Hepatic Phase I, Phase II and Antioxidant Enzymes

The methods for phase I, phase II enzyme assays were recently described (Canistro et al., 2016). Antioxidant enzyme activities were measured as defined by Melega et al. (2013). All details relating to the procedure are reported here.

NADPH-(CYP)-c-reductase (CYP-red) activity. The analytical method is based on the determination of the reduction rate of cytochrome c at 550 nm (𝜀 = 19.1 mM^-1 cm^-1), according to previously defined procedures (Bruce, 1967; Canistro et al., 2012b). The incubation mixture contained 1.6 ml of 0.05 M Tris-HCl buffer (pH = 7.7) with 0.1 mM EDTA + 0.5 mg cytochrome c + 0.2 ml of microsomes. The reaction begins with the addition of 0.2 ml NADPH. Specific reaction was read at 550 nm against buffer plus cytochrome c.

Aminopyrine N-demethylase (APND) activity-CYP3A1/2. Activity was determined by the quantification of CH₂O release, according to Mazel (1971). The total incubation volume was 3 ml, composed of 0.5 ml water solution of 50 mM aminopyrine and 25 mM MgCl₂, 1.48 ml of 0.60 mM NADP⁺, 3.33 mM G6P in 50 mM Tris-HCl buffer (pH 7.4), 0.02 ml G6PDH (0.93 U/ml) and 0.125 ml of sample. After 5 min of incubation at 37°C, the yellow color developed by the reaction of the released of CH₂O with the Nash reagent was read at 412 nm, and the molar absorptivity of 8,000 used for calculation (Melega et al., 2013).

p-nitrophenol hydroxylase (p-NPH) activity-CYP2E1. Activity was determined in a final volume of 2 ml: 2 mM p-nitrophenol in 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl₂, and a NADPH-generating system consisting of 0.4 mM NADP⁺, 30 mM isocytrate, 0.2 U of isocytrate dehydrogenase and 1.5 mg of proteins. After 10 min of incubation at 37°C, the reaction was terminated by addition of 0.5 ml of 0.6 N perchloric acid. Precipitated proteins were removed by centrifugation and 1 ml of the resultant supernatant was mixed with 1 ml of 10 N NaOH. Absorbance at 546 nm was immediately recorded and 4-nitrocathecol determined (𝜀 = 10.28 mM^-1 cm^-1) (Canistro et al., 2012a).

Pentoxyresorufin O-dealkylase (PROD) activity-CYP2B1/2, ethoxyresorufin O-deethylase, (EROD)-CYP1A1 and methoxyresorufin O-demethylase (MROD)-CYP1A2. Reaction mixture (PROD) consisted of 0.025 mM MgCl₂, 200 mM pentoxyresorufin, 0.32 mg of proteins and 130 mM NADPH in 2.0 ml 0.05 M Tris-HCl buffer (pH 7.4). Resorufin formation at 37°C was calculated by comparing the rate of increase in relative fluorescence to the fluorescence of known amounts of resorufin (excitation 563 nm, emission 586 nm) (Lubet et al., 1985). EROD and MROD activities were measured in exactly the same manner as described for the pentoxyresorufin assay, except that substrate concentration was 1.7 mM for ethoxyresorufin and 5 mM for methoxyresorufin (Sapone et al., 2016).

Ethoxycoumarin O-deethylase (ECOD) activity-CYP1A1/2, CYP2A, CYP2B, CYP2E1. ECOD was determined by the quantification of umbelliferone formation, according to Aitio (1978). Incubation mixture consisted of 2.6 ml, composed of 1 mM ethoxycoumarin, 5 mM MgCl₂, NADPH-generating system (see aminopyrine assay) and 0.25 ml of sample. After 5 min of incubation at 37°C, the reaction was stopped by the addition of 0.85 ml of trichloroacetic acid (TCA) 0.31 M. The pH of the mixture was brought to about 10 by adding 0.65 ml of 1.6 M NaOH-glycine buffer (pH 10.3); the amount of umbelliferone was measured fluorimetrically (excitation 390 nm; emission 440 nm) (Vivarelli et al., 2016b).

The incubation mixture for measuring overall GST activity contained 1 mM glutathione + 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) in methanol + 0.025 mL of sample in a final volume of 2.5 mL 0.1 M phosphate Na⁺/K⁺ buffer (pH 6.5). The product of the reaction of the thiol group of glutathione with the electrophilic group of CDNB was read at 340 nm (𝜀 = 9.6 mM^-1 cm^-1) (Vivarelli et al., 2016a).

The overall UDPGT activity was determined kinetically using 1-naphthol as substrate (final concentration, 50 mM) by the continuous fluorimetric (excitation 390 nm; emission 440 nm) monitoring of 1-naphtholglucuronide production in the presence of 1 mM uridine-5′-diphosphoglucuronic acid (Mackenzie and Hänninen, 1980). Experiments were performed in the presence or absence of Triton X-100 (0.2%) as a detergent, in order to improve the assay sensitivity (Canistro et al., 2008).

Catalase (CAT) activity. The reaction was started in a quartz cuvette containing 50 mM potassium phosphate buffer and cytosol sample by adding 30 mM H₂O₂. The decomposition of the substrate was measured at 240 nm and catalase activity was expressed as mol of H₂O₂ consumed per minute per mg protein using a molar extinction coefficient of 43.6 mM^-1 cm^-1 (Bonamassa et al., 2016).

NAD(P)H:quinone reductase (NQO1) activity. NQO1 activity was assayed spectrophotometrically at 600 nm by monitoring the reduction of the blue redox dye of DCPIP (𝜀 = 9.6 mM^-1 cm^-1), and expressed as mol of DCPIP reduced per minute per mg protein (Bonamassa et al., 2016).

Oxidized glutathione reductase activity (GSSG-red). GSSG-red activity was measured by adding 1.5 mM NADPH to an assay cuvette containing 50 mM potassium phosphate buffer, 1 mM EDTA, cytosol sample and 20mM GSSG. The generation of NADP⁺ from NADPH during the reduction of GSSG was recorded at 340 nm for 5 min at 37°C. GSSG-red activity was calculated using the extinction coefficient of 6.22 per mM x cm, and expressed as mol of NADPH consumed/min per mg protein (Bonamassa et al., 2016).

Superoxide dismutase activity (SOD): Superoxide dismutase activity activity was determined according to Misra and Fridovich’s assay (Misra and Fridovich, 1972). Briefly, the activity was assayed spectophotometrically at 320 nm by monitoring the generation of adenochrome, one of the main products of epinephrine autoxidation at pH 10.2. The dejection of autoxidation was used to calculate SOD activity using the extinction coefficient of 4.02 per mM × cm, and expressed as mol of epinephrine oxidized/min per mg protein, derived by subtracting each test curve from the epinephrine autoxidation standard curve (Bonamassa et al., 2016).

For each animal, blood samples were collected in both heparinized and non-heparinized tubes. Plasma from heparinized samples was obtained after tabletop centrifugation at 2,000 rpm, while blood in non-heparinized tubes was centrifuged at 1,500 rpm for 10 min after complete coagulation, to obtain serum. Biochemical and hematological analyses were assessed by the Department of Veterinary Medical Science, University of Bologna.

Protein concentration was determined according to the Lowry method revised in Bailey (1967) using bovine serum albumin as standard and diluting samples 200 times to provide a suitable protein concentration.