Modulation of Intracellular Glutathione and Cysteine Metabolism in Bovine Oviduct Epithelial Cells Cultured In Vitro¹

^a Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, Ann Arbor, Michigan 48109–2029

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/µg protein for Cys and from 5.3 to 6.4 pmol/µg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 µM. Both compounds depleted GSH in a dose-dependent manner, and 500 µM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 µM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 µM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.

¹ Research supported by NIH grants ES05235 and ES07062.

² Correspondence: Craig Harris, Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, 1420 Washington Heights, Ann Arbor, MI 48109–2029. FAX: (734) 763–8095.

³ Current address: Tamara L. McNutt-Scott, Biology Department, 2500 Walton Way, Augusta State University, Augusta, GA 30904–2200.

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