EROD activity

The 7-ethoxyresorufin-O-deethylase (EROD) activity assay was conducted according to previous studies with slight modifications (e.g. buffer volume, employed basic instruments) [31]. The liver tissues of individual fish were homogenized in 0.25 M sucrose and centrifuged at 9,000 × g for 20 min at 4°C. To sample the microsomal fraction, the supernatant was transferred and centrifuged at 105,000 × g for 60 min at 4°C. Approximately 35 μg of microsomal fraction was transferred into the EROD buffer (0.1 M NaPO4, pH 7.6) with the addition of 7-ethoxyresorufin (ER) solution (0.4 mM ER in DMSO) and NADPH solution (10 mM NADPH in distilled water). The background subtraction was prepared with the same mixed buffer with the absence of the microsomal fraction. The resourufin production was measured with 530 nm excitation and 595 nm emission filters with a Varioskan Flash fluorometer (Thermo Fisher Scientific, Tewksbury, MA, USA). Results were represented as μmol min/mg protein.