Reference Details

Regimen description

Aromatase activity of fathead minnow whole brain and ovary homogenates was determined by measuring the release of tritiated water from the C-1 carbon of a tritiated androstenedione substrate (Thompson and Siiteri, 1974; Lephart and Simpson, 1991). Whole fathead minnow brains or ovaries were thawed, weighed, and homogenized in phosphate buffer (10mM K2HPO4, 100mM KCl, 1mM EDTA, 1mM dithiothreitol, pH 7.4; 20 ul per mg tissue for brains, 4 ul per mg tissue for ovary). Homogenates were centrifuged for 10 min at 4 ◦C, 10,000×g. Aromatase assays were performed using 50ul of the resulting post-mitochondrial supernatant (PMS, S9 fraction). Extra aliquots of randomly selected samples were placed in an 80 ◦C waterbath for 10 min and used as heat-inactivated blanks. Reactions were initiated by adding 150
l of phosphate buffer containing 160nM 1-3H-androstenedione (Perkin- Elmer NET-926, Boston, MA, USA; specific activity 25.3 Ci/mmol) and 1mM NADPH (Sigma N1630). Samples were incubated for 90 min (brains) or 4 h (ovary) at 25 ◦C. Following incubation, the samples were extracted and analyzed as described elsewhere (Ankley et al., 2005b). 

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