2,3,7,8-Tetrachlorodibenzo-p-dioxin and Diindolylmethanes Differentially Induce Cytochrome P450 1A1, 1B1, and 19 in H295R Human Adrenocortical Carcinoma Cells

J. Thomas Sanderson*,1, Lennert Slobbe*, Gideon W. A. Lansbergen*, Stephen Safe{dagger} and Martin van den Berg*

* Research Institute for Toxicology, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands; and {dagger} Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466

Received September 12, 2000; accepted December 28, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen receptor cross-talk. DIM and various structural analogs were examined in H295R cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase activity was measured by conversion of 1ß-3H-androstenedione to estrone and 3H2O. H295R cells were exposed to the test chemicals dissolved in dimethyl sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (0–30 nM) and DIM (0–10 µM) induced ethoxyresorufin-O-deethylase (EROD) activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC50 values of about 0.3 nM and 3 µM, respectively. DIM, but not TCDD, induced aromatase activity with an apparently maximal 2-fold increase at 10 µM; higher concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM) significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for CYP19. DIM (3 µM) significantly increased mRNA levels for all three CYPs. DIM analogs with substitutions on the 5 and 5' position (3 µM) induced aromatase and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that were substituted on the central carbon of the methane group showed little or no inductive activity toward the CYPs. In conclusion, DIM and several of its analogs appear to induce CYPs via multiple yet distinct pathways in H295R human adrenocortical carcinoma cells.

Key Words: CYP1B1; CYP1A1; aromatase; CYP19; TCDD; diindolylmethane.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3,3'-Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol (I3C), a constituent of cruciferous vegetables, and is formed in the stomach (Bradfield and Bjeldanes, 1987Go; De Kruif et al., 1991Go). I3C and other dietary indoles exhibit anti-carcinogenic properties in laboratory animals (Boone et al., 1990Go; Bradlow et al., 1991Go; Grubbs et al., 1995Go; Kojima et al., 1994Go; Wattenberg and Loub, 1978Go) and in epidemiological studies (Graham, 1983Go). The antitumor properties of I3C in estrogen-dependent breast cancers (Wattenberg and Loub, 1978Go) are related, in part, to its ability to alter estrogen metabolism in vitro and in vivo (Bradlow et al., 1991Go; Jellinck et al., 1993Go). I3C is only active orally and rapidly oligomerizes in the stomach (De Kruif et al., 1991Go). Thus, the anticarcinogenic properties have been attributed to some of its condensation products, including the dimer DIM (Chen et al., 1998Go; Grubbs et al., 1995Go). In rats, DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor (Bjeldanes et al., 1991Go; Jellinck et al., 1993Go) and induces hepatic cytochrome P450 (CYP) 1A1, 1A2, and their associated catalytic activity ethoxyresorufin-O-deethylase (EROD). The binding of DIM to the Ah receptor is associated with Ah-estrogen receptor cross-talk, resulting in an antiestrogenic effect on the estrogen-mediated transcription of certain genes (Safe et al., 1998Go). It has been further shown that DIM increases the 2-hydroxylation of estradiol in liver microsomes of rats exposed in vivo (Jellinck et al., 1993Go). In vitro studies show that I3C induces 2-hydroxylation of estrogens in MCF-7 cells without significantly influencing competing routes of estrogen metabolism, such as 4- or 16{alpha}-hydroxylation (McDougal and Safe, 1998Go; Sepkovic et al., 1994Go;). The 4- and 16{alpha}-hydroxyestrogens are potentially genotoxic, and the 16{alpha}-hydroxyestrogens are intrinsically estrogenic, and have been implicated in both initiation and promotion of mammary carcinogenesis (Bradlow et al., 1985Go; Liehr, 1990Go). Increased 2-hydroxylation of estrogens is thought to be a protective effect in the development of breast cancer by converting estradiol and estrone to the less estrogenic 2-hydroxyestrogens, rather than the more reactive 4- and 16{alpha}-hydroxyestrogens (Bradlow et al., 1996Go). In MCF-7 and T47D human breast cancer cells the 16{alpha}-hydroxyestrogens exhibit relatively high estrogenic and mitogenic activity, whereas the 2-OH estrogens are only weakly estrogenic and act as antiestrogens in the presence of 17ß-estradiol (Gupta et al., 1998Go). In human liver, the main enzymes responsible for both 2- and 4-hydroxylation of 17ß-estradiol appear to be CYP3A4 and 3A5 (Kerlan et al., 1992Go), with a smaller contribution of CYP1A2 to 2-hydroxylation (Martucci and Fishman, 1993Go). However, in human extrahepatic tissues such as the mammary gland, CYP3A enzymes are less highly expressed (De Waziers et al., 1990Go); here, 17ß-estradiol 2- and 4-hydroxylase activities are catalyzed by CYP1A1 and 1B1 (Hayes et al., 1996Go; Spink et al., 1994Go), respectively.

