SIGNIFICANCE OF GLUTATHIONE DEPLETION AND OXIDATIVE STRESS IN EARLY EMBRYOGENESIS IN GLUCOSE-INDUCED RAT EMBRYO CULTURE

Recent studies have demonstrated the protective effects of supplementi ng free oxygen radical scavenging enzymes against hyperglycemia-induce d embryonic malformations. in this study, the glutathione (GSH)-depend ent protection system in hyperglycemia-induced embryopathy was investi gated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of G SH and activity of the rate-limiting GSH-synthesizing enzyme, gamma-gl utamylcysteine synthetase (gamma-GCS), significantly decreased in embr yos exposed to hyperglycemia compared with controls (7.9 +/- 0.6 vs. 1 2.5 +/- 0.9 nmol/mg protein, P < 0.01 and 13.3 +/- 1.9 vs. 22.6 +/- 1. 1 mu U/mg protein, P < 0.01, respectively). Decreased activity of gamm a-GCS in embryos exposed to hyperglycemia was associated with decrease d expression of gamma-GCS mRNA levels. However, the activities of supe roxide dismutase and glutathione peroxidase did not significantly chan ge in these embryos. Extracellular and intracellular free oxygen radic al formations estimated by Lucigenin-dependent chemoluminescence and f low cytometric analysis using 2',7'-dichlorofluorescein diacetate incr eased in isolated embryonic cells taken from embryos cultured under hy perglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglyce mic culture nearly restored GSH concentration in these embryos (11.9 /- 0.5 vs. 12.5 +/- 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization o f growth retardation and embryonic dysmorphogenesis. These results ind icate that the GSH-dependent protection system has a central role agai nst oxidative stress in embryos cultured under hyperglycemia and that GSH depletion in embryonic cells during the critical periods of organo genesis plays a role in hyperglycemia-induced embryopathy.