PARENT SESSION

PW08 Aquatic Ecotoxicology II
Exhibit Hall
8:00 AM - Wednesday

(PW120) Cytochrome P4503A- mediated hydroxy estrogen formation by largemouth bass (Micropterus salmoides) liver microsomes.

Hughes, E.¹, Gallagher, E.², ¹ Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida, USA² Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA

ABSTRACT- Hydroxy estrogens such as 2-hydroxy estrogen (2-OH-E₂) and 4-hydroxy estrogen (4-OH-E₂) are generated during cytochrome P450 (CYP)-mediated hydroxylation of E₂ and can lead to the formation of DNA-reactive catechol estrogen quinones. In the current study, we examined the in vitro formation of 2-OH-E₂ and 4-OH-E₂ by hepatic CYP1A- and 3A- dependent pathways in largemouth bass, a higher order predatory game fish that may inhabit polluted waterways. CYP-dependent oxidation of E₂ by bass liver microsomal fractions produced predominantly 2-OH-E₂, as compared to 4-OH-E₂. The initial rates of 4-OH-E₂ production were not increased in the presence of high substrate concentrations (1 mM E₂) or on increasing microsomal protein concentrations. Bass liver microsomes exhibited high catalytic activity toward 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC, 50 uM), a CYP3A4 substrate in rodents, and CYP3A-dependent BFC activity was effectively inhibited by azamulin, a mammalian CYP3A- inhibitor. Inhibition of bass liver CYP3A- by azamulin caused a 60% reduction in the initial rates of formation of 2-OH- E₂. Pretreatment of bass with the CYP1A- inducer beta-naphthoflavone (NF), caused an induction of bass hepatic CYP1A-dependent EROD activities, but did not alter the formation of either 2-OH-E₂ or 4-OH-E₂. Our results indicate that bass liver microsomes catalyze the formation of 2-OH- E₂ through a CYP3A- dependent pathway, and that 4-OH- E₂ is not likely to be of toxicological significance in bass liver. Furthermore, CYP1A-does not appear to be an active catalyst of the formation of hydroxy estrogen metabolites in this species. Collectively, these data indicate that environmental exposure to compounds that alter the expression level or activity of CYP3A- may be a determinant of hydroxy estrogen concentrations in bass liver.

Key words: hydroxy estrogen, largemouth bass, cytochrome P450, azamulin