- Email: [email protected]
Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes
MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 58 (2004) 463–467 www.elsevier.com/locate/marenvrev
Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes Adria A. Elskus
*
Department of Biology, 101 Morgan Building, University of Kentucky, Lexington, KY 40506-0225, USA
Abstract Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17b-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 107 M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Estrogen; Trout; CYP1A; Suppression; Gender
*
Tel.: +1-859-257-7751; fax: +1-859-257-1717. E-mail address: [email protected] (A.A. Elskus).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.029
464
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
1. Introduction Strong sexual dimorphism of CYP1A expression occurs in fish, with CYP1A levels over 40-fold lower in spawning females than in males or juvenile fish of some species (Gray, Woodin, & Stegeman, 1991). While in vivo treatment with the female hormone estradiol (E2) can suppress both basal expression and chemical induction of this pollutant-metabolizing enzyme, even very high doses do not provoke the strong sex differences observed during spawning (Gray et al., 1991). Recent work demonstrates that estriol (E3), but not other estrogen metabolites, suppresses CYP1A1 in a mammalian cell line (Son, Roby, Rozman, & Terranova, 2002), prompting our interest in determining if this metabolite contributes significantly to estrogenic suppression of CYP1A in fish. To evaluate the contribution of estriol to CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) (188–215 g b.w; GSI <0.1%) with vehicle (DMSO, 0.5%), 17b-estradiol [1,3,5[10]-estratriene3,17b-diol] (0.5, 5, 50 lM E2), or estriol [1,3,5[10]-estratriene-3,16a, 17b-triol] (0.8, 8, 80 lM E3), alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (0.5 lM B[a]P). Cells were dosed in 96 well plates (n ¼ 2–3 fish, 4 replicate wells per treatment) incubated at 16 °C. Estradiol doses were similar to those used by others with this model (Anderson, Olsen, Matsumura, & Hinton, 1996; Navas & Segner, 2001), but higher than those observed in spawning female rainbow trout (0.22 lM, Van Bohemen & Lambert, 1981), and were used to determine dose-response. Estriol doses were chosen to mirror those of estradiol, and to fall within the range used in other studies of estriol suppression of CYP1A1 (Son et al., 2002). Forty-eight hours after dosing, CYP1A activity was evaluated (as ethoxyresorufin-O-deethylase, EROD) on a CytoFluorTM 4000 fluorescent plate reader (Applied Biosystems) essentially as described (Hahn, Woodward, Stegeman, & Kennedy, 1996). Hepatic viability for all treatments remained high at 48 h (>95%), as assessed using the lactate dehydrogenase viability assay of Scholz and Segner (1999). Statistical differences between treatments were determined by one-way ANOVA, p < 0:05. We found significant, dose-related suppression of B[a]P-induced CYP1A activity by estradiol and estriol relative to vehicle controls. Estradiol (0.5, 5, 50 lM) suppressed B[a]P-induced EROD by 3-fold, 5-fold, and to non-detectable levels, respectively, relative to B[a]P treatment alone (Fig. 1). The two higher doses of estriol (8, 80 lM) suppressed B[a]P-induced EROD by 11, and 40-fold, respectively, relative to B[a]P treatment alone (Fig. 2). Although basal EROD activities tended to be lower, 15-, 19- and 120-fold in the presence of estradiol (0.5, 5.0 and 50 lM, respectively) or 12-, 713-fold and non-detectable in the presence of estriol (0.8, 8.0 and 80 lM, respectively), these activity levels were not statistically different from vehicle controls due to high variability in the vehicle treatment groups. Although not significant, co-exposure to the lowest doses of estradiol and estriol tended to suppress basal (5-fold), but not B[a]P-induced, EROD relative to exposure to either steroid alone (data not shown). EROD activities were lower than measured in other studies in our laboratory (example, in other studies
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
465
Fig. 1. Estradiol suppression of CYP1A catalytic activity in primary rainbow trout hepatocytes. Hepatocytes were untreated or treated with DMSO (0.5%), 17b-estradiol (0.5, 5, 50 lM), B[a]P (0.5 lM), or combinations, and CYP1A catalytic activity (ethoxyresorufin O-deethylase, EROD) measured after 48 h. Bars represent means SD (n ¼ 2–3 fish). Mean EROD activity of steroid-treated cells is significantly different from BaP (#) at P < 0:05.
