Transplacental Exposure to Indole-3-carbinol Induces Sex-Specific Expression of CYP1A1 and CYP1B1 in the Liver of Fischer 344 Neonatal Rats

Shelley A. Larsen-Su and David E. Williams,1

Department of Environmental and Molecular Toxicology and the Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, Oregon 97331-6512

Received January 18, 2001; accepted August 27, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Indole-3-carbinol (I3C), a naturally occurring component of broccoli, cabbage, and other members of the family Cruciferae, is a tumor modulator in several animal models that demonstrates significant chemoprevention against development of both spontaneous and chemically induced cancers while conversely eliciting tumor promoter effects in others. This study examines the disposition of I3C in the pregnant rat model, specifically to determine whether I3C can traverse the maternal placenta, and what effects, if any, are elicited in the neonate. We now report that dietary I3C treatment of pregnant female rats results in appearance of I3C acid condensation products in both maternal and neonatal livers. Livers from I3C-fed maternal rats showed CYP1A1 protein induction; however, no CYP1B1 protein was detected. No CYP1A1 or CYP1B1 protein was detected in the livers of pregnant controls or their offspring. We also report a sex-specific induction of CYP1A1 and CYP1B1 protein in livers from newborns born to I3C-fed dams. CYP1A1 protein was significantly induced in male neonatal liver, but not in females. Conversely, hepatic CYP1B1 protein was induced to high levels in female neonates, with no CYP1B1 protein detected in male littermates. Our results demonstrate that dietary I3C acid condensation products can cross the maternal placenta and differentially induce neonatal hepatic CYP1A1 and CYP1B1 in a sex-specific manner. The results highlight the potential of I3C to effect changes in the overall metabolic profile of xenobiotics to which the fetus is exposed transplacentally and indicate the possible involvement of sex-specific modulators in Ah receptor-mediated responses in this model.

Key Words: indole-3-carbinol; CYP1A1; CYP1B1; sex-dependent; transplacental.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Indole-3-carbinol (I3C), a naturally occurring plant alkaloid present in significant concentrations in brussels sprouts and other cruciferous vegetables (McDanell et al., 1988Go), when administered through the diet, induces a host of phase I enzymes in various species and tissues, including CYP1A1, CYP1A2, CYP2B1/2, and CYP3A1/2 (Bjeldanes et al., 1991Go; Staack et al., 1998Go; Stresser et al., 1994aGo; Wortelboer et al., 1992Go). The Phase II enzymes UDP-glucuronosyl transferase, NAD(P)H:quinone oxidoreductase, and glutathione S-transferase are also induced by dietary I3C (Hayes et al., 1998Go; Salbe and Bjeldanes, 1986Go; Staack et al., 1998Go; Stresser et al., 1994bGo; Wortelboer et al., 1992Go). Modulation of these xenobiotic-metabolizing enzymes is associated with both decreased tumor incidence, and protection against formation of preneoplastic lesions in several animal models (Bradlow et al., 1991Go; Grubbs et al., 1995Go; Guo et al., 1995Go; Jin et al., 1999Go; Manson et al., 1997Go; 1998; Nixon et al., 1984Go; Wattenberg and Loub, 1978Go; Xu and Dashwood, 1999Go). Studies have shown that I3C alters the metabolism of steroid hormones in vitro and in vivo, such as the upregulation of the CYP1A2-dependent 2-hydroxylation of estrogens (Bradlow et al., 1991Go; Michnovicz et al., 1997Go; Sepkovic et al., 1994Go). For these reasons, I3C is currently being evaluated in human clinical trials, especially for protection against breast, endometrial, and other estrogen-related carcinomas.

Other mechanisms of chemoprevention by I3C not associated with induction of detoxication enzymes have also been observed. Several researchers have reported I3C-mediated electron scavenging, intervention in free radical hepatotoxicity and lipid peroxidation (Fong et al., 1990Go; Shertzer et al., 1988Go), and effects on control of the cell cycle and programmed cell death (Chang et al., 1999Go; Cover et al., 1998Go; 1999; Ge et al., 1996Go, 1999Go; Katdare et al., 1998Go).

Chemoprevention is not always observed with dietary I3C administration. Several studies indicate I3C can act as a promoter of carcinogenesis, especially when administered after a carcinogen (Dashwood et al., 1991Go; Oganesian et al., 1999Go). Our laboratory reported dietary administration of I3C and the dimer of I3C, 3,3`-diindolylmethane, dramatically down-regulated both the expression and activity of flavin-containing monooxygenase (FMO), form 1 (Katchamart et al., 2000Go; Larsen-Su and Williams, 1996Go). This down-regulation is associated with the concomitant induction of CYP, with the predicted result of altered metabolism of drugs that are substrates for both monooxygenases, including nicotine and tamoxifen (Katchamart et al., 2000Go).

