2.4. EROD, MROD, ECOD, APND, and ERND Activity Measurements

D. magna protein extraction to test biochemical biomarkers to assess CYP450 enzymes’ activity consisted of adding 450 µL of crushing buffer in stocked samples placed in the Bertin Cryolis evolutionTM for 1 min (7500 rpm). The crushing buffer was prepared with phosphate buffer (100 mM, pH 7.8) and 0.1% of protease inhibitor (details of protease inhibitor component available in Appendix A, “List of chemicals”). Then, the homogenate was centrifuged at 9000× g for 15 min (4 °C). The supernatant was collected to proceed with the protein assay using the QuantiProTM BCA Assay Kit (Sigma-Aldrich) and enzymes’ activity measurements. All manipulations were performed on ice to avoid products’ denaturation.

Total protein concentration was measured using the bicinchoninic acid assay (BCA assay). Protein content was quantified using bovine serum albumin (BSA, 0.5 mg/mL) standard dilution series (0–200 µg/mL BSA in phosphate buffer 100 mM, pH 7.8). Diluted samples were incubated with a BCA mix in a transparent 96-well plate for 1 h at 60 °C (350 rpm). Absorbance was read at 562 nm. All assays were performed in triplicate in the following enzyme activity tests, and absorbance or fluorescence was measured using the Tecan Spark^® multi-mode microplate reader.

A black 96-well fluorometric plate was used to measure EROD, MROD, and ECOD activities. Based on existing methods, protocols of EROD [40,41], MROD [16], and ECOD [21] were optimized. We adapted the existing methods by adjusting the standard range of resorufin and hydroxycoumarin as well as the manual gain setting, which determines the amplification of a detected signal during the fluorescence endpoint measurement.

EROD and MROD activities were determined by measuring the formation of the resorufin and its fluorescence (excitation wavelength of 535 nm and emission wavelength of 590 nm). The assay consisted of the transformation of 7-ethoxyresorufin (EROD) or 7-methoxyresorufin (MROD) into resorufin by CYP450 enzymes present in the microsome samples (i.e., supernatant of the protein fraction extracted from D. magna). Regarding ECOD activity, the formation of hydroxycoumarin and its fluorescence was determined as its activity (excitation wavelength of 380 nm and emission wavelength of 480 nm). The ECOD assay consisted of the transformation of 7-ethoxycoumarin into 7-hydroxycoumarin (see Appendix A for EROD, MROD, and ECOD stock solution details and reaction illustrations).

D. magna microsome (30 µL) was added with either 7-ethoxyresorufin (concentration in well: 2 µM, EROD), 7-methoxyresorufin (concentration in well: 2 µM, MROD), or 7-ethoxycoumarin (concentration in well: 0.45 µM, ECOD) and NADPH (concentration in well: 0.167 mM) for a final reaction volume of 200 µL. A standard curve of resorufin products (0–0.018 µM in phosphate buffer 100 mM, pH 7.8) was used to convert relative fluorescence units to pM/min/mg for EROD and MROD assays. The standard curve for the ECOD assay was realized using 7-hydroxycoumarin (0–0.075 µM in phosphate buffer 100 mM, pH 7.8) to convert relative fluorescence units to pM/min/mg. NADPH was put just before the microplate reading, because of its fast reactivity on reaction. Fluorescence levels of resorufin and 7-hydroxycoumarin were measured 30 min after adding NADPH to determine the EROD, MROD, and ECOD activities. The manual gain setting was adjusted to 60 on the Tecan Spark^® multi-mode microplate reader.

In a 96-well plate colorimetric assay, APND and ERND activities were measured as described by Peng et al. (2013) [23] and Nash (1953) [42], with some modifications. Their activities were determined by measuring the formation of formaldehyde (see Appendix A for ERND and APND stock solution details and reaction illustrations).

D. magna microsome (10 µL) was mixed with NADPH (concentration in well: 83.3 µM) and aminopyrine (concentration in well: 1.16 mM) or erythromycin (concentration in well: 57.6 µM). The plate was incubated for 30 min at 37 °C (350 rpm). The reaction was then stopped by adding Nash solution (0.3 M). Finally, the plate was incubated for 10 min at 60 °C (350 rpm), and absorbance was read at 420 nm. A standard curve of formaldehyde products (0–20 µM in phosphate buffer 100 mM, pH 7.8) was used to convert absorbance units to APND or ERND activities in nM/min/mg.