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Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish
Citation:
Fay, K., P. Fitzsimmons, A. Hoffman, AND J. Nichols. Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, 44(4):345-351, (2014).
Impact/Purpose:
This manuscript examines the use of cryopreserved hepatocytes isolated from male and female sexually immature trout as an in vitro alternative to the whole animal bioconcentration factor (BCF) studies used for chemical bioaccumulation assessment. Previous groups have shown that trout hepatocytes can be used successfully to improve BCF predictions. However, all of the previous work has been conducted using hepatocytes from male fish only. In contrast, whole animal BCF studies use mixed-sex, juvenile trout. The results of this study indicate that hepatocytes isolated from male and female sexually immature trout may be used interchangeably for bioaccumulation predictions, improving the basis of comparison to determined laboratory BCF values, and preventing the waste of stock fish. Furthermore, scaling factors for male and female trout were determined for extrapolation of the in vitro data to whole animal values. Additionally, two methods of cryopreservation are compared. These results have implications in the evaluation of chemicals for their potential to bioaccumulate.
Description:
Measured rates of biotransformation by cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of predicting metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be standardized and optimized. Specifically, questions exist concerning gender differences in metabolic clearance, cryopreservability of cells, and the accuracy of in vitro-in vivo scaling factors. In the present study, we evaluated hepatocytes from juvenile male and female trout. No gender differences in cell size, protein abundance, cytochrome P450 content, EROD activity, UGT activity or intrinsic clearance of pyrene were observed for freshly isolated hepatocytes. Measured GST activity was greater in males than females, but this difference was relatively small (< 25%). Cells were cryopreserved by two methods: direct placement into the vapor phase of liquid N2 and a controlled, slow-rate freezing program. 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 GST activity (frozen < fresh). Hepatocellularity values did not differ between the sexes. These results suggest that hepatocytes obtained from male and female juvenile trout may be used interchangeably and are suitable for in vitro-in vivo metabolism extrapolation.