Effects of DIM on steroid metabolizing CYPs have not been investigated in detail and no information is available on potential effects of DIM on steroid synthesizing CYPs. An important mechanism by which some antitumor chemicals exert their therapeutic effect in the treatment of estrogen-responsive breast cancer is the inhibition of aromatase (CYP19) activity. Aromatase is the rate-limiting enzyme responsible for the conversion of androgens to estrogens. The present study was based on the working hypothesis that aromatase inhibition may present an alternative or additional mechanism by which DIM exerts its antiestrogenic properties and protects against certain tumors. The effects of DIM and several structurally related analogs on aromatase activity and mRNA expression were investigated in the H295R human adrenocortical carcinoma cell line. This cell line was chosen because, unlike MCF-7 cells, it produces estrogens de novo (Gazdar et al., 1990Go) and stably expresses a large number of steroidogenic enzymes, including aromatase (Sanderson et al., 2000Go; Staels et al., 1993Go). In addition to aromatase, the effects of the DIM compounds are also reported on two other enzymes involved in extrahepatic estrogen metabolism and found to be present in H295R cells, namely CYP1A1 and 1B1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
TCDD (> 99% pure) was obtained from Cambridge Isotope Laboratories (Woburn, MA). DIM, three 5,5'-substituted analogs, 5,5'-dibromo-DIM (B-DIM), 5,5'-dimethyl-DIM (M-DIM) and 5,5'-dimethoxy-DIM (MO-DIM), and 10 C-substituted DIMs (DIM-1 to 10) were synthesized in the laboratory of Dr. S. Safe (Texas A&M University, College Station, TX) and were > 98% pure as determined by gas chromatography; the structures were confirmed by nuclear magnetic resonance spectroscopy (for structures please refer to Table 1Go). Spectroscopic details and antitumor/antiestrogenic activities of some of these DIM analogs have been published recently (McDougal et al., 2000Go) or will be published separately. The DIM compounds were considered light-sensitive, thus stocks (10 mM in dimethyl sulfoxide) and working solutions were stored at –20°C in the dark. Cell culture reagents and media were purchased from GIBCO-BRL (Breda, The Netherlands) and cell culture plasticware from Greiner (Frickenhause, Germany). All other chemicals were purchased from Sigma (St. Louis, MO).


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TABLE 1 Structures of Diindolylmethane (DIM) and Its Analogs, and a Summary of Their Ability to Induce the Catalytic Activity (% Control ± Standard Deviation) and/or mRNA of CYP1A1, 1B1, and 19 in H295R Cells after a 24-h Exposure to a Concentration of 3 µM
 
Cell culture conditions.
H295R cells were obtained from the American Type Culture Collection (ATCC #CRL-2128) and grown in culture under conditions published previously (Rainey et al., 1993Go, 1994Go) with modifications (Sanderson et al., 2000Go). Cell culture plates (24-well) were seeded with 1 ml of cell suspension per well, resulting in a cell density of about 1 x 105 cells/well. The culture medium was changed 24 h after seeding, during which time attachment of the cells had occurred. Under these conditions the cells were almost confluent in each well (about 2 x 105 cells/well). The cells were then exposed to the test chemicals added to the wells (under subdued light) at various concentrations dissolved in 1 µl of dimethyl sulfoxide (DMSO; D4540, Sigma), resulting in a 0.1% (v/v) or 14 mM concentration of DMSO in the culture medium. Negative control cells received 1 µl of DMSO (0.1%). As positive controls for CYP19 induction, cells were exposed to 100 µM of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP; B5386, Sigma) dissolved in medium containing 0.1% DMSO. As positive controls for CYP1A1 and 1B1 induction, cells were exposed to 30 nM TCDD dissolved in DMSO. Unexposed cells were included as further controls. DMSO at 0.1% had no effect on the expression of the various CYPs compared with unexposed cells. Exposures were for 24 h unless stated otherwise, such as in the case of the direct catalytic inhibition experiments.

