Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line

Transcription

1 Marine Environmental Research 58 (2004) MARINE ENVIRONMENTAL RESEARCH Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line Jennifer M. Keller a,c, Patricia McClellan-Green a,b, a Marine Laboratory, Duke University, 135 Duke Marine Lab Road, Beaufort, NC 28516, USA b Department of Environmental and Molecular Toxicology, North Carolina State University, P.O. Box 7633, Raleigh, NC , USA c National Institute of Standards and Technology, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA Abstract Many classes of environmental contaminants affect the reproductive function of animals through interactions with the endocrine system. The primary components affected by endocrine active compounds (EACs) are the steroid receptors and the enzymes responsible for steroidogenesis. This study sought to develop an in vitro model for assessing EAC effects in sea turtles by examining their ability to alter cytochrome P450 aromatase (CYP19) activity. Aromatase is the enzyme responsible for the conversion of testosterone to estradiol. This enzyme is critical in the sexual differentiation of reptiles which demonstrate temperature-dependent sex determination. An immortal testis cell line GST-TS from a green sea turtle was grown in culture at 30 C in RPMI 1640 media. The cells were exposed to three known aromatase inducers; dexamethasone (Dex), 8Br-cyclic AMP, or human chronic gonadotropin (HCG) and one aromatase inhibitor 4-androstenol-dione (4-OHA). In addition, the GST-TS cells were exposed to lm atrazine and lm 4,4 0 -DDE. The inducing compounds that have been shown to increase aromatase activity in other systems failed to induce aromatase activity in the GST-TS cells, yet exposure to the inhibiting compound, 4-OHA, did result in a significant reduction. Atrazine (0.1, 1.0 and 10 lm) significantly induced aromatase activity following a 24 h exposure, and 4,4 0 -DDE inhibited the activity but only at cytotoxic concentrations (100 lm). Based on these results, this in vitro model can be useful in examining the endocrine effects of EACs in sea turtles. Ó 2004 Elsevier Ltd. All rights reserved. Corresponding author. Tel.: ; fax: address: pmcclell@duke.edu (P. McClellan-Green) /$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi: /j.marenvres

2 348 J.M. Keller, P. McClellan-Green / Marine Environmental Research 58 (2004) Keywords: Sea turtle; Endocrine; Aromatase; Cell line; Testis; Atrazine; DDE Cytochrome P450 aromatase (CYP19) is the key enzyme responsible for the conversion of testosterone to 17b-estradiol. Little is known about the expression and activity of this enzyme in endangered sea turtles or how contaminants affect its activity. Many reptiles have a unique mechanism of sexual differentiation termed temperature-dependent sex determination (TSD). During a critical embryonic stage the nest temperature cues steroid production which then directs gonadal differentiation. The mechanism behind TSD involves, in part, temperature-influenced expression and activity of cytochrome P450 aromatase. Aromatase has been shown to be regulated by temperature in the embryonic brain and gonads of several reptiles, including leatherback sea turtles (i.e., Desvages, Girondot, & Pieau, 1993; Milnes, Roberts, & Guillette, 2002). Sex reversal by estrogens has been shown in the olive ridley sea turtle, and exposure to organochlorine (OC) contaminants such as PCBs, 4,4 0 -DDE and chlordanes has resulted in sex reversal of red eared slider turtles (Merchant-Larios, Ruiz-Ramirex, Moreno-Mendoza, & Marmolejo-Valencia, 1997; Willingham & Crews, 2000). Likewise, atrazine, a heavily-used herbicide, has been shown to demasculinize amphibians and alligators (Crain, Guillette, Rooney, & Pickford, 1997; Hayes et al., 2002). These feminizing/demasculinizing effects have been linked to changes in the level of aromatase expression or activity (Willingham & Crews, 2000; Hayes, personal communication). Examining the effects of EACs on sea turtles is important as our recent studies have shown that significant concentrations of PCBs and chlorinated pesticides are present in their tissues (Keller, Kucklick, Harms, & McClellan-Green, 2004). Thus, the presence of these endocrine disrupters could contribute to their population decline. It was the goal of this study to examine the effects of EACs on aromatase activity in sea turtles through the use of an immortal cell line derived from testes of a green sea turtle (GST-TS). GST-TS cells were maintained in RPMI 1640 media without phenol red and supplemented with 10% charcoal-stripped FBS in 5% CO 2 at 30 C (Lu et al., 1999). Cells were plated into Costar 6-well plates at cells/well. The cells were washed and the media was replaced the following day using serum-free media containing either vehicle, the inducers dexamethasone (Dex), human chorionic gonadotropin (HCG), or 8Br-cAMP, an inhibitor 4-androsten-4-ol-3,17-dione (4-OHA), or the pesticides atrazine or 4,4 0 -DDE. Cells were incubated with 100 nm [1b- 3 H]-androstenedione for 6 h and aromatase activity was measured by the 3 H- water release assay (Sanderson, Boerma, Lansbergen, & van den Berg, 2002). Dexamethasone significantly induced aromatase activity at an exposure level of 1 lm compared to controls (Fig. 1(a)). Lower concentrations of dexamethasone, 0.01 and 0.1 lm, failed to induce aromatase activity. 8Br-cAMP (100 lm) and HCG ( IU/ml) failed to induce aromatase activity (data not shown). A known inhibitor, 4-OHA, significantly inhibited aromatase activity during a 6 h co-incubation with substrate at both 10 and 100 lm compared to controls (Fig. 1(b)).

