Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus

Abstract

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux {NAD(H) and NAD(P)H} in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-μM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-μM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 μM produced fluorescence decreases similar to that of 500 μM diamide, but the decreases were attenuated at 1000 μM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 μM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.