TOXICOLOGICAL HIGHLIGHT Redox Redux: A Closer Look at Conceptal Low Molecular Weight Thiols

Glutathione (GSH) is present as the most abundant low molecular weight thiol (LMWT) in virtually all mitochondriabearing eucaryotic cells, often at millimolar concentrations (Meister, 1988). Functions of GSH include roles in DNA and protein synthesis, maintenance of cell membrane integrity, drug and chemical metabolism, and protection from oxidative stress. The role of GSH in normal reproduction and development, as well as its role in protecting against reproductive toxicants, has been studied extensively, but remains poorly understood. Sources of reducing equivalents other than GSH provide unique as well as redundant functions and include the thioredoxin pathway, superoxide dismutase, catalase, and cysteine. The presence of functional redundancy, as well as the ability of many mammalian cells to tolerate substantial decreases in intracellular GSH, has made studies of the specific roles of glutathione in reproductive and developmental toxicity difficult, and results have been discordant. In the feature article, Beck et al. take a novel and important approach to studying the content and distribution of GSH and cysteine in organogenesis-stage embryos, using acetaminophen (APAP) as a modulator of GSH and mercury orange staining to localize LMWT. Their results provide insight on the redox status and capacity of these embryos, including compartmentalization and intracellular distribution. Previous studies have demonstrated that APAP is toxic to both preimplantation mouse (Laub et al., 2000) and organogenesis-stage rat embryos (Harris et al., 1989) in vitro but not in vivo. One possibility for this difference is that APAP reduced embryonal GSH in vitro but not in vivo. The study by Beck et al. shows that APAP does indeed lower GSH in embryos in vivo, despite its lack of developmental toxicity. Thus, it appears that embryos at this stage have a substantial buffer of reducing equivalents in vivo, and that either this buffer is not present in vitro, or the degree of GSH depletion achieved in vitro cannot be attained in vivo. These results serve to highlight redox metabolism as a critical factor in assessing developmental effects, especially when comparing in vivo and in vitro results. Equally important is the intracellular distribution of LMWT. Exposure to APAP did not produce a uniform effect on LMWT, but rather the depletion was tissue-specific and may represent loss of cytoplasmic stores in affected tissues. Beck et al. demonstrate that following APAP treatment, some subcellular LMWT remain, possibly in mitochondria. As the authors point out, differences in the localization of LMWT depletion at the subcellular level may explain why APAP is not teratogenic, while ethanol, which depletes mitochondrial GSH, is embryotoxic and teratogenic (Beck, 2000). The importance of GSH during development has been well established. In mature mouse and hamster oocytes, GSH concentrations are high, comparable to those in hepatocytes (Meister and Andersen, 1983; Perreault et al., 1988). During fertilization, GSH appears to aid in development of the male pronucleus (Calvin et al., 1986; Perreault et al., 1988; Sutovsky and Schatten, 1997), as well as in oocyte spindle formation (Zuelke et al., 1997). Induction of GSH synthesis in bovine (de Matos and Furnus, 2000) or porcine (Abeydeera et al., 1999) oocytes with beta-mercaptoethanol and cysteine during in vitro maturation improved cleavage rates and embryo development. Conversely, reduced GSH levels contributed to the impaired growth of bovine embryos from oocytes matured in high-glucose media (Hashimoto et al., 2000). Developmental delay is a consistent observation in mammalian preimplantation embryos grown in vitro. Exposure to atmospheric oxygen tension (;20%) is thought to be a major contributing factor (Wright and Bondioni, 1981); indeed, the maternal reproductive tract has been reported to have reduced oxygen (e.g., about 8% in rabbit and hamster oviduct and 5.3 and 3.5%, respectively, in the hamster and rabbit uterus at the time of implantation [Fischer and Bavister, 1993]). Yet, the use of reduced oxygen tension has produced mixed results. Improved blastulation rates have been reported in the mouse (Gardner and Lane, 1996; Orsi 1 To whom correspondence should be addressed. E-mail: rogers.john@epamail.epa.gov. TOXICOLOGICAL SCIENCES 62, 1–3 (2001)