Isolation and amplification of RNA.
RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the enclosed instructions and stored at –70°C. RT-PCRs were performed using the Access RT-PCR System (Promega, Madison, WI). RNA preparations were considered acceptable for RT-PCR when their A260 (nm)/A280 (nm) ratios were greater than 1.8. The purity of the RNA preparations was verified by denaturing agarose gel electrophoresis. Suitable primer pairs were obtained by entering the human CYP cDNA sequences obtained from the European Molecular Biology Laboratories database (accession numbers for CYP1A1, 1B1, and 19 were K013191, U03688, and M22246, respectively) into the software program Geneworks (version 2.4; IntelliGenetics, Mountain View, CA). The primer pair for CYP1A1 was chosen in such a way that it would not recognize cDNA from CYP1A2 mRNA. The primer pairs used for CYP mRNA amplification were the following: CYP1A1: 5'-GAT-GAG-AAC-GCC-AAT-GTC-C-3' and 5'-TCT-GGT-CAT-GGT-TGA-TCT-GC-3', resulting in an amplification product of 373 base pairs; CYP1B1: 5'-TAT-CAG-TGA-CAT-CTT-CGG-CG and 5'-TCC-TTG-TCC-AAG-AAT-CGA-GC-3' (378 base pair product); CYP19: 5'-TTA-TGA-GAG-CAT-GCG-GTA-CC-3'; 5'-CTT-GCA-ATG-TCT-TCA-CGT-GG-3' (314 base pair product). PCR conditions, such as annealing temperature and Mg2+ concentration were optimized empirically. The conditions of the RT-PCR using the Access RT-PCR system kit were adapted as follows: RT-PCRs for CYP1A1 and 1B1 mRNA (300 ng/reaction) were performed in the presence of 0.5 and 1 mM MgSO4, respectively. After reverse transcription at 48°C for 45 min and denaturing at 94°C for 2 min, the resultant cDNA underwent 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 1 min. A final extension of 7 min completed the amplification. RT-PCR conditions for CYP19 mRNA (300 ng/reaction) were slightly different: 40 cycles were used and annealing and extension temperatures of 57°C and 68°C, respectively. As reference, RT-PCR was performed on ß-actin mRNA using the primer pair 5'-AAA-CTA-CCT-TCA-ACT-CCA-TC-3' and 5'-ATG-ATC-TTG-ATC-TTC-ATT-GT-3'. ß-actin mRNA (100 ng/reaction) was amplified according to the procedures for CYP1A1 and 1B1 with slight modifications: 2 mM MgSO4, an annealing temperature of 54°C and 25 cycles were used. Serial dilutions of RNA were amplified using each primer pair to determine the linear range of the PCR reaction, so semi-quantitative inferences could be made. ß-actin mRNA was found not to be affected by any of the test chemicals and could be used reliably as a reference amplification response. To further enhance the reproducibility and comparability of the RT-PCR method, we included in each DIM-exposure experiment, apart from a vehicle control (cells exposed to DMSO), 2 positive controls (cells exposed to 8Br-cAMP or TCDD). Further detail of the reproducibility and ability of the RT-PCR method to be used (semi)quantitatively was published previously (Sanderson et al., 2000Go). Amplification products were detected using agarose gel electrophoresis and ethidium bromide staining. Intensity of the ethidium bromide stains were quantified using a FluorImager (Molecular Dynamics, city, state).

Aromatase assay.
The catalytic activity of aromatase was determined based on the tritiated water-release method of Lephart and Simpson (1991) with modifications described by Sanderson et al. (2000). The specificity of the aromatase assay based on the release of tritiated water was verified by measuring the production of estrone (the aromatization product of androstenedione), using a 125I-labeled double-antibody radioimmunoassay kit (DSL-8700; ICN, Costa Mesa, CA), and by using 4-hydroxyandrostenedione, an irreversible inhibitor of the catalytic activity of aromatase, to block the formation of tritiated water from 1ß-3H-androstenedione (Brodie et al., 1977Go).