The parent compound I3C may not be responsible for any of the above-mentioned effects. Indole-3-carbinol is a light-sensitive, unstable compound, especially under acid conditions. On exposure to the acid environment of the stomach, I3C undergoes hydrolysis, yielding a variety of dimers, linear and cyclic trimers (Fig. 1Go), as well as a host of other as yet unidentified high-molecular-weight oligomers (Stresser et al., 1995Go). A number of acid condensation products of I3C (notably 3,3`-diindolylmethane [I33`] and indolocarbazole [ICZ]) have been shown to be potent Ah receptor agonists (Bjeldanes et al., 1991Go; Chen et al., 1996Go), which provides a partial explanation for the relative potency I3C exhibits in the induction of CYP1A1 and CYP1A2. In contrast to the adults (Katchamart et al., 2000Go; Larsen-Su and Williams, 1996Go) in the present study, we find that fetal transplacental exposure to I3C does not affect hepatic levels of FMO1 (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1. Examples of some commonly identified I3C acid condensation products. These structures represent previously identified oligomers of I3C, produced in an acid environment: parent I3C, indole-3-carbinol; two dimers I33`, 3,3`-diindolylmethane and HI-IM, 1-(3hydroxymethyl)-indolyl-3-indolylmethane; ICZ, indolo-[3,2-b]carbazole; a linear trimer LT1, 2,3-bis-[3-indolylmethyl]indole; a cyclic trimer CT, 5,6,11,12,17,18-hexahydrocyclononal-[1,2-b:4,5b`:7,8b``]triindole; and two tetramers, one linear (LTET) and one cyclic (CTET).

 
As I3C is both a naturally occurring dietary component and currently being evaluated as a chemopreventative agent for women, we examined the disposition of I3C in the pregnant rat model, specifically to determine whether I3C can traverse the maternal placenta, and what effects, if any, are elicited on expression of CYP1A1 and CYP1B1 in the neonatal liver.

This study demonstrates that some I3C oligomers have the ability to traverse the maternal placenta in the rat and can alter the overall metabolic profile of the neonate. The observed differential induction of xenobiotic-metabolizing enzymes in the exposed neonates could have profound effects on the metabolism of endogenous substrates, as well as the potential for selective activation of xenobiotics. Previous studies have shown I3C acid condensation products capable of eliciting both pro- and antiestrogenic effects (Chang et al., 1999Go; Chen et al., 1998Go; Liu et al., 1994; Riby et al., 2000Go; Shilling et al., 2001Go). Such biological effects may be critical in the developing fetus. These results indicate the possible involvement of sex-specific modulators in the early developmental expression of Ah receptor-mediated responses, and underscores the need for caution in the use of I3C in ongoing chemoprevention trials.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Indole-3-carbinol was obtained from Aldrich (Milwaukee, WI). I3C acid condensation products, I33`, ICZ, CT (5,6,11,12,17,18-hexa-hydrocyclononal[1,2-b:4,5-b`:7,8-b"]triindole), and LT, were generated by the method of Bjeldanes et al. (1991).

Animals.
Eight timed-pregnant Fischer 344 rats were purchased from Simonsens (Gilroy, CA) at day 10 of gestation. Animals were randomly divided into two groups and fed either AIN93G powdered semisynthetic control diet, or control diet containing 2000 ppm I3C for the next 11 days until parturition. Both water and diet were available ad libitum throughout the study. Animals nearing parturition were monitored closely, and at birth, neonates were immediately separated from the mother before they could nurse to avoid any possible lactational exposure. One female assigned to the I3C-fed group was found to not be pregnant after 21 days, did not give birth, and was removed from the study, resulting in a total of four controls and three I3C-fed dams. The four control-fed dams gave birth to a total of 40 pups, 19 males and 21 females. The three I3C-fed dams gave birth to a total of 26 pups, 11 males and 15 females. Immediately after parturition all pups were separated by sex (as determined by measurement of anogenital distance). Animals were then killed by CO2 asphyxiation (pups were sacrificed by CO2 asphyxiation plus decapitation), and their livers removed. These protocols were approved by the Oregon State University Institutional Animal Care and Use Committee and are in accordance with the Guiding Principles in the Use of Animals in Toxicology. A portion of the maternal liver was removed for extraction of I3C acid condensation products. Pup livers from each experimental group were also randomly selected and set aside for extraction of I3C acid condensation products. All liver samples were quick-frozen in liquid nitrogen and stored at –80°C until analysis. (Note: one rat pup was excluded from the study. The pup, from control dam no.2, was delivered normally, but was incompletely formed, born dead, and of indeterminate sex.).

