Buthionine sulfoximine embryotoxicity is associated with prolonged AP‐1 activation

Abstract

Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.