V., 1998. Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroid and growth hormone in co-cultured adult male rat hepatocytes. Biochem. Pharmacol. 56, 1047᎐1051. Cover, C.M., Hsieh, S.J., Tran, S.H. et al., 1998. Indole3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J. Biol. Chem. 273, 3838᎐3847. Cover, C.M., Hsieh, S.J., Cram, E.J. et al., 1999. Indole3-carbinol and tamoxifen cooperate to arrest the cell
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