Microsome preparation.
Maternal livers were individually homogenized by hand in glass homogenizers kept on ice, using four volumes of homogenization buffer (0.1 M potassium phosphate [pH 7.25] containing 0.15 M potassium chloride, 1.0 mM EDTA, 0.1 mM phenyl-methylsulfonylfluoride, 1.0 mM dithiothreitol, and 20 µM butylated hydroxytoluene (BHT). Liver microsomes were then prepared by ultracentrifugation (Guengerich, 1989Go). Protein levels were determined by the method of Lowry et al. (1951). Ten neonatal livers of each sex were randomly selected from the four control litters and also from the three litters from I3C-fed mothers, combined into five pools of two livers each, and microsomes prepared as described above.

Tissue extraction.
Maternal and neonatal livers were initially homogenized as described above. Homogenates were then extracted three times each with three volumes of ice-cold ethyl acetate (containing 0.001% BHT). The extract was kept on ice, shielded from light, and evaporated to near dryness under a gentle stream of nitrogen. The final residue was redissolved in THF (to a total volume of 30 µl) and stored at –80°C until analysis.

HPLC analysis.
A standard I3C acid reaction mixture was prepared as previously described (Bjeldanes et al., 1991Go) and used to generate a sample chromatogram to establish retention time of I3C oligomers dissolved in THF. HPLC separation of the I3C oligomers was achieved at 30° with a Beckman Ultrasphere C-18 analytical column, 4.6 mm x 25 cm, 5-µm pore size, and peaks detected by UV absorbance utilizing a Shimadzu SPD-6AV spectrophotometer, monitored at 280 nm by a UV/VIS detector (Kyoto, Japan). The flow rate was held constant at 1 ml/min. throughout the analysis. Initial conditions were 20% acetonitrile (solvent A), 80% water (solvent B). These conditions were held constant for 0.5 min, then a linear gradient to 15% solvent B was run over the next 29.5 min, held for 5 min, then returned to starting conditions over the next 10 min. At t = 45 min, the mobile phase was then gradually returned to the starting composition over the next 10 min. The identity of individual peaks was tentatively assigned based on identical retention times with standards we have previously characterized by GC-MS (Stresser et al., 1995Go).

Electrophoresis and immunoblotting.
Microsomal proteins were resolved by SDS-PAGE (Laemmli, 1970Go), then electrophoretically transferred to nitrocellulose membranes (Towbin et al., 1979Go). The membranes were incubated with rabbit antibody raised against either rat CYP1A1 (purchased from Human Biologics, Phoenix, AZ) or mouse CYP1B1 (a generous gift from Dr. Colin Jefcoate, University of Wisconsin). The blots were probed with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Biorad, Richmond, CA), then visualized by a chemiluminescence detection kit (Amersham Corporation, Arlington Heights, IL).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maternal Liver
A Western blot of maternal liver microsomal protein, probed with antibodies to CYP1A1, demonstrates that no CYP1A1 protein was detected in hepatic tissues of dams fed AIN93G control diet (Fig. 2Go). However, high levels of CYP1A1 protein were detected in the liver microsomes from I3C-fed dams. Rat liver microsomes containing high levels of BNF-induced CYP1A1 were used as a positive control. Maternal liver microsomes were also probed for CYP1B1 protein. No CYP1B1 protein was detected in either control or I3C-treated liver microsomes (data not shown).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. Western blot of maternal rat liver microsomes for CYP1A1. Microsomal protein (20 µg) from livers of control (lanes 1–4) and I3C-fed dams (lanes 5–7) were resolved by SDS-PAGE and probed with rabbit anti-CYP 1A1 as described in Materials and Methods section. Lanes 9–11, representing positive controls for CYP1A1 expression, are liver microsomes from male rats fed 0.04% ß-naphthoflavone (BNF) for 7 days.

 
Neonatal Liver
Microsomes were prepared from five pools of two livers each from neonates separated into four groups: control male, control female, I3C-fed male, and I3C-fed female. Hepatic microsomes from male and female neonates were probed with antibody to CYP1A1 protein (Fig. 3Go). No CYP1A1 protein was detected in any of the control male or female neonate liver microsome samples. Only a slight trace amount of CYP1A1 was detected in the microsomes of female pups from I3C-treated dams (Fig. 3AGo). However, CYP1A1 was easily detected in all male pup liver microsomes from I3C-treated dams (Fig. 3BGo).



