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Commercial assay kits were used to measure catalase activity (Cayman Chemical, USA; Catalogue Number 707002), 8-isoprostanes (RayBitotech Inc, USA; Catalogue Number EIA-IPF2A), TBARS (Cayman Chemical, USA; Item No. 700870), and nitrate+nitrite concentrations ([NOx] Cayman Chemical, USA; Item No. 780001) in serum. An inter-assay CV of 6.95% and intra-assay CV of 5.25% was calculated for catalase activity. An inter-assay CV of 7.40% and intra-assay CV of 2.75% was calculated for 8-isoprostanes. An inter-assay CV of 4.79% and intra-assay CV of 2.59% was calculated for TBARS. An inter-assay CV of 2.71% and intra-assay CV of 1.38% was calculated for NOx (nitrate+nitrite), and an inter-assay CV of 1.90% and intra-assay CV of 1.00% was calculated for nitrite alone. Nitrate was calculated as NOx (nitrate+nitrite) minus nitrite, as per the manufacturer’s instructions.

Nicotinamide (NA), streptozotocin (STZ), p-nitrophenyl α-D-glucopyranoside (pNPG), and α-glucosidase were purchased from Sigma-Aldrich®, St. Louis, MO, USA. Rat TNF-α, rat IL-6, and rat IL-1β ELISA kits were purchased from eBioscience (San Diego, CA USA). Rat insulin ELISA kit was purchased from Mercodia AB (Uppsala, Sweden). TBARS, Glutathione, and Glycogen assay kits were purchased from Cayman chemical company (Ann Arbor, MI, USA). All of the other chemicals are analytical grade purchased from Sigma and Merck Chemical Co.

Three biomarkers of oxidative stress were measured from first morning urine samples^{22 (link)}. All analyses were conducted using commercial ELISA kits in accordance with manufacturer procedure in Yale Reproductive Ecology Lab by the same researcher (8-OHdG, Enzo Life Sciences, catalogue ADI-EKS-350; Cu-Zn SOD, Enzo Life Sciences, catalog ALX-850-033; TBARS, Cayman Chemical Company, catalogue 10009055).
8-OHdG is a modified nucleotide base and a by-product of repaired DNA damage that is excreted in urine. Levels of 8-OHdG depict the amount of oxidative damage accumulated in cellular DNA through repaired lesions on the guanine base pair caused by ROS. Cu–Zn SOD catalyzes the dismutation of superoxide anion radicals to free oxygen and hydrogen peroxide. Levels of Cu–Zn SOD represent a primary line of enzymatic defense produced in the cytoplasm to neutralize ROS. Levels of TBARS reflect peroxidation of cellular lipids resulting from oxidative stress.
The average inter-assay variability for 8-OHdG, Cu–Zn SOD, and TBARS analysis was 14%, and intra-assay variability was 5%. All results were corrected for urine concentration based on samples’ specific gravity measured with refractometer (model PAL-10S ATAGO, U.S.A., Inc).

Malondialdehyde (MDA) was determined using a TBARS (Thiobarbituric acid reactive substances) Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's recommendations. The absorbance was read at 540 nm in a spectrophotometer (Spectra MAX 190, Molecular Devices).

The tissue samples were washed in neutral ice-cold phosphate-buffered saline 10% (w/v). The homogenate liver tissues were prepared via Teflon homogenizer (performed on ice), and then centrifuged at 4500 rpm for 15 min at 4 °C excluding cell debris. The collected supernatant was examined for antioxidant actions by using superoxide dismutase (SOD) and catalase (CAT) kits (Cayman Chemical Company, Ann Arbor, MI, USA) [39 (link)]. Malondialdehyde (MDA), as a lipid peroxidation biomarker, was also assessed to analyze the level of oxidative stress. Thus, the MDA assay kits were purchased and the reacted amount of thiobarbituric acid was determined [40 (link)] (TBARS, Cayman Chemical Company).

Brain concentrations of total and reduced GSH (tGSH and rGSH) were measured by ultraperformance liquid chromatography (Waters ACQUITY UPLC System) as reported by us previously [13 (link),14 (link),64 (link)]. Oxidized glutathione (GSSG) levels were calculated as the difference between tGSH and rGSH. OxS was measured using a TBARS (thiobarbituric acid reducing substances) assay kit (Cayman Chemicals, Ann Arbor, MI, USA) [70 (link)].