EROD assay.
Ethoxyresorufin-O-deethylation (EROD) activity was determined in H295R cells using a modification of the method described by Burke and Mayer (1974). Medium was removed from H295R cells in 24-well plates, and the cells were washed twice with warm (37°C) phosphate-buffered saline (PBS). Cells were then exposed to 0.5 ml Tris buffer (50 mM, pH 7.8) containing 0.9% (w/v) NaCl, 6.25 mM MgCl2, 5 µM 7-ethoxyresorufin, and 10 µM dicumerol. The formation of resorufin was followed over time and was linear for 60 min, at 37°C. The specificity of several CYP1A inhibitors to block EROD activity was examined by exposing the cells to {alpha}-naphthoflavone (ANF) and ellipticine, added directly to the assay medium in 0.5 µl of DMSO. The inhibitory effect of PCB-169, a relatively selective CYP1B1 inhibitor (Pang et al., 1999Go), was also examined.

MTT reduction.
Cell viability, as an indicator of cytotoxicity, was determined by measuring the capacity of H295R cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Denizot and Lang, 1986Go). MTT is reduced to the blue-colored formazan by the mitochondrial enzyme succinate dehydrogenase, which is considered a reliable and sensitive measure of mitochondrial function. The cells in each well on the 24-well plate were incubated for 30 min, at 37°C with 0.5 ml of MTT (1 mg/ml) dissolved in KREBS buffer. Then, the MTT solution was removed, after which the cells were washed twice with PBS. The formazan formed in the cells was extracted by adding 1 ml of isopropanol and incubating for 10 min at room temperature. The isopropanol was added directly to a plastic cuvette for spectrophotometric analysis at an absorbance wavelength of 560 nm. MTT reduction was linear with time for about 45 min and was not affected by DMSO treatment.

Data analysis.
All experiments were performed in triplicate; per experiment each concentration was tested in triplicate. All responses are presented as the mean with its standard deviation (n = 3). Statistically significant differences were determined by a two-tailed t-test using a correction for multiple comparisons and a significance level of 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of DIMs in H295R Cells
Prior to examining effects on enzyme activities, the DIM compounds and TCDD were tested for potential cytotoxicity in H295R cells, after a 24-h exposure. DIM, B-DIM, M-DIM, and MO-DIM were cytotoxic at 30 µM, demonstrated by various degrees of inhibition of MTT reduction, indicating loss of mitochondrial function (Fig. 1Go). Of the 10 C-substituted DIMs, DIM-10, 7, 6 and 8 were cytotoxic (in order of decreasing potency) at 30 µM (data not shown). TCDD did not inhibit MTT reduction at concentrations as high as 0.3 µM (Fig 1Go). Of the DIM compounds, only M-DIM was cytotoxic at 3 µM.



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FIG. 1. Effect of 30 or 300 nM TCDD, and 3 or 30 µM of diindolylmethane (DIM) and three 5,5'-substituted DIM analogs on MTT reduction, as an index of cytotoxicity, in H295R human adrenocortical carcinoma cells, after a 24-h exposure. Each concentration was tested in triplicate. Bars depict means with standard deviations. *Significantly lower than control (Bonferroni t-test; p < 0.05).

 
Induction of EROD Activity in H295R Cells by TCDD and DIMs
Incubation of H295R cells for 24 h with TCDD or DIM resulted in a concentration-dependent induction of EROD activity above basal values (Fig. 2Go). Basal EROD activity was barely detectable in H295R cells and an EROD activity value of about 0.3 pmole resorufin/h/mg cellular protein was the lowest detectable activity under our experimental conditions. DIM appeared to be as efficacious, but more than 3000-fold less potent (apparent EC50 = 1 µM) than TCDD (EC50 = 0.3 nM). DIM was not tested at concentrations greater than 10 µM due to its cytotoxicity. At a test concentration of 3 µM, which was the approximate EC75 of DIM for induction of EROD activity, B-DIM, M-DIM and MO-DIM induced EROD activity 4- to 8-fold above control (Fig. 3Go). B-DIM and M-DIM were about as efficacious as DIM at this concentration; MO-DIM appeared somewhat less potent. DIM-1 to DIM-10 were poor inducers of EROD activity (see summary in Table 1Go); at 3 µM only DIM-4 and DIM-5 induced the activity above 3-fold. To examine the ability of several of the DIM compounds to interfere directly with the catalytic activity of EROD, H295R cells were first exposed to 30 nM TCDD for 24 h, after which the TCDD-containing medium was removed. This induction step was necessary to obtain cells with detectable EROD activities (about 4 pmole resorufin/h/mg cellular protein). Then the cells were exposed to 3 or 30 µM of the 5,5'-substituted DIM compounds, only for the duration of the EROD assays. These experiments demonstrated a concentration-dependent catalytic inhibition of (TCDD-induced) EROD activity by DIM, B-DIM, M-DIM and MO-DIM without marked differences in inhibitory potency (Fig. 4Go); the C-substituted DIMs were not tested. In these experiments the exposure of the cells to the various DIMs was 60 min, too short to cause cytotoxicity measured by the MTT test.