View larger version (45K):
[in this window]
[in a new window]
 
FIG. 3. Western blot of neonatal rat liver microsomes for CYP1A1. Microsomal liver proteins (20 µg) from female (A) or male (B) neonates were analyzed by immunoblotting as described in Figure 2Go. Lanes 1–5 were offspring (each lane represents a pool of two neonatal livers each or a total of 10 pups) from dams fed control diet, lanes 6–10 were offspring (each lane represents a pool of two neonatal livers each or a total of 10 pups) from dams fed I3C-containing diets. Lanes 11–14 contained liver microsomes from BNF-fed male rats as a positive control for CYP1A1.

 
Liver microsomes from neonates of control and I3C-treated dams were also probed for CYP1B1 protein expression. Whereas no CYP1B1 expression was seen in either the male pups from control dams, the male pups from I3C-treated dams, or the female pups from control dams, the livers of female pups from I3C-treated dams showed marked induction of CYP1B1 (Fig. 4Go). Interestingly, no CYP1B1 protein was detected in the livers of either control or I3C-fed control dams themselves.



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 4. Western blot of neonatal liver microsomes for CYP1B1. Microsomal liver proteins (20 µg) from female (A) or male (B) neonates were analyzed by immunoblotting as described in Figure 2Go. Lanes 1–5 were offspring (each lane represents a pool of two neonatal livers each or a total of 10 pups) from dams fed control diet; lanes 6–10 were offspring (each lane represents a pool of two neonatal livers each or a total of 10 pups) from dams fed I3C-containing diets. Lanes 11–13 contained the recombinant mouse CYP1B1 standard from Dr. Colin Jefcoate. The antibody used was a polyclonal rabbit anti-mouse CYP1B1 IgG, also obtained from Dr. Jefcoate.

 
Samples of liver from control and I3C-treated dams, as well as livers from corresponding neonates, were extracted with ethyl acetate and analyzed for the presence of I3C oligomers by the method of Stresser et al. (1995). In all pups examined, only a single peak was apparent by HPLC analysis. This HPLC peak was present in the livers of both male and female pups from I3C-fed dams. Results indicated an HPLC retention time consistent with LT1, linear trimer. No other peaks were detected in any pup liver extracts. Figure 5Go depicts a typical acid reaction mixture generated in vitro (Fig. 5AGo), and the ethyl acetate extracts of both an I3C-treated dam (Fig. 5BGo) and a representative chromatogram from an I3C-exposed pup (Fig. 5CGo). Approximately a dozen 13C acid condensation products are readily resolved in the ethyl acetate extract from the adult rat liver. Prominent peaks are tentatively identified: I33` (3,3`diindolyl-methane); CT (5,6,11,12,17,18-hexa-hydrocyclononal[1,2-b:4,5-b`:7,8-b"]triindole) and LT (2,3-bis-[3-indolylmethyl]indole), based on co-elution with known standards (Stresser et al., 1995Go).



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 5. HPLC profile of I3C oligomers extracted from maternal and neonatal livers. The panels depict HPLC chromatographic resolution of the major I3C oligomers formed in vitro (A) or recovered by extraction from livers of dams fed I3C (B) or their offspring (C). Peak identities were tentatively assigned based on co-elution with standard previously identified by GC-MS: I33` (3,3`-diindolyl-methane); LT (2,3-bis-[3-indolylmethyl]indole); CT (5,6,11,12,17,18-hexa-hydrocyclononal-[1,2-b;-4,5-b`:7,8-b``]triindole).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A diet rich in cruciferous vegetables has been associated with a lower incidence of cancer (McDanell et al., 1988Go), and this chemoprotection is thought to be partially mediated by I3C and its ability to induce Phase I and Phase II enzymes (Hayes et al., 1998Go; Staack et al., 1998Go; Stresser et al., 1994aGo,bGo). Although the mechanisms of induction of these enzymes is not completely understood, certain I3C derivatives are known to bind with relatively high affinity to the aryl hydrocarbon receptor (AhR; Bjeldanes et al., 1991Go; Chen et al., 1996Go, 1998Go), with subsequent translocation of the liganded AhR to the nucleus and binding of the AhR/AhR nuclear translocator (Arnt) complex to specific xenobiotic response elements (XREs) in the upstream region of CYP genes, modulating their expression (Denison et al., 1989Go).

We previously have isolated 3H-I3C derivatives from the livers of 3H-I3C-fed or gavaged animals (Dashwood et al., 1989Go; Stresser et al., 1995Go). We now report the detection of at least one I3C derivative in the neonatal rat liver from I3C-fed mothers. Because animals were removed from the mother at birth and were not allowed to nurse, it is assumed that I3C derivatives are capable of traversing the maternal placenta. Further, we have shown that this transplacental exposure results in a sex-specific induction of CYP enzymes. CYP1A1 was induced to significant levels in male neonates only, whereas CYP1B1 was selectively induced in female litter mates.

