Pyridine nucleotide concentrations in the developing rat conceptus: Relationships to embryotoxicity and glutathione status.

Abstract

This dissertation determined concentrations and alterations of the pyridine nucleotides (nicotinamide adenine dinucleotide (NAD(H)) and (NADP(H)) during development. Embryotoxicants were found to selectively modulate NAD(H) and NADP(H) pools while producing toxicity. Using the microfiberoptic perifusion system, fluorescence decreases were observed in the visceral yolk sac (VYS) upon exposure to <italic>tert</italic>-butyl hydroperoxide (tBH), diamide, and ethanol (EtOH). Decreases were attributed to utilization of NADPH by glutathione disulfide reductase (GSSG-Rd) during restoration of thiol homeostasis. Quantitative analyses showed that NAD<super>+</super> levels increased 3- and 4-fold in embryos and VYSs respectively, from gestational day (GD) 10 to GD 11 while NADH levels decreased 1.6- and 7-fold in embryos and VYSs, respectively. NADP(H) levels were below limits of detection on GD 10--11. Older conceptuses and fetal livers were analyzed for NADP(H) levels because these tissues are important for biotransformation and intermediary metabolism and utilize pyridine nucleotides as cofactors. NADP<super>+</super> was first detected in GD 20 fetal livers at concentrations of 3.1 +/- 0.2 pmol/mug protein. NADPH was detected in GD 12 embryos and VYSs at concentrations of 4.4 +/- 0.4 and 5.2 +/- 0.5 pmol NADPH/mug protein, respectively. Low NAD(P)H levels during organogenesis limit the ability of conceptuses to maintain thiol redox status, increasing susceptibility to toxicity. Temporal and spatial variations in NADP(H) levels correlated with fluctuations in NAD<super>+</super> kinase (NADK) activity, an enzyme that catalyzes the phosphorylation of NAD<super> +</super>. The embryotoxicant methanol (MeOH) rapidly decreased NAD<super>+</super> to 13% of controls in GD 10 conceptuses at 30-minutes. Pretreatment with 3-aminobenzamide, prevented MeOH-induced NAD<super>+</super> decrease, suggesting that ADP ribosylation may play a role in MeOH-induced NAD<super>+</super> decrease and subsequent embryotoxicity. tBH decreased NADH to 52% and 30% of controls in embryos and VYSs respectively, without altering NAD<super>+</super>. EtOH and MeOH increased NADH to 128 and 138% of controls, respectively. While tBH altered NADP(H) pool, MeOH altered NAD(H) pool. These studies show for the first time that differential spatial and temporal alterations occur in conceptual pyridine nucleotide pools in response to chemically-induced stress. Understanding changes in pyridine nucleotide status during development will help define their critical role in embryo protection and maintenance of homeostasis.