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FIG. 2. Concentration-response curves for induction of ethoxyresorufin O-deethylase (EROD) activity (pmole resorufin/h/mg cellular protein) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0–30 nM) and diindolylmethane (DIM; 0–10 µM) in H295R human adrenocortical carcinoma cells, after a 24 h exposure. Points depict means with standard deviations. Each concentration was tested in triplicate.

 


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FIG. 3. Effect of 30 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3 µM of diindolylmethane (DIM), 3 µM of three 5,5'-substituted DIM analogs, or 100 µM of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) on ethoxyresorufin O-deethylase (EROD) activity (pmole resorufin/h/mg cellular protein) in H295R human adrenocortical carcinoma cells, after a 24 h exposure. Each concentration was tested in triplicate. Bars depict means with standard deviations. *Significantly greater than control (Bonferroni t-test; p < 0.05).

 


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FIG. 4. Catalytic inhibition of ethoxyresorufin O-deethylase (EROD) activity by 3 or 30 µM of diindolylmethane (DIM) or three 5,5'-substituted DIM analogs in H295R human adrenocortical carcinoma cells induced with 30 nM TCDD 24 h prior. Each concentration was tested in triplicate. Bars depict means with standard deviations. *Significantly lower than control (Bonferroni t-test; p < 0.05).

 
Effects of TCDD and DIMs on Aromatase Activity in H295R Cells
DIM, but not TCDD, caused a concentration-dependent induction of aromatase activity in H295R cells after a 24-h exposure, achieving about 2-fold induction at 3 µM (Fig. 5Go). At concentrations of 10 µM and above DIM was cytotoxic and aromatase activities decreased. Concentration-dependent induction of aromatase activity was also observed for the other 5,5'-substituted DIM compounds (Fig. 5Go). M-DIM and MO-DIM produced dose-response curves similar to that of DIM, whereas B-DIM was somewhat less efficacious. 8Br-cAMP induced aromatase activity between 4- and 5-fold in these experiments (data not shown). In all situations where induction of aromatase activity was observed according to the tritiated water-release method, a corresponding increase in estrone formation was measured independently by radioimmunoassay (see Materials and Methods). Tritiated water and estrone were formed in a 1:1 molar ratio (data not shown). The decrease in aromatase activities observed at higher concentrations was due to the cytotoxicity of the DIM compounds. Of the C-substituted DIMs, only DIM-1 and DIM-2 were weak inducers (Table 1Go). The ability of the 5,5'-substituted DIM compounds to inhibit the catalytic activity of aromatase directly, was examined in untreated H295R cells by exposing them to 3 or 30 µM of the DIM compounds for the duration of the aromatase assays. None of the compounds directly inhibited aromatase activity under these conditions (data not shown).



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FIG. 5. Concentration-response curves for induction of aromatase activity (pmole androstenedione/h/mg cellular protein) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), diindolylmethane (DIM) and three 5,5'-substituted DIM analogs in H295R human adrenocortical carcinoma cells, after a 24 h exposure. 8-bromo-cyclic adenosine monophosphate (8Br-cAMP: 100 µM) induced aromatase between 4- and 5- fold in these experiments. Each concentration was tested in triplicate. Points depict means with standard deviations.