The basis for the differential inducibility of these CYP1A1 and CYP1B1 enzymes in rat neonates is unknown. In addition to AhR and Arnt, a number of transacting coactivators and co-repressors could be expressed in a cell-, tissue-, and sex-specific manner, explaining the results observed in this study (Alexander et al., 1997Go; Bhattacharyya et al., 1995Go; Eltom et al., 1999Go; Kress and Greenlee, 1997Go; Shehin et al., 2000Go). Hakkola et al. (1997) reported expression of CYP1B1 in human placenta to be regulated independently from CYP1A1, although a sex-dependence was not investigated. Walker et al. (1995) noted a sex-dependent expression of CYP1B1 in the kidneys and livers of TCDD-treated adult Sprague-Dawley rat; however, no sex-dependent difference in CYP1A1 expression was noted. TCDD has been shown to be more hepatocarcinogenic in female rats than in males (Kociba et al., 1978Go). Estrogen has been implicated as an important mediator of these sex-specific phenomena (Lucier et al., 1991Go). Evidence suggests that CYP1A1 and CYP1B1 metabolize estrogen to 2-hydroxyestradiol and 4-hydroestradiol, respectively (Badawi et al., 2000Go; Hanna et al., 2000Go; Hayes et al., 1996Go; Li et al., 2000Go), which can then undergo free-radical generation by redox cycling (Bolton et al., 1998Go; Zhu and Conney, 1998Go). The resultant increase in oxidative stress from production of these catechol estrogens may contribute to the observed sex-dependent carcinogenicity of TCDD in exposed female rats. The induction of CYP1A1 and CYP1B1 in early development may play a significant role in the response of the fetus and neonate to the toxicity of xenobiotics. CYP1A1 and CYP1B1 metabolize a number of procarcinogens to reactive intermediates (Crespi et al., 1997Go; Guengerich, 1992Go; Kim et al., 1998Go; Larsen et al., 1998Go; Luch et al., 1998Go; Shimada et al., 1996Go). Transplacental and/or lactational exposure to I3C may enhance the risk (upon subsequent or concurrent exposure to such xenobiotics) for the development of disease, including cancer. Previous studies by Wilker et al. (1996) demonstrated that transplacental exposure of Sprague-Dawley rats to I3C resulted in alterations in reproductive parameters in adult males in a manner that was both similar and distinct from that exhibited by the potent AhR agonist TCDD. The results from this study support our finding of I3C bioavailability to the fetus with the potential for alterations in AhR-mediated signal transduction pathways.

Further studies are required to determine the possible role of sex-specific modulators of CYP1A1 and CYP1B1 expression. In addition, examination of the developmental patterns of induction of these enzymes in fetal tissues should be done to determine the tissue-specific potential for metabolic activation in the developing fetus. The fetus may be exposed to a large number of transplacental carcinogens (Autrup, 1993Go). One major class of rodent transplacental carcinogens is polycyclic aromatic hydrocarbons (Anderson et al., 1995Go). Anderson, Miller, and coworkers have documented the critical role of fetal CYP1A1 induction during late gestational PAH exposure in the tumorigenic response (Anderson et al., 1985Go; Miller et al., 1989Go; 1990). This CYP1A1 induction is mediated by the fetal AhR in the mouse. To date, little information is available on the response of CYP1B1 to transplacental exposure to PAHs and the impact on tumor development. The data from our study would indicate that both CYP1A1 and CYP1B1 could play a role in the metabolism of xenobiotics to which the fetus is transplacentally exposed and, additionally, that the response could be sex dependent.


    ACKNOWLEDGMENTS
 
The authors would like to express their thanks to Dr. Colin Jefcoate of the University of Wisconsin at Madison for his generous gifts of polyclonal rabbit anti-mouse CYP1B1 and the recombinant mouse CYP1B1 standard. This work was supported by PHS grants HL38650 and ESO3850, ESO4766 and ES00210. This is publication no. 11,750 issued from the Oregon Agricultural Experiment Station.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (541) 737-5077. E-mail: david.williams{at}orst.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alexander, D. L., Eltom, K. E., and Jefcoate, C. R. (1997). Ah Receptor regulation of CYP1B1 expression in primary mouse embryo-derived cells. Cancer Res. 57, 4498–4506.[Abstract]

Anderson, L. M., Jones, A. B., Riggs, C. W., and Ohshima, M. (1985). Fetal mouse susceptibility to transplacental lung and liver carcinogenesis by 3-methylcholanthrene: positive correlation with responsiveness to inducers of aromatic hydrocarbon metabolism. Carcinogenesis 5, 1389–1393.

