Glutathione oxidation and embryotoxicity elicited by diamide in the developing rat conceptus in vitro.
This study was performed in the rat whole-embryo culture system to investigate the effects of glutathione oxidation by diamide, a thiol oxidant, in developing rat conceptuses during early organogenesis. The effects of diamide on reduced glutathione (GSH), glutathione disulfide (GSSG), and embryotoxicity were found to be concentration and time dependent. Diamide at concentrations of 75 and 100 microM produced abnormal axial rotation (62-89%), decreased viability (to 69% by 100 microM diamide), and reduced protein and DNA content in the embryo and visceral yolk sac (VYS) when evaluated on Day 11. High concentrations of diamide (250-500 microM) resulted in 100% mortality. GSH and GSSG levels in the conceptuses were not significantly affected during 2 hr following diamide addition at concentrations of 50 to 100 microM. At concentrations of 250 and 500 microM, rapid GSH depletion (50% of control) was seen within 5 min of exposure and was followed at 5-30 min by a significant increase in GSSG relative to control values. Diamide (500 microM) exposure for only 15 min on Gestational Day 10 was sufficient to elicit malformations (53% of exposed conceptuses with abnormal axial rotation) without significant loss of viability. After 30 min of exposure to the high concentration (500 microM), viability was decreased to 71% and defects of axial rotation increased to 87% in surviving conceptuses. This indicates that events associated with initial exposure are critical for expression of toxicity. Inhibition of glutathione disulfide reductase (GSSG reductase) activities in embryo and VYS with 1,3-bis(2-chloroethyl)-1-nitro-sourea prior to diamide addition potentiated the embryotoxicity of diamide (75 microM) and resulted in corresponding reductions in GSH/GSSG ratios as determined during the first 2 hr of exposure. Inhibition of new GSH synthesis with L-buthionine-[S,R]-sulfoximine during diamide (75 microM) exposure also exacerbated toxicity compared to diamide treatment alone. These results implicate the involvement of GSH synthesis and GSSG reductase activity in mediating the embryotoxicity of diamide.
Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor 48109.
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Hiranruengchok
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Harris
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D019328:Buthionine Sulfoximine; D002330:Carmustine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003958:Diamide / Q000633:toxicity*; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D054477:Glutaredoxins; D005978:Glutathione / Q000031:analogs & derivatives / Q000187:drug effects* / Q000378:metabolism; D019803:Glutathione Disulfide; D066298:In Vitro Techniques; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000502:physiology; D011490:Protein Disulfide Reductase (Glutathione); D051381:Rats; D017207:Rats, Sprague-Dawley
D000963:Antimetabolites; D054477:Glutaredoxins; D003958:Diamide; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D002330:Carmustine; D019803:Glutathione Disulfide