 
Induction of CYP mRNA Expression in H295R Cells by TCDD and DIMs
TCDD (30 nM) significantly induced CYP1A1 and 1B1 mRNA levels above control values, but did not affect the expression of CYP19, after a 24-h exposure (Fig. 6Go). DIM, as well as its 5,5'-substituted analogs B-DIM, M-DIM and MO-DIM (3 µM), increased mRNA levels for all three CYPs significantly above control values; of the C-substituted DIMs, DIM-1 and 2 increased CYP19 mRNA only. The induction profiles produced by TCDD, DIM, and the three 5,5'-substituted DIM compounds were similar after exposures of 72 h: induction of CYP1A1 and 1B1 mRNA levels by TCDD was somewhat greater after 72 h than 24 h. In contrast, the induction of CYP19 by DIM and its 5,5'-substituted analogs was somewhat lower after 72 h than 24 h (data not shown). This probably reflects differences in the mechanism of induction of CYP1A1/1B1 (Ah receptor-mediated) and CYP19 (cAMP-mediated), and/or differences in mRNA stability.



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FIG. 6. Effect of 30 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3 µM of diindolylmethane (DIM), 3 µM of three 5,5'-substituted DIM analogs, or 100 µM of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) on the levels of mRNA for CYP1A1, 1B1, and 19. Each concentration was tested in triplicate. Bars depict means with standard deviations. *Significantly greater than control (Bonferroni t-test; p < 0.05).

 
Effects of Known CYP Inhibitors on EROD and Aromatase Activity in H295R Cells
The relative selectivity of several CYP inhibitors was examined in H295R cells by measuring their ability to inhibit aromatase and EROD activities, as described in Materials and Methods. ANF and ellipticine were potent inhibitors of EROD activity in TCDD-induced H295R cells, causing 50% inhibition at about 20 nM (Fig. 7Go, top panel). (As explained earlier, basal EROD activity in H295R cells was too low to perform inhibition studies.) PCB-169 and 4-HA also inhibited EROD activity with about 40 and 30% inhibition, respectively, at 10 µM. ANF and ellipticine also inhibited aromatase activity, causing 50% inhibition at about 2 and 0.5 µM, respectively (Fig. 7Go, bottom panel). The inhibitory effects of these compounds on the catalytic activity of aromatase were verified by measuring estrone formation (see Materials and Methods), which was reduced correspondingly (data not shown). 4-HA was a potent inhibitor of aromatase, causing 52% inhibition at the lowest tested concentration of 10 nM, whereas PCB-169 had no effect.



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FIG. 7. Concentration-response curves for catalytic inhibition of ethoxyresorufin O-deethylase (EROD) activity (top panel) and aromatase activity (bottom panel) by {alpha}-naphthoflavone (ANF), ellipticine (Ellipt), 4-hydroxyandrostenedione and PCB-169 in H295R human adrenocortical carcinoma cells. EROD inhibition was examined in cells induced 24 h prior with 30 nM TCDD; aromatase inhibition was examined in untreated cells. Each inhibitor concentration was tested in triplicate. Points depict means with standard deviations.TABLE 1Go

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of DIMs
Cytotoxicity was observed in H295R human adrenocortical carcinoma cells treated for 24 h with 30 µM of DIM or several DIM analogs. DIM has been reported to cause cell death by apoptosis in several human cancer cell lines, causing about 10–15% apoptotic cell death in either MCF-7, T47D or Saos-2 cells, after a 48-h exposure to a concentration of 50 µM (Ge et al., 1996Go). In H295R cells we observed about 20% loss of metabolically functional cells with DIM, and about 30, 60, and 70% loss with MO-DIM, B-DIM, and M-DIM (30 µM), respectively. Whether the cytotoxicity caused by the DIM compounds in H295R cells is mediated via apoptosis remains to be investigated. Significant loss of cells as determined by lactate dehydrogenase leakage was not observed in cultured primary rat hepatocytes exposed to 40 µM DIM for 48 h (Wortelboer et al., 1992Go). These reports do not necessarily indicate differences in sensitivity among cell types toward DIM, because the MTT test used in the present study, which measures mitochondrial function, is likely to be a more sensitive indicator of decreased cell function than, for example, LDH leakage, which is not observed until significant loss of membrane integrity occurs.