Anderson, L. M., Ruskie, S., Carter, J., Pittinger, S., Kovatch, R. M., and Riggs, C. W. (1995). Fetal mouse susceptibility to transplacental carcinogenesis: differential influence of Ah receptor phenotype on effects of 3-methylcholanthrene, 12-dimethylbenz[a]anthracene and benzo[a]pyrene. Pharmacogenetics 5, 364–372.[ISI][Medline]

Autrup, H. (1993). Transplacental transfer of genotoxins and transplacental carcinogenesis. Environ. Health Perspect. 101 (Suppl.), 33–38.

Badawi, A. F., Cavalieri, E. L., and Rogan, E. G. (2000). Effect of chlorinated hydrocarbons on expression of cytochrome P450 1A1, 1A2 and 1B1 and 2- and 4-hydroxylation of 17ß-estradiol in female Sprague-Dawley rats. Carcinogenesis 21, 1593–1599.[Abstract/Free Full Text]

Bhattacharyya, K. K., Brake, P. B., Eltom, S. E., Otto, S. A., and Jefcoate, C. R. (1995). Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. J. Biol. Chem.270, 11595–11602.[Abstract/Free Full Text]

Bjeldanes, L. F., Kim, J.-Y., Grose, K. R., Bartholomew, J. C., and Bradfield, C. A. (1991). Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. U.S.A. 88, 9543–9547.[Abstract]

Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998). Role of quinoids in estrogen carcinogenesis. Chem. Res. Toxicol. 11, 1113–1127.[ISI][Medline]

Bradlow, H. L., Michnovicz, J. J., Telang, N. T., and Osborne, M. P. (1991). Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12, 1571–1574.[Abstract]

Chang, Y. C., Riby, J., Chang, G. H., Peng, B. C., Firestone, G., and Bjeldanes, L. F. (1999). Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3`-diindolylmethane, a major in vivo product of dietary indole-3-carbinol. Biochem. Pharmacol. 58, 825–834.[ISI][Medline]

Chen, I., McDougal, A., Wang, F., and Safe, S. (1998). Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19, 1631–1639.[Abstract]

Chen, I., Safe, S., and Bjeldanes, L. (1996) Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells. Biochem. Pharmacol. 51, 1069–1076.[ISI][Medline]

Cover, C. M., Hsieh, S. J., Cram, E. J., Hong, C., Riby, J. E., Bjeldanes, L. F., and Firestone, G. L. (1999). Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. Cancer Res. 59, 1244–1251.[Abstract/Free Full Text]

Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F., and Firestone, G. L. (1998). Indole-3-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.[Abstract/Free Full Text]

Crespi, C. L., Penman, B. W., Steimel, D. T., Smith, T., Yang, C. S., and Sutter, T. R. (1997). Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: substrate specificity with model substrates and promutagens. Mutagenesis 12, 83–89.[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, 2362–2365.[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, 385–392.[ISI][Medline]

Denison, M. S., Fisher, J. M., and Whitlock, J. P., Jr. (1989). Protein-DNA interactions at recognition sites for the dioxin Ah-receptor complex. J. Biol. Chem. 264, 16478–16482.[Abstract/Free Full Text]

Eltom, S. E., Zhang, L., and Jefcoate, C. R. (1999). Regulation of cytochrome P-450 (CYP) 1B1 in mouse Hepa-1 variant cell lines: a possible role for aryl hydrocarbon receptor nuclear translocator (ARNT) as a suppressor of CYP1B1 gene expression. Mol. Pharmacol. 55, 594–604.[Abstract/Free Full Text]

Fong, A. T., Swanson, H. I., Dashwood, R. H., Williams, D. E., Hendricks, J. D., and Bailey, G. S. (1990). Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.Biochem. Pharmacol. 39, 19–26.[ISI][Medline]

Ge, X., Yannai, S., Rennert, G., Gruener, N., and Fares, F. A. (1996).3,3`-Diindolylmethane induces apoptosis in human cancer cells. Biochem. Biophys. Res. Commun. 228, 153–158.[ISI][Medline]

Ge, X., Fares, F. A., and Yannai, S. (1999). Induction of apoptosis in MCF-7 cells by indole-3-carbinol is independent of p53 and bax. Anticancer Res. 19, 3199–3203.[ISI][Medline]

Grubbs, C. J., Steele, V. E., Casebolt, T., Juliana, M. M., Eto, I., Whitaker, L. M., Dragnev, K. H., Kelloff, G. J., and Lubet, R. L. (1995). Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Res. 15, 709–716.[ISI][Medline]

Guengerich, F. P. (1989). Analysis and characterization of enzymes. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp 777–814. Raven Press, New York.

