Antioxidant enzymes assay
SOD activity in the cell lysate was assayed using a method based on the capability of the enzyme to inhibit the autoxidation of pyrogallol. Briefly, 1 ml Tris-EDTA buffer 5 mM (pH=8.0) was mixed with 50 ?l of the cell lysate supernatant. The unit was blanked at 420 nm and then 50 ?l of pyrogallol (0.2 mM) was added to the above solution and swiftly the absorbance of samples was evaluated at 420 nm every 15 s, up to 10 minutes. The inhibition of pyrogallol autoxidation is commensurate with the activity SOD present in the sample. Enzyme inhibitory capacity is determined as one unit of SOD19, 20. The enzyme activity was reported as IU/mg of protein.
Catalase activity was determined by Aebi method, that monitoring the decomposition of hydrogen peroxide21. 1 ml Tris-EDTA buffer 5 mM (pH=7.0) and 25 ?l hydrogen peroxide (10 mM) added to cuvette. Absorbance decrease based on the decomposition of H2O2 was measured at a wavelength of 240 nm for 30 seconds.
Determination of total thiol content
Total -SH groups are indicators of free radical damages. This sensitive factor decreases in oxidative damage conditions. Thiol groups were evaluated using DTNB (5, 5?-dithiobis- 2- nitrobenzoic acid) as the Ellman’s reagent, in which it reacts with the SH groups and produces a yellow color which has a peak absorbance at 412 nm22. To perform this assay, 1 ml Tris EDTA buffer (pH 8.6) was added to 50 ?l cell lysate and the absorbance of the sample was read at 412 nm against Tris-EDTA buffer alone (A1). Then, 20 ?l DTNB reagents (10 mM in methanol) was added to the cuvette mixture and after 15 min (stored at laboratory temperature), the sample absorbance was read again (A2). The absorbance of DTNB reagent was also read as a blank (B). Total thiol concentration (mM) was calculated using the following equation:
Total thiol concentration (mM)= (A2-A1-B) × 1.07/0.05 × 13.6
Measurement of MDA level
The concentration of MDA as a marker of lipid peroxidation was measured via thiobarbituric acid reactive substances (TBARS) assay in the KG1a cells lysate. This technique is based on the reaction of two molecules of thiobarbituric acid (TBA) with one molecule of MDA. The ultimate soluble including the substances which are responsible for the pink color23. Absorption of the samples was read at 532 nm using a UV–vis spectrophotometer.