* Research Institute for Toxicology, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands; and
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466
Received September 12, 2000; accepted December 28, 2000
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ABSTRACT |
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Key Words: CYP1B1; CYP1A1; aromatase; CYP19; TCDD; diindolylmethane.
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INTRODUCTION |
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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., 1990) and stably expresses a large number of steroidogenic enzymes, including aromatase (Sanderson et al., 2000
; Staels et al., 1993
). 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.
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MATERIALS AND METHODS |
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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., 2000). 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., 1977).
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 -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., 1999
), 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, 1986). 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.
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RESULTS |
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DISCUSSION |
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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., 1996; Stresser et al., 1995
) and acetanilide 4-hydroxylase (Stresser et al., 1995
). 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. 5) can be explained by their cytotoxicity (Fig. 1
). 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., 1996; Chen et al., 1998
; McDougal et al., 2000
). 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., 1997
; J. T. Sanderson et al., manuscript submitted), although conflicting reports exist that show detectable aromatase activities and CYP19 mRNA expression (Castagnetta et al., 1997
; Ciolino et al., 2000
). 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., 1993
).
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. 6). This induction response has also been reported in other human cell lines, such as MCF-7 (Christou et al., 1994
), 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., 1993
) (Fig. 6
). 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, 1995
). 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., 2000
). 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 1
) 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 15) and those without (DIM 610). 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 15.
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. 7). 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., 1980
). 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., 1999). 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
-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.
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ACKNOWLEDGMENTS |
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NOTES |
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