2.3.1. Glutathione S-Transferases (GSTs) Activity Assay

GST activity was determined according to Habig et al. [27] using 1-chloro 2,4-dinitrobenzene as a substrate with some modifications. The samples were thawed on ice, homogenized, and centrifuged at 9205 rpm for 15 min at 4 °C (5417R Eppendorf centrifuge, Germany). After removing the supernatant, the pellets were resuspended in 200 μL of DPBS buffer and centrifuged again to collect as much enzyme as possible. The combined supernatant (from two centrifugation times) was used for the GSTs and protein assay. The substrate (reaction solution) was a mixture of 100 mM DPBS buffer (pH 6.5), 200 mM GSH and 100 mM CDNB. The reaction was started by mixing 0.95–0.98 mL of the reaction mixture with 0.02–0.05 mL of samples, and the absorbance was measured every minute (for 8 min) at 340 nm using a Thermo Scientific^TM Biomate spectrophotometer. A blank sample containing 1 mL of the substrate was included. The specific activities of GSTs were calculated and expressed as nmoles of GSH-CDNB conjugate formed/min/mg protein.