Glutathione synthesis in the rat embryo and visceral yolk sac: Differential regulation and response to embryotoxicants.
Lee, Eunyong
1996
Abstract
Embryotoxicities induced by various therapeutic and environmental agents have been associated with decreases in intracellular glutathione (GSH) concentrations and have been shown to be modulated by intracellular GSH status. However, the mechanisms regulating GSH metabolism are not yet clearly understood in the developing embryo. This dissertation provides important evidence that biosynthesis of GSH is differentially regulated in the rat embryo and visceral yolk sac (VYS) during organogenesis. The differential capacity of GSH synthesis may influence the cell-selective pathology elicited by various embryotoxicants. The initial study on GSH depletion by diethylmaleate (DEM) and subsequent repletion of the GSH pools provided insights showing that GSH synthesis may be differentially regulated in the embryo and VYS. It was hypothesized that either enzyme activities for GSH synthesis are lower in the embryo when compared to the VYS, or that the supply of precursor amino acids may not be adequate in the embryo. The hypothesis was investigated by determining the relative activities of $\gamma$-glutamylcysteine synthetase (GCS) in the two compartments and evaluating the kinetic properties of GCS in the utilization of the two precursors, glutamate and cysteine. Inducibility of GCS was examined using agents selected for their ability to enhance GCS activity in other mature organs as a means to stimulate GSH synthesis during development. The enzyme activities and kinetic parameters of GCS were determined by measuring the formation of $\gamma$-glutamylcysteine in crude tissue homogenates. The specific activities of GCS were found to be 61 and 119 pmol/mg/min in the gestational day 10 rat embryo and VYS, respectively, and 23 and 71 pmol/mg/min in the respective day 11 tissues, showing two to three-fold greater GCS activity in the VYS. The apparent $K\sb{\rm m}$, of cysteine was 0.03 mM in both conceptal tissues, and the apparent $K\sb{\rm m}$, of glutamate was 1.38 and 0.75 mM in the embryo and VYS, respectively, suggesting that the availability of glutamate can also be a rate-limiting factor for GSH synthesis in the embryo. $\gamma$-Glutamylcysteine synthetase activity was increased to 150-240% of control in the VYS by exposures to DEM and diamide in vitro, and GCS mRNA levels were also elevated approximately two-fold by DEM, suggesting that GCS activity can be induced in the developing conceptus by agents which deplete GSH or induce oxidative stress. High degrees of chemical exposure can occur during development and may result in depletion of intracellular GSH in the conceptus. These studies imply that the embryo may be more susceptible to the deleterious effects of toxicants for longer periods of time than the VYS because replenishment of embryonic GSH may be limited by lower enzyme activities and inadequate supply of precursors for GSH synthesis. The greater activity of GCS in the VYS may enhance the ability of GSH synthesis and play a critical role in maintaining adequate detoxification capacity to protect the embryo from environmental and therapeutic exposures.Other Identifiers
(UMI)AAI9635548
Subjects
Health Sciences, Toxicology Health Sciences, Pharmacology Health Sciences, Human Development
Types
Thesis
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