title: Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish.
abstract: Biotransformation rates measured using cryopreserved trout hepatocytes can be extrapolated to the whole animal to predict metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be optimized and standardized. Specifically, questions exist concerning gender differences in metabolism, cryopreservability of cells, and the accuracy of in vitro-in vivo scaling factors. 2. In this study, we evaluated hepatocytes from juvenile male and female trout. No gender differences in cell size, protein abundance, cytochrome P450 content, ethoxyresorufin-O-deethylase activity, uridine diphosphate glucuronosyltransferase activity or intrinsic clearance of pyrene were observed for freshly isolated hepatocytes. There was a small difference in measured glutathione-S-transferase activity (<25%; males > females). 3. Cells were cryopreserved by two methods: direct placement into liquid N2 vapor and controlled, slow-rate freezing. Comparable live recovery and enzymatic activity were observed regardless of freezing method or gender. Cells cryopreserved in liquid N2 vapor exhibited activity levels similar to those of freshly isolated cells, although there were small but significant differences in pyrene clearance and glutathione-S-transferase activity (frozen < fresh). Hepatocellularity values did not differ by sex. 4. These results suggest that hepatocytes from male and female juvenile trout may be used interchangeably for in vitro-in vivo metabolism extrapolations.
authors.affiliation: Division of Mid-Continent Ecology, US Environmental Protection Agency (US EPA), ORD, NHEERL , Duluth, MN , USA.
authors.forename: Kellie A
authors.initials: KA
authors.lastname: Fay
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Fitzsimmons
authors.forename: Alex D
authors.initials: AD
authors.lastname: Hoffman
authors.forename: John W
authors.initials: JW
authors.lastname: Nichols
mesh_terms: D000818:Animals; D001711:Biotransformation; D002470:Cell Survival; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects*; D008297:Male; D009584:Nitrogen / Q000737:chemistry; D017686:Oncorhynchus mykiss; D018570:Risk Assessment
chemical_list: D003577:Cytochrome P-450 Enzyme System; D009584:Nitrogen