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METHOXYCHLOR METABOLISM AND VITELLOGENESIS IN MALE RAINBOW TROUT LIVER SLICES
Citation:
Sheedy, B R., M A. Tapper, M. W. Hornung, R C. Kolanczyk, AND P. K. Schmieder. METHOXYCHLOR METABOLISM AND VITELLOGENESIS IN MALE RAINBOW TROUT LIVER SLICES. Presented at Joint Regional SETAC/SOT Annual Meetings, USEPA, Duluth, MN, April 9-10, 2002.
Description:
Induction of vitellogenin (VTG) in male fish has become an accepted biomarker to xenoestrogenicity. This study utilized the male rainbow trout liver slice model to determine the estrogenicity of parent compound, methoxychlor (MXC) and metabolites, di-hydroxy methoxychlor (HPTE) and mono-hydroxy methoxychlor (MHMXC). Tissue slices were exposed to concentrations (1-10000 nM) of MXC, HPTE or MHMXC for 72 h at 11 C in 12-well tissue culture plates. VTG induction was assessed by measurement of VTG mRNA (Northern dot blot and RPA analysis) and VTG protein (ELISA). Slices exposed to estradiol (E2; 0.1-1000 nM) served as a positive control. There was a significant induction at 72 h at both VTG mRNA and VTG protein in slices exposed to 10,000 nM MXC or 10,000 nM HPTE (with suggestion of induction at 100 and 1000nM concentrations) for 72 h. Exposure of slices to MHMXC resulted in no detectable induction of VTG mRNA or protein at the concentrations tested. Chemical concentrations were measured in the exposure media at 0 and 72 h. Initial concentrations of HPTE or MHMXC were undetectable after 72 h, presumably due to Phase II metabolic conjugation as seen with disappearance of E2 observed previously in trout liver slices. However, while MXC concentrations decreased from initial exposure concentrations, detectable levels were measured at 72 H. This indicated that the rate of MXC metabolism in liver slices was significantly less than that observed for E2, although adequate to support VTG induction. These results are contrasted with lack of metabolic conversion of MXC, HPTE, or MHMXC in a trout VTG reporter gene assay using RTH-149 cells cultured at 11 C. The VTG reporter yielded positive results only upon exposure to HPTE. The ability of the liver slices to metabolize MXC, presumably to HPTE prior to conjugation, is associated with the VTG induction noted in slices but not in the RTH-149 gene assay where metabolism was absent. This demonstrates the advantage of using a metabolism was absent. This demonstrates the advantage of using a metobolically competent assay system to detect importance of biotransformation in screening of chemicals for potential endocrine disruption.