Guengerich, F. P. (1992). Metabolic activation of carcinogens. Pharmacol. Ther. 54, 17–61.[ISI][Medline]

Guo, D., Schut, H. A. J., Davis, C. D., Snyderwine, E. G., Bailey, G. S., and Dashwood, R. H. (1995). Protection by chlorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat. Carcinogenesis 16, 2931–2937.[Abstract]

Hakkola, J., Pasanen, M., Pelkonen, O., Hukkanen, J., Evisalmi, S., Anttila, S., Rane, A., Mantyla, M., Purkunen, R., Saarikoski, S., Tooming, M., and Raunio, H. (1997). Expression of CYP1B1 in human adult and fetal tissues and differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands in human placenta and cultured cells. Carcinogenesis 18, 391–397.[Abstract]

Hanna, I. H., Dawling, S., Roodi, N., Guengerich, F. P., and Parl, F. F. (2000). Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity. Cancer Res. 60, 3440–3444.[Abstract/Free Full Text]

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, 9776–9781.[Abstract/Free Full Text]

Hayes, J. D., Pulford, D. J., Ellis, E. M., McLeod, R., James, R. F., Seidegard, J., Mosialou, E., Jernstrom, B., and Neal, G. E. (1998). Regulation of rat glutathione S-transferase A5 by cancer chemopreventive agents: mechanisms of inducible resistance to aflatoxin B1. Chem. Biol. Interact. 111–112, 51–67.

Jin, L., Qi, M., Chen, D. Z., Anderson, A., Yang, G. Y., Arbeit, J. M., and Auborn, K. J. (1999). Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice. Cancer Res. 59, 3991–3997.[Abstract/Free Full Text]

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, 930–936.[Abstract/Free Full Text]

Katdare, M., Osborne, M. P., and Telang, N. T. (1998). Inhibition of aberrant proliferation and induction of apoptosis in pre-neoplastic human mammary epithelial cells by natural phytochemicals. Oncol. Rep. 5, 311–315.[ISI][Medline]

Kim, J. H., Stansbury, K. H., Walker, N. J., Trush, M. A., Strickland, P. T., and Sutter, T. R. (1998). Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 1847–1853.[Abstract]

Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. N., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978). Results of a two year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo- p-dioxin in rats. Toxicol. Appl. Pharmacol. 46, 279–303.[ISI][Medline]

Kress, S., and Greenlee, W. F. (1997). Cell-specific regulation of human CYP1A1 and CYP1B1 genes. Cancer Res. 57, 1264–1269.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685.[ISI][Medline]

Larsen, M. C., Angus, W. G. R., Brake, P. B., Eltom, S. E., Sukow, K. A., and Jefcoate, C. R. (1998). Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res. 58, 2366–2374.[Abstract]

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, 927–931.[Abstract]

Li, D. N., Seidel, A., Pritchard, M. P., Wolf, C. R., and Friedberg, T. (2000). Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10, 343–353.[ISI][Medline]

Liu. H., Wormke, M., Safe, S. H., and Bjeldanes, L. F. (1994). Indolo[3,2-b]carbazole: a dietary-derived factor that exhibits both antiestrogenic and estrogenic activity. J. Natl. Cancer Inst. 86, 1758–1765.[Abstract]

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, 265–275.[Free Full Text]

Luch, A., Coffing, S. L., Tang, Y. M., Schneider, A., Soballa, V., Greim, H., Jefcoate, C. R., Seidel, A., Greenlee, W. F., Baird, W. M., and Doehmer, J. (1998). Stable expression of human cytochrome P450 1A1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene. Chem. Res. Toxicol. 11, 686–695.[ISI][Medline]

Lucier, G. W., Tritscher, A., Goldsworthy, T., Foley, J., Clark, G., Goldstein, J., and Maronpot, R. (1991). Ovarian hormones enhance 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated increases in cell proliferation and preneoplastic foci in a two-stage model for rat hepato-carcinogenesis. Cancer Rest 51, 1391–1397.

Manson, M. M., Ball, H. W., 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, 1729–1738.[Abstract]

Manson, M. M., Hudson, E. A., Ball, H. W. L., Barrett, M. C., Clark, H. L., Judah, D. J., Verschoyle, R. D., and Neal, G. E. (1998). Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver—predicting the outcome using early biomarkers.Carcinogenesis 19, 1829–1836.[Abstract]

McDanell, R., McLean, A. E. M., Hanley, A. B., Heaney, R. K., and Fenwick, G. R. (1988). Chemical and biological properties of indole glucosinolates (glucobrassicins): a review. Food Chem. Toxicol. 26, 59–70.[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, 718–723.[Abstract/Free Full Text]