EROD Induction and Inhibition by DIMs
This study has demonstrated that several synthetic analogs of DIM can induce EROD activity and mRNA levels for CYP1A1 and 1B1. It is the first time that expression and induction of these CYPs have been described in H295R cells. H295R cells are therefore not only capable of de novo cholesterol and estrogen synthesis, but also capable of estrogen and xenobiotic metabolism (albeit with relatively low activities). Dependent on the activities of these xenobiotic-metabolizing CYPs and the manner in which their expression is regulated, this observation may expand the range of potentially useful applications for this cell line. After TCDD, the compounds DIM, B-DIM, M-DIM and to a slightly lesser extent MO-DIM appeared to be the most efficacious inducers, although considerably less potent than TCDD. DIM and its 3 analogs also directly inhibited (TCDD-induced) EROD activity during the catalytic assays, indicating that they bind to or may even be substrates for one or more of the inducible enzymes involved in the O-deethylation of ethoxyresorufin, most prominently CYP1A1. Previous studies have demonstrated that DIM inhibits the catalytic activities of EROD (Chen et al., 1996Go; Stresser et al., 1995Go) and acetanilide 4-hydroxylase (Stresser et al., 1995Go). According to Stresser and coworkers, this inhibition appeared to be noncompetitive for the CYP1A1-associated EROD activity (Kis = 7.4 µM; Kii = 13 µM) and competitive for the CYP1A2-associated acetanilide 4-hydroxylase activity (Ki = 7.6 µM); DIM had no effect on NADPH-dependent cytochrome P450 reductase. Our study thus indicates that DIM, B-DIM, M-DIM, and MO-DIM interact with CYP1A catalytic activity, as well as being Ah receptor agonists in human adrenocortical carcinoma cells.

Aromatase Induction and Inhibition by DIMs
This study is the first to demonstrate the ability of DIM and several structural analogs to induce human aromatase activity in vitro. It is important to note that the decreased aromatase activities by the DIM compounds at concentrations of above 10 µM (Fig. 5Go) can be explained by their cytotoxicity (Fig. 1Go). None of the DIM compounds directly inhibited the catalytic activity of CYP19.

The induction of aromatase activity by DIM and some of its analogs in H295R cells is in contrast to the observed anti-estrogenic effects in MCF-7 cells (Chen et al., 1996Go; Chen et al., 1998Go; McDougal et al., 2000Go). This can be explained by the fact that aromatase activity is generally not expressed or expressed at very low levels in MCF-7 cells (Jorgensen et al., 1997Go; J. T. Sanderson et al., manuscript submitted), although conflicting reports exist that show detectable aromatase activities and CYP19 mRNA expression (Castagnetta et al., 1997Go; Ciolino et al., 2000Go). It appears that the expression of aromatase in MCF-7 cells is strongly dependent on the source of the cells and the culture conditions used. The results of the present study indicate that the effects of DIM compounds on estrogen synthesis and metabolism are cell-specific and dependent on the ability of the cells to express various CYPs. The lack of aromatase induction by TCDD further indicates that the DIM compounds exert their effects on aromatase independent of their anti-estrogenic activities mediated by the Ah receptor through cross-talk with the estrogen receptor. The physiological significance of the 2-fold increase in aromatase activity observed in vitro requires further study in animal models in vivo. However, this induction response is of interest because CYP19 is responsible for the final step in estrogen synthesis from androgens, and has recently been shown also to catalyze the 2-hydroxylation of estradiol with a Km of 1.6 µM (Osawa et al., 1993Go).

