McNutt-Scott T L, Harris C
Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, Ann Arbor, Michigan 48109-2029, USA.
Biol Reprod. 1998 Aug;59(2):314-20. doi: 10.1095/biolreprod59.2.314.
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/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg 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 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-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 microM) 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.
本研究的目的是确定输卵管上皮细胞内硫醇水平如何响应可能导致氧化应激的化学或环境条件而发生改变。根据卵巢的生殖阶段，将牛输卵管分为卵泡期（F）或黄体期（L）。从每个输卵管的壶腹部（AMP）和峡部（ISTH）区域收集上皮细胞，悬浮于培养基中，然后接种到胶原包被的培养板中，培养至汇合。测定输卵管上皮细胞内半胱氨酸（Cys）和谷胱甘肽（GSH）的基础水平，发现Cys为0.36至0.46 pmol/μg蛋白，GSH为5.3至6.4 pmol/μg蛋白。氧化型Cys值占总Cys的21%至39%，而观察到的氧化型GSH水平占总GSH的21%至28%，但黄体期ISTH除外，其水平显著较低（6%）。将汇合细胞暴露于GSH消耗剂，L-丁硫氨酸-S，R-亚砜亚胺（BSO）或马来酸二乙酯（DEM），剂量范围为10至5000 microM。两种化合物均以剂量依赖性方式消耗GSH，后续每种化合物的研究均选择500 microM浓度。在最初5小时的孵育期间，BSO（500 microM）处理的输卵管上皮细胞中的Cys水平短暂高于对照值；然后AMP处的输卵管上皮细胞中的Cys水平下降，但ISTH处的未下降。在孵育期间，BSO处理的输卵管上皮细胞中的GSH持续消耗，到24小时时消耗了38%至61%。这些结果表明，与生殖阶段相关的输卵管上皮细胞中GSH周转存在差异。暴露于DEM（500 microM）导致Cys和GSH水平下降，去除DEM后部分恢复。一般来说，观察到黄体期的输卵管上皮细胞在补充硫醇储备方面比卵泡期的输卵管上皮细胞更有能力。本研究结果表明，生殖阶段和区域会影响输卵管上皮细胞内的硫醇状态，因此可能会影响该组织对导致氧化应激的化学物质或环境条件的反应方式。