Miller, M. S., Jones, A. B., Chauhan, D. P., Park, S. S., and Anderson, L. M. (1989). Differential induction of fetal mouse liver and lung cytochromes P-450 by ß-naphthoflavone and 3-methylcholanthrene. Carcinogenesis10, 875–891.[Abstract]

Miller, M. S., Jones, A. B., Park, S. S., and Anderson, L. M. (1990). The formation of 3-methyl-cholanthrene-initiated lung tumors correlates with induction of cytochrome P4501A1 by the carcinogen in fetal but not adult mice. Toxicol. Appl. Pharmacol. 104, 235–245.[ISI][Medline]

Nixon, J. E., Hendricks, J. D., Pawlowski, N. E., Pereira, C., Sinnhuber, R. O., and Bailey, G. S. (1984). Inhibition of aflatoxin B1 carcinogenesis in rainbow trout by flavone and indole compounds. Carcinogenesis 5, 615–619.[Abstract]

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 aflatoxin B1-initiated hepatocarcinogenesis: results from a 9000 animal tumor study. Carcinogenesis 20, 453–458.[Abstract/Free Full Text]

Riby, J. E., Chang, G. H., Firestone, G. L., and Bjeldanes, L. F. (2000). Ligand-independent activation of estrogen-receptor function by 3,3`-diindolylmethane in human breast cancer cells. Biochem. Pharmacol. 60, 167–177.[ISI][Medline]

Salbe, A. D., and Bjeldanes, L. F. (1986). Dietary influences on rat hepatic and intestinal DT-diaphorase activity. Food Chem. Toxicol. 24, 851–856.[ISI][Medline]

Sepkovic, D. W., Bradlow, H. L., Michnovicz, J., Murtezani, S., Levy, I., and Osborne, M. P. (1994). Catechol estrogen production in rat microsomes after treatment with indole-3-carbinol, ascorbigen, or ß-naphthoflavone: a comparison of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods. Steroids 59, 318–323.[ISI][Medline]

Shehin, S. E., Stephenson, R. O., and Greenlee, W. F. (2000). Transcriptional regulation of the human CYP1B1 gene. Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression. J. Biol. Chem. 275, 6770–6776.[Abstract/Free Full Text]

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, 333–338.[ISI][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, 191–200.[ISI][Medline]

Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P., and Sutter, T. R. (1996). Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979–2984.[Abstract]

Staack, R., Kingston, S., Wallig, M. A., and Jeffery, E. H. (1998). A comparison of the individual and collective effects of four glucosinolate breakdown products from brussels sprouts on induction of detoxification enzymes. Toxicol Appl. Pharmacol. 149, 17–23.[ISI][Medline]

Stresser, D. M., Bailey, G. S., and Williams, D. E. (1994a). Indole-3-carbinol and ß-naphtho-flavone induction of aflatoxin B1 metabolism and cytochromes P-450 associated with bioactivation and detoxication of aflatoxin B1 in the rat. Drug Metab. Dispos. 22, 383–391.[Abstract]

Stresser, D. M., Williams, D. E., McLellan, L. I., Harris, T. M., and Bailey, G. S. (1994b). Indole-3-carbinol induces a rat liver glutathione transferase subunit (Yc2) with high activity toward aflatoxin B1 exo-epoxide: association with reduced levels of hepatic aflatoxin-DNA adducts in vivo. Drug Metab. Dispos. 22, 392–399.[Abstract]

Stresser, D. M., Williams, D. E., Griffin, D. A., and Bailey, G. S. (1995). Mechanisms of tumor modulation by indole-3-carbinol. Disposition and excretion in male Fischer 344 rats. Drug Metab. Dispos. 23, 965–975.[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, 4350–4354.[Abstract]

Walker, N. J., Gastel, J. A., Costa, L. T., Clark, G. C., Lucier, G. W., and Sutter, T. R. (1995). Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals. Carcinogenesis 16, 1319–1327.[Abstract]

Wattenberg, L. W., and Loub, W. D. (1978). Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 38, 1410–1413.[Abstract]

Wilker, C., Johnson, L., and Safe, S. (1996). Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring. Toxicol. Appl. Pharmacol. 141, 68–75.[ISI][Medline]

Wortelboer, H. M., van der Linden, E. C. M., de Kruif, C. A., Noordhoek, J., Blaauboer, B. J., van Bladeren, P. J., and Falke, H. E. (1992). Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures. Food Chem. Toxicol. 30, 589–599.[ISI][Medline]

Xu, M., and Dashwood, R. H. (1999). Chemoprevention studies of heterocyclic amine-induced colon carcinogenesis. Cancer Lett. 143, 179–183.[ISI][Medline]

Zhu, B. T., and Conney, A. H. (1998). Functional roles of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19, 1–27.[Abstract]