Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): Comparison of basal‐level enzyme activities with rat and determination of xenobiotic intrinsic clearance in support of bioaccumulation assessment

Abstract

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.

Toward improved models for predicting bioconcentration of well‐metabolized compounds by rainbow trout using measured rates of in vitro intrinsic clearance

“…Similar models have been given previously by other authors [11][12][13][14][15]. The goal of this effort was to update these procedures using new information and develop models to serve a specific purpose-that of predicting measured levels of accumulation in small trout commonly used in BCF testing efforts.…”

“…To date, in vitro-in vivo metabolism extrapolations with fish have been performed using a variety of chemical substrates including herbicides (atrazine [11], trifluralin [11], and molinate [11,12]), pesticides (chlorpyrifos and zoxamide [13]), pharmaceuticals (ibuprofen, norethindrone, and propranolol [14]), alcohol ethoxylates (C16EO8 [13] and C13EO8 [14]), a benzophenone derivative (4,4-bis[dimethylamino]-benzophenone [11,12]), a surfactant (4-nonylphenol [11,12]), an alkylphenol (2,4-di-tert-butylphenol [11,12]), a polycyclic aromatic hydrocarbon (benzo[a]pyrene [11,12]), and an alkylbenzene sulfonate (C12LAS [13]). The biotransformation pathways responsible for metabolic clearance of these compounds in mammals are well known (see, for example, Table 1 in Han et al [11]).…”

“…The binding term f U,S9 is calculated using an empirical relationship given by Han et al [12] f U;S9 ¼ 1= C S9 Â 10 0:694 log KowÀ2:158 þ 1:0…”

In vitro biotransformation rates in fish liver S9: Effect of dosing techniques

“…However, the lack of a priori consideration of biotransformation rates is a major limitation in current bioaccumulation assessments. The other approach is the application of experimental in vitro biotransformation tests [3][4][5][6][7][8]. Empirical BCFs and BAFs do not exist for the vast majority of commercial chemicals.…”

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

“…For example, from an environmental perspective, if a substance is suspected to have effects on a fish species, then the use of a fish liver S9 system may be most appropriate. Recent sources of fish S9 are described in Han et al (2009) and Gomez et al (2011). Trout S9 fractions have been used as metabolic "activating" systems in different assays such as logical variability, a potential solution is a concordance analysis with assays for the same endpoints, both in vitro and then in vivo where data already exists, or for subsequent in vivo screening and test prioritization purposes (e.g., ER binding assays first with estrogen proliferation assays and ER reporter gene assays, and then with uterotrophic assay) to discern whether any respond with sufficient precision to allow biologically significant changes to be identified and thus inform the AOP.…”