Ethoxyresorufin-O-Deethylase Assay

Activity of the Cyp1a1 enzyme was analyzed using the fluorometric ethoxyresorufin-O-deethylase (EROD) assay. The fluorescent EROD assay for Cyp1a1 activity was performed in 6-well plates according to the method described by Kennedy et al. (1993). The total protein concentration in each well was measured using fluorescamine according to the method described by Kennedy and Jones (1994). The measurement of Cyp1a1 activity was performed after 24 and 48 h of exposure to 1 to 100 nM and 1 to 100 μM DEHP or TCDD as a positive control. The EROD assays were carried out in multiwell plates, and the fluorescent product, resorufin, and the total amount of protein were quantified within the same wells using a fluorescence plate reader (Bio-Tek Instruments, Biokom). The ethoxyresorufin metabolite, resorufin, was measured using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Protein concentrations were measured using fluorescamine at an excitation wavelength of 400 nm and an emission wavelength of 460 nm.