Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos.
To examine the importance of reduced intracellular glutathione (GSH) in the modulation of dysmorphogenesis and to gain insight into the electrophilic character of the embryotoxic intermediates generated in the rat embryo from N-acetoxy-2-acetylaminofluorene (AAAF) and acetaminophen (APAP) in cultured embryos, the effects of GSH depletion on the embryotoxicity, dysmorphogenesis and covalent binding of these agents were examined. Both AAAF (90 microM) and APAP (500 microM) produced concentration-dependent, statistically significant (P less than or equal to 0.05) decreases in embryonic length as well as embryonic and visceral yolk sac protein content when rat embryos were exposed in vitro between days 10 and 11 of gestation. The predominant malformations observed upon exposure to AAAF and APAP were prosencephalic hypoplasia and abnormal neurulation respectively. Exposure of conceptuses to [3H]APAP followed by separation and fractionation of the cellular RNA, DNA and protein via density gradient centrifugation resulted in detectable binding in fractions that contained protein, but not DNA or RNA. This suggested that the rat conceptus is capable of bioactivating APAP to a soft electrophile that selectively arylates protein. In contrast, conceptuses exposed to [3H]AAAF exhibited detectable binding to RNA, DNA and protein, indicative of conversion to both hard and soft electrophiles. Depletion of GSH was accomplished by pretreating conceptuses with 500 microM L-buthionine-S,R-sulfoximine (BSO) from the start of the culture period (day 9.5) until the morning of day 10. When conceptuses were depleted previously of GSH by BSO, exposure to APAP resulted in significant potentiation (relative to APAP alone) of the observed embryotoxicity. These conceptuses displayed further decreases in both embryonic size and protein content of the embryo and yolk sac, as well as increased incidence of abnormally open anterior neuropores and increased binding (3-fold) of [3H]APAP to protein. In contrast, pretreatment with BSO did not potentiate the AAAF-elicited decreases in embryonic size or protein content, nor the severity of prosencephalic hypoplasia, although a slight increase in binding of [3H]AAAF to DNA was observed. Taken together, these data are consistent with the concept that abnormal neurulation elicited by APAP results from the generation of one or more soft electrophilic species, whereas elicitation of prosencephalic hypoplasia by AAAF appears to be a consequence of conversion to a relatively hard electrophile(s).
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
K L
KL
Stark
C
C
Harris
M R
MR
Juchau
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives*; D000082:Acetaminophen / Q000378:metabolism / Q000633:toxicity*; D000099:Acetoxyacetylaminofluorene / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001711:Biotransformation; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D005326:Fetal Proteins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D009024:Morphogenesis / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains
D005326:Fetal Proteins; D000082:Acetaminophen; D000099:Acetoxyacetylaminofluorene; D015073:2-Acetylaminofluorene; D005978:Glutathione