Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction

Abstract

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.

Benzo(a)pyrene Metabolism and EROD and GST Biotransformation Activity in the Liver of Red- and White-Blooded Antarctic Fish

“…The slope of BaP depletion in rainbow trout, measured in S9 fractions of frozen liver tissue, 32 is in a comparable range to the values measured in the present study. Our results demonstrate that also Antarctic fish possess a capacity for xenobiotic metabolism at their physiological optimum temperature, although the rates are very low.…”

“…32 Liver weights were not available for all fish, thus the intrinsic hepatic clearance per gram of liver could not be calculated. All data were tested for normality (Kolmogorov−Smirnov) and homogeneity of variance.…”

Concentration dependence of in vitro biotransformation rates of hydrophobic organic sunscreen agents in rainbow trout S9 fractions: Implications for bioaccumulation assessment

“…The apparent in vitro concentration dependence of modeled BCFs may also help explain the tendency of IVIVE methods to overestimate BCFs relative to empirical data (Han et al ; Laue et al ). The past use of substrate concentrations ranging from 1 to 10 µM (Han et al ; Cowan‐Ellsberry et al ; Johanning et al ) may have saturated biotransformation enzymes, resulting in underestimates of k MET and overestimates of the BCF. In the present study, BCFs calculated from both k dep, C →0 and the k dep measured at initial concentrations of 0.05 µM were up to 2‐ and 7‐fold lower than those generated from reaction rates measured at 1 and 10 µM, respectively.…”

“…Incubations performed without added cofactors (test 2) were designed to test for biotransformation by cofactor‐independent enzymes such as carboxylesterases, whereas those performed in the presence of β‐NADPH (test 3) and β‐NADPH plus phase II cofactors (test 4) were included to evaluate the contribution of CYP and phase II reaction pathways, respectively. Three independent incubations, using a multitube approach (Johanning et al ), were conducted. Each experiment included one replicate of each incubation mixture (tests 1–4) to determine a mean in vitro depletion rate constant ( n = 3) for each UVF for the tested set of conditions.…”

In vitro metabolism and bioavailability tests for endocrine active substances: What is needed next for regulatory purposes?

“…Recent efforts also have focused on standardizing in vitro S9 systems to measure metabolism in fish species (Johanning et al, 2012). Related work to measure effects on chemical bioavailability in these systems has been shown to be an important component of improving predictions of in vivo metabolism (Escher et al, 2011).…”