3 J.M. Keller, P. McClellan-Green / Marine Environmental Research 58 (2004) fmol/mg/hr fmol/mg/hr (a) (b) µm Dexamethasone µm 4-OHA Fig. 1. GST-TS aromatase activity after exposure to: (a) dexamethasone for 21 h prior to substrate incubation, and to (b) 4-androsten-4-ol-3,17-dione (4-OHA) during the 6 h incubation with 100 nm 3 H-androstenedione. Mean ± standard deviation. Difference from control; (1a) ANOVA p ¼ 0:128 with Least Significant Difference (LSD) test p ¼ 0:025; (1b) ANOVA p < 0:001 with Bonferroni multiple comparison test p < 0:005. GST-TS cells were exposed to two different organochlorine pesticides. Atrazine induced aromatase activity at 1.0 and 10 lm following a 24 h exposure (Fig. 2(a)). Atrazine was also tested at 0.1 and 0.5 lm for a 24 h exposure. The 0.1 lm atrazine exposure resulted in significant induction of aromatase, but the 0.5 lm atrazine treatment did not (data not shown). All other atrazine exposure regimes did not significantly alter aromatase activity. It is important to note that basal aromatase activity was lower in the control for the 6 h atrazine treatment than in the controls of the other atrazine treatments. This difference was likely caused by the use of a different passage of cells for the 6 h treatment compared to the other treatments. 4,4 0 -DDE significantly inhibited aromatase activity but only at a cytotoxic concentration (Fig. 2(b)). The atrazine and 4,4 0 -DDE results are similar to what has been observed previously by Sanderson et al. (2002) in a human cell line. In addition, previous studies have demonstrated that 4,4 0 -DDE failed to reverse the sexual differentiation of reptiles, including green sea turtle embryos (Podreka, Georges, Maher, & Limpus, 1998). Failure of the known inducers to significantly increase aromatase activity suggests that part of the pathway necessary for induction may be missing in this

4 350 J.M. Keller, P. McClellan-Green / Marine Environmental Research 58 (2004) Fig. 2. GST-TS aromatase activity after exposure to: (a) atrazine, and to (b) 4,4 0 -DDE. The 6 h direct exposures were co-incubated with 100 nm 3 H-androstenedione. All other exposure durations were prior to substrate incubation. Mean ± standard deviation. Difference from control; (2a) ANOVA p ¼ 0:032 with Least Significant Difference (LSD) test p < 0:05; (1b) ANOVA for 6 h direct p ¼ 0:128 with LSD test p ¼ 0:048 and ANOVA for 24 h prior p ¼ 0:026 with LSD test p ¼ 0:012. cell line. However, this cell line may still be useful in screening EACs for their effects on sea turtles, especially compounds that function in a similar fashion as 4-OHA. Acknowledgements We thank Yuanan Lu for providing the GST-TS cells and our funding sources: Morris Animal Foundation, Disney Wildlife Conservation Fund, Duke University Marine Biomedical Center, and Oak Foundation.

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