ToxSci Advance Access published online on April 28, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh154
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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1 Department of Biochemistry, Emory University, Atlanta, GA 30322
* To whom correspondence should be addressed. E-mail: charris{at}umich.edu.
Glutathione (GSH) synthesis is differentially regulated in the embryo and visceral yolk sac (VYS) of the developing rat conceptus. The innate capacity to respond to environmental insult and chemical exposure by inducing de novo GSH synthesis may help to determine overall cell sensitivity and/or resistance to chemically-induced malformation. Specific activities of glutamatecysteine ligase (GCL), the rate limiting enzyme in GSH synthesis, were determined by measuring the formation of
Accepted April 13, 2004
Reproductive and Developmental Toxicology
Spatial Activities and Induction of Glutamate-Cysteine Ligase (GCL) in the Postimplantation Rat Embryo and Visceral Yolk Sac
2 Toxicology Program, Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029
Abstract 
-glutamylcysteine (GC) in homogenates prepared from rat embryos and VYSs. GC formation increased linearly with time and with relative protein concentration. Specific activities were found to be 60.5 ± 3.2 and 118.9 ± 4.2 pmol GC/mg protein/min in the GD 10 embryo and VYS, respectively, and 22.7 ± 0.4 and 71.3 ± 0.6 pmoles GC/mg protein/min in the respective GD 11 embryo and VYS. Apparent kinetic constants determined from embryo and VYS homogenates gave respective apparent Km values for glutamate of 0.75 and 1.38 mM and for cysteine 0.03 mM in both tissues. Apparent Vmax values were higher in the VYS in each case, corresponding with a lower apparent Km and higher GCL activity. GCL specific activities increased significantly following a 24 hr in vitro exposure to diethyl maleate (DEM) and diamide, but remained unchanged following exposure to prostaglandin A2 (PGA2) and t-butylated hydroxytoluene (BHT). Basal expression of GCL catalytic subunit (GCLC) and regulatory subunit (GCLR) was 59 and 25-fold higher in VYS, respectively, compared to the embryo. Quantitative real-time fluorescence RT-PCR showed that following DEM and diamide treatment, GCLC expression increased up to 19-fold in embryonic tissues but was not induced in the VYS. Only DEM increased the expression of the light/regulatory subunit GCLR in the embryo (8-fold). Densitometry of immunoblots revealed approximately 75% more GCLC in the VYS than in the embryo. Following treatments, a marked increase was induced in embryonic GCLC content with both DEM (85%) and diamide (19%), but in the VYS, only DEM caused an increase in GCLC protein (38%).
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