Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

JM216-, JM118-, and cisplatin-induced cytotoxicity in relation to platinum-DNA adduct formation,glutathione levels and p53 status in human tumour cell lines with different sensitivities to cisplatin

The aim of this study is to establish anti-tumour potency of the new oral platinum drug JM216 and its metabolite JM118 in relation to the platinum (Pt)-DNA adduct formation, glutathione (GSH)-levels, and p53 status in human cancer cell lines with different sensitivities to cisplatin (CDDP). These parameters were studied in the CDDP sensitive human germ cell cancer cell line Tera and the small-cell lung cancer cell line GLC4 and their sublines with in vitro acquired CDDP resistance, Tera-CP and GLC4-CDDP, in a human ovarian cancer cell line transfected with mutant p53 (A2780/mt273) and with an empty vector as control (A2780/cmv), and in the intrinsic CDDP resistant human non-small-cell lung cancer cell line SW1573/S1 and colon carcinoma cell line Caco-2. Cytotoxicity was tested with the microculture tetrazolium (MTT)-assay. Pt-DNA adduct levels