Fig. 2. Estriol suppression of CYP1A catalytic activity in primary rainbow trout hepatocytes. Hepatocytes were untreated or treated with DMSO (0.5%), estriol (0.8, 8, 80 lM), B[a]P (0.5 lM), or combinations and CYP1A catalytic activity (measured as ethoxyresorufin O-deethylase) measured after 48 h. Bars represent means SD (n ¼ 2–3 fish). Mean EROD activity of steroid-treated cells is significantly different from BaP (#) at P < 0:05.
466
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
EROD ranged from basal 10–20 to B[a]P-induced 80–140 pmol/min/mg). The lower EROD levels observed in the present study may reflect the substantial interindividual variation in EROD activity in rainbow trout primary hepatocyte preparations noted by others (Sadar & Andersson, 2001). Catalytic activity did not differ between DMSO-treated and untreated cells (Figs. 1 and 2). While estradiol suppression of basal CYP1A activity has been reported in vivo and in vitro, in vitro evidence for estrogenic suppression of CYP1A induction is inconsistent. We found 48 h exposures to doses of 0.5 lM E2 or higher significantly suppressed induction of EROD by 0.5 lM B[a]P. In other studies with rainbow trout hepatocytes a 48 h exposure to estradiol at 1 lM or higher significantly suppressed induction of EROD by 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Anderson et al., 1996), but failed to suppress EROD induction by 0.78 lM b-naphthoflavone (b-NF, Navas & Segner, 2001). The mechanism by which estrogens suppress CYP1A expression is not known. Field studies with spawning fish suggest estrogenic suppression of CYP1A occurs pretranslationally (Elskus, Pruell, & Stegeman, 1992). In vitro treatment with estradiol lowered basal levels of CYP1A mRNA in rainbow trout hepatocytes, but did not block induction (Navas & Segner, 2001). Fish liver expresses three estrogen receptor (ER) subtypes (Hawkins et al., 2000), and studies in trout with the ER antagonist, tamoxifen, suggest CYP1A suppression by E2 is ER-mediated (Navas & Segner, 2001). Estradiol is completely metabolized to water soluble conjugates within 1 h in cell culture (Anderson et al., 1996) yet effects persist for at least 48 h. This suggests estradiol may act on CYP1A indirectly, by initially altering estradiolregulated genes, which, in turn, suppress CYP1A expression, or that estrogen metabolites are the main suppressive agents. Preliminary time-course data indicate suppression of basal CYP1A mRNA expression in rainbow trout hepatocytes does not occur until 24 h after estradiol treatment (unpublished data), lending support to an indirect mechanism for E2 suppression of CYP1A. Further support for this mechanism is suggested by the lag period between the fall in plasma estradiol levels and increased CYP1A expression in post-spawning female fish (Elskus and Stegeman, unpublished data). Possible indirect mechanisms include promoter methylation, an epigenetic process shown to silence CYP1A expression (Takahashi, Suzuki, & Kamataki, 1998), and estradiol induction of transcription factors that suppress CYP1A induction (e.g., Hohne, Becker-Rabbenstein, Kahl, & Taniguchi, 1990). Cross-talk between the aryl hydrocarbon receptor and ER in regulation of CYP1A is also a possibility (Thomsen, Wang, Hines, & Safe, 1994). Clearly, further work on the molecular mechanism of estrogen regulation of CYP1A is needed.