Structure-Activity Relationship and Mechanisms of CYP Induction
We observed induction of human CYP1A1 and 1B1 in vitro in H295R cells after exposure of cells to known Ah receptor agonists such as TCDD and DIM (Fig. 6Go). This induction response has also been reported in other human cell lines, such as MCF-7 (Christou et al., 1994Go), and indicates that H295R cells express a functional Ah receptor. We also demonstrated that CYP1A1 and 1B1 are not induced via the cAMP-mediated protein kinase A pathway that results in induction of steroidogenic enzymes, such as CYP19, in H295R cells (Staels et al., 1993Go) (Fig. 6Go). This observation is in contrast to the induction of CYP1B1 by ACTH, 8Br-cAMP or forskolin seen in freshly isolated rat adrenocortical cells in culture, which in turn were poorly inducible by TCDD (Brake and Jefcoate, 1995Go). These differences may be related to species- or cell type-specific differences in regulation of CYP1B1. Other investigators have also observed a lack of induction of CYP1B1 after cAMP-stimulation in H295R cells (Jefcoate, personal communication). In any case, the present study provides indirect evidence that the induction of CYP1A1/1B1 and CYP19 gene expression by the various DIM compounds proceeds via 2 distinct mechanisms in H295R cells, the Ah receptor-mediated and protein kinase A-mediated pathways, respectively. Regarding a structure-activity relationship for induction of the various CYPs, it appears that 5,5'-substituted DIM analogs resemble DIM as good inducers of CYP1A1 and 1B1. B-DIM, which appeared at least as potent and efficacious as DIM, may be of particular interest as it has been shown to be a potent antitumor agent in carcinogen-induced rat mammary tumors (McDougal et al., 2000Go). These 5,5'-subsituted DIM compounds also induced aromatase activity with potencies and efficacies similar to that of DIM. Substitution on the central carbon atom (see Table 1Go) strongly decreased the ability of the DIM analogs to induce EROD activity. This is likely to be due to the bulky groups on the central carbon which would prevent the molecule from achieving the coplanar structure required for interaction with the Ah receptor. Within the group of 10 C-substituted DIM analogs, 2 subgroups could be distinguished, namely those with 1,1'-substitution with methyl groups (DIM 1–5) and those without (DIM 6–10). DIMs that did not contain N-methyl substituents did not induce EROD activity, possibly related to the greater polarity of the secondary amine group relative to the more lipophilic tertiary amines of DIMs 1–5.

Selective CYP Inhibitors
We initially used several selective CYP inhibitors to verify that the basal and induced catalytic activities in the H295R cell line were indeed selective for the CYPs in question. We found that ANF and ellipticine were excellent inhibitors of EROD activity. However, they also inhibited aromatase activity at concentrations commonly used to selectively inhibit CYP1A (Fig. 7Go). In addition, the aromatase inhibitor 4-HA inhibited EROD activity at concentrations above 1 µM. The ability of ellipticine to inhibit CYPs other than CYP1A (such as aromatase) can be explained by its inhibitory effect on NADPH-dependent cytochrome P450 reductase activity (Guenthner et al., 1980Go). Ellipticine decreases electron transfer to CYPs at concentrations above 100 nM, resulting in "non-selective" inhibition of all cytochrome P450 reductase-dependent CYP activities, without necessarily interacting with the catalytic sites on the specific CYPs. The mechanism by which ANF inhibits aromatase activity and 4-HA inhibits EROD activity requires further study.

Concentration-response experiments were carried out to select suitable inhibitor concentrations for selective inhibition of either EROD or aromatase activity in H295R cells. For inhibition of EROD activity by ANF and ellipticine these were 10 and 100 nM respectively, with 100 nM ellipticine having less effect on aromatase activity than 10 nM ANF. Aromatase inhibition by 100 nM 4-HA was 90% effective without any significant inhibition of EROD activity. PCB-169 has been reported to selectively inhibit CYP1B1-mediated 4-hydroxylation of estradiol in MCF-7 cells at concentrations below 100 nM, while also inhibiting CYP1A1-mediated estradiol 2-hydroxylation above 100 nM (Pang et al., 1999Go). In H295R cells, PCB-169 did not affect aromatase activity, and only inhibited EROD activity above 1 µM. This latter result suggests that TCDD-induced EROD activity in H295R cells is primarily due to CYP1A and not CYP1B1. Preliminary unpublished results from our laboratory indicate that the catalytic activity of CYP1A1 and 1B1 toward the 2- and 4-hydroxylation of estradiol, respectively, is very low, and 16{alpha}-hydroxylation (a CYP3A-type activity) undetectable. This is likely due to the relatively low basal expression and inducibility of these enzymes in H295R cells, which may render these cells less useful for the study of estrogen hydroxylation reactions.

Future experiments will determine how the observed induction and/or inhibition of the various CYPs by TCDD, the DIM compounds and the various CYP inhibitors, may affect estradiol synthesis and metabolism in H295R cells and other human cell systems. Continued efforts will be made to delineate Ah receptor-dependent and -independent interactions with other hormone-mediated responses in H295R and other cell lines.


    ACKNOWLEDGMENTS
 
This study was financially supported by NIH (ESO 9106, CA64081), the DAMD (17–99–1–93–96), and the EU (ENV4-CT97-0581).


    NOTES
 
1 To whom correspondence should be addressed. Fax: 011–31–30–253–5077. E-mail: t.sanderson{at}ritox.vet.uu.nl. Back


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