Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

DNA oxidative damage analysis. The 8-OHdG was measured in staged adults C. elegans based on a published protocol. 11 In brief, staged first-day adults were harvested, washed, and resuspended in TE buffer followed by sonication and centrifugation at 12 000 r.p.m. for 15 min at 4 1C. The supernatant was incubated with 0.7 mg/ml RNase A, 0.46% SDS, and 0.1 mM deferoxamine mesylate at 37 1C for 1 h. It was followed by the addition of 0.36 mg/ml of proteinase K for [16] [17] [18] h in dark at room temperature on a rotating roller drum. The DNA was extracted by phenol/ chloroform/isoamylalcohol (25 : 24 : 1) and precipitated with 3 M sodium acetate (pH 5.2). The DNA pellet was sequentially washed by 99.5 and 70% ethanol. The washed DNA was resuspended in 20 mM sodium acetate (pH 5.2) and digested to nucleotide level for 2 h at 37 1C with 20 units of nuclease P1. Subsequently, 6 units of alkaline phosphatase in 1 M Tris buffer (pH 8.5) were added for 1.5 h at 37 1C. Prior to HPLC analysis, hydrolysate was subjected to filtration with Microcon YM-10 (EMD Millipore, Billerica, MA, USA) to remove proteins and and other macromolecules. The filtrate containing nucleosides was separated by a reverse-phase HPLC system (ESA, Inc., Chelmsford, MA, USA) using a C8 column (3 mm, 4.6 Â 150 mm; YMC-BD) and was eluted at a flow rate of 1.0 ml/min with 10% methanol in 20 mM sodium acetate buffer (pH 5.2) as mobile phase. The amount of 8-OHdG and dG were detected with an ESA Coulochem II electrochemical detector (ESA, Inc., USA). The standards 8-OHdG and dG were used in this assay. The 8-OHdG level was represented as the number of 8-OHdG molecules per 10 6 dG.