Acknowledgements This work was supported by a University of Kentucky Research Committee Grant. The rainbow trout were generously provided by Mr. James Gray of the Wolf Creek National Fish Hatchery, Jamestown, KY.
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
467
References Anderson, M. J., Olsen, H., Matsumura, F., & Hinton, D. E. (1996). Aquatic Toxicology, 34, 327–350. Elskus, A. A., Pruell, R. J., & Stegeman, J. J. (1992). Marine Environmental Research, 34, 97–101. Gray, E. S., Woodin, B. R., & Stegeman, J. J. (1991). Journal of Experimental Zoology, 259, 330–342. Hahn, M. E., Woodward, B. L., Stegeman, J. J., & Kennedy, S. W. (1996). Environmental Toxicology and Chemistry, 15, 582–591. Hawkins, M. B., Thorton, J. W., Crews, D., Skipper, J. K., Dotte, A., & Thomas, P. (2000). Proceedings of the National Academy of Sciences of the United States of America, 97, 19751–19756. Hohne, M., Becker-Rabbenstein, V., Kahl, G., & Taniguchi, H. (1990). FEBS, 273, 219–222. Navas, J. M., & Segner, H. (2001). Chemico-Biological Interactions, 138, 285–298. Sadar, M. D., & Andersson, T. B. (2001). In Vitro Cellular and Developmental Biology – Animal, 37, 180– 184. Scholz, S., & Segner, H. (1999). Ecotoxicology and Environmental Safety, 43, 252–260. Son, D.-S., Roby, K., Rozman, K., & Terranova, P. F. (2002). Toxicology, 176, 229–243. Takahashi, Y., Suzuki, C., & Kamataki, T. (1998). Biochemical and Biophysical Research Communications, 247, 383–386. Thomsen, J. S., Wang, X., Hines, R. N., & Safe, S. (1994). Carcinogenesis, 15, 933–937. Van Bohemen, C., & Lambert, J. (1981). General and Comparative Endocrinology, 45, 105–114.
Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes Adria A. Elskus
*
Department of Biology, 101 Morgan Building, University of Kentucky, Lexington, KY 40506-0225, USA
Abstract Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17b-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 107 M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Estrogen; Trout; CYP1A; Suppression; Gender
*
Tel.: +1-859-257-7751; fax: +1-859-257-1717. E-mail address: [email protected] (A.A. Elskus).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.029
464
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
1. Introduction Strong sexual dimorphism of CYP1A expression occurs in fish, with CYP1A levels over 40-fold lower in spawning females than in males or juvenile fish of some species (Gray, Woodin, & Stegeman, 1991). While in vivo treatment with the female hormone estradiol (E2) can suppress both basal expression and chemical induction of this pollutant-metabolizing enzyme, even very high doses do not provoke the strong sex differences observed during spawning (Gray et al., 1991). Recent work demonstrates that estriol (E3), but not other estrogen metabolites, suppresses CYP1A1 in a mammalian cell line (Son, Roby, Rozman, & Terranova, 2002), prompting our interest in determining if this metabolite contributes significantly to estrogenic suppression of CYP1A in fish. To evaluate the contribution of estriol to CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) (188–215 g b.w; GSI <0.1%) with vehicle (DMSO, 0.5%), 17b-estradiol [1,3,5[10]-estratriene3,17b-diol] (0.5, 5, 50 lM E2), or estriol [1,3,5[10]-estratriene-3,16a, 17b-triol] (0.8, 8, 80 lM E3), alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (0.5 lM B[a]P). Cells were dosed in 96 well plates (n ¼ 2–3 fish, 4 replicate wells per treatment) incubated at 16 °C. Estradiol doses were similar to those used by others with this model (Anderson, Olsen, Matsumura, & Hinton, 1996; Navas & Segner, 2001), but higher than those observed in spawning female rainbow trout (0.22 lM, Van Bohemen & Lambert, 1981), and were used to determine dose-response. Estriol doses were chosen to mirror those of estradiol, and to fall within the range used in other studies of estriol suppression of CYP1A1 (Son et al., 2002). Forty-eight hours after dosing, CYP1A activity was evaluated (as ethoxyresorufin-O-deethylase, EROD) on a CytoFluorTM 4000 fluorescent plate reader (Applied Biosystems) essentially as described (Hahn, Woodward, Stegeman, & Kennedy, 1996). Hepatic viability for all treatments remained high at 48 h (>95%), as assessed using the lactate dehydrogenase viability assay of Scholz and Segner (1999). Statistical differences between treatments were determined by one-way ANOVA, p < 0:05. We found significant, dose-related suppression of B[a]P-induced CYP1A activity by estradiol and estriol relative to vehicle controls. Estradiol (0.5, 5, 50 lM) suppressed B[a]P-induced EROD by 3-fold, 5-fold, and to non-detectable levels, respectively, relative to B[a]P treatment alone (Fig. 1). The two higher doses of estriol (8, 80 lM) suppressed B[a]P-induced EROD by 11, and 40-fold, respectively, relative to B[a]P treatment alone (Fig. 2). Although basal EROD activities tended to be lower, 15-, 19- and 120-fold in the presence of estradiol (0.5, 5.0 and 50 lM, respectively) or 12-, 713-fold and non-detectable in the presence of estriol (0.8, 8.0 and 80 lM, respectively), these activity levels were not statistically different from vehicle controls due to high variability in the vehicle treatment groups. Although not significant, co-exposure to the lowest doses of estradiol and estriol tended to suppress basal (5-fold), but not B[a]P-induced, EROD relative to exposure to either steroid alone (data not shown). EROD activities were lower than measured in other studies in our laboratory (example, in other studies
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
465
Fig. 1. Estradiol suppression of CYP1A catalytic activity in primary rainbow trout hepatocytes. Hepatocytes were untreated or treated with DMSO (0.5%), 17b-estradiol (0.5, 5, 50 lM), B[a]P (0.5 lM), or combinations, and CYP1A catalytic activity (ethoxyresorufin O-deethylase, EROD) measured after 48 h. Bars represent means SD (n ¼ 2–3 fish). Mean EROD activity of steroid-treated cells is significantly different from BaP (#) at P < 0:05.
Fig. 2. Estriol suppression of CYP1A catalytic activity in primary rainbow trout hepatocytes. Hepatocytes were untreated or treated with DMSO (0.5%), estriol (0.8, 8, 80 lM), B[a]P (0.5 lM), or combinations and CYP1A catalytic activity (measured as ethoxyresorufin O-deethylase) measured after 48 h. Bars represent means SD (n ¼ 2–3 fish). Mean EROD activity of steroid-treated cells is significantly different from BaP (#) at P < 0:05.
466
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
EROD ranged from basal 10–20 to B[a]P-induced 80–140 pmol/min/mg). The lower EROD levels observed in the present study may reflect the substantial interindividual variation in EROD activity in rainbow trout primary hepatocyte preparations noted by others (Sadar & Andersson, 2001). Catalytic activity did not differ between DMSO-treated and untreated cells (Figs. 1 and 2). While estradiol suppression of basal CYP1A activity has been reported in vivo and in vitro, in vitro evidence for estrogenic suppression of CYP1A induction is inconsistent. We found 48 h exposures to doses of 0.5 lM E2 or higher significantly suppressed induction of EROD by 0.5 lM B[a]P. In other studies with rainbow trout hepatocytes a 48 h exposure to estradiol at 1 lM or higher significantly suppressed induction of EROD by 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Anderson et al., 1996), but failed to suppress EROD induction by 0.78 lM b-naphthoflavone (b-NF, Navas & Segner, 2001). The mechanism by which estrogens suppress CYP1A expression is not known. Field studies with spawning fish suggest estrogenic suppression of CYP1A occurs pretranslationally (Elskus, Pruell, & Stegeman, 1992). In vitro treatment with estradiol lowered basal levels of CYP1A mRNA in rainbow trout hepatocytes, but did not block induction (Navas & Segner, 2001). Fish liver expresses three estrogen receptor (ER) subtypes (Hawkins et al., 2000), and studies in trout with the ER antagonist, tamoxifen, suggest CYP1A suppression by E2 is ER-mediated (Navas & Segner, 2001). Estradiol is completely metabolized to water soluble conjugates within 1 h in cell culture (Anderson et al., 1996) yet effects persist for at least 48 h. This suggests estradiol may act on CYP1A indirectly, by initially altering estradiolregulated genes, which, in turn, suppress CYP1A expression, or that estrogen metabolites are the main suppressive agents. Preliminary time-course data indicate suppression of basal CYP1A mRNA expression in rainbow trout hepatocytes does not occur until 24 h after estradiol treatment (unpublished data), lending support to an indirect mechanism for E2 suppression of CYP1A. Further support for this mechanism is suggested by the lag period between the fall in plasma estradiol levels and increased CYP1A expression in post-spawning female fish (Elskus and Stegeman, unpublished data). Possible indirect mechanisms include promoter methylation, an epigenetic process shown to silence CYP1A expression (Takahashi, Suzuki, & Kamataki, 1998), and estradiol induction of transcription factors that suppress CYP1A induction (e.g., Hohne, Becker-Rabbenstein, Kahl, & Taniguchi, 1990). Cross-talk between the aryl hydrocarbon receptor and ER in regulation of CYP1A is also a possibility (Thomsen, Wang, Hines, & Safe, 1994). Clearly, further work on the molecular mechanism of estrogen regulation of CYP1A is needed.
Acknowledgements This work was supported by a University of Kentucky Research Committee Grant. The rainbow trout were generously provided by Mr. James Gray of the Wolf Creek National Fish Hatchery, Jamestown, KY.
A.A. Elskus / Marine Environmental Research 58 (2004) 463–467
467
References Anderson, M. J., Olsen, H., Matsumura, F., & Hinton, D. E. (1996). Aquatic Toxicology, 34, 327–350. Elskus, A. A., Pruell, R. J., & Stegeman, J. J. (1992). Marine Environmental Research, 34, 97–101. Gray, E. S., Woodin, B. R., & Stegeman, J. J. (1991). Journal of Experimental Zoology, 259, 330–342. Hahn, M. E., Woodward, B. L., Stegeman, J. J., & Kennedy, S. W. (1996). Environmental Toxicology and Chemistry, 15, 582–591. Hawkins, M. B., Thorton, J. W., Crews, D., Skipper, J. K., Dotte, A., & Thomas, P. (2000). Proceedings of the National Academy of Sciences of the United States of America, 97, 19751–19756. Hohne, M., Becker-Rabbenstein, V., Kahl, G., & Taniguchi, H. (1990). FEBS, 273, 219–222. Navas, J. M., & Segner, H. (2001). Chemico-Biological Interactions, 138, 285–298. Sadar, M. D., & Andersson, T. B. (2001). In Vitro Cellular and Developmental Biology – Animal, 37, 180– 184. Scholz, S., & Segner, H. (1999). Ecotoxicology and Environmental Safety, 43, 252–260. Son, D.-S., Roby, K., Rozman, K., & Terranova, P. F. (2002). Toxicology, 176, 229–243. Takahashi, Y., Suzuki, C., & Kamataki, T. (1998). Biochemical and Biophysical Research Communications, 247, 383–386. Thomsen, J. S., Wang, X., Hines, R. N., & Safe, S. (1994). Carcinogenesis, 15, 933–937. Van Bohemen, C., & Lambert, J. (1981). General and Comparative Endocrinology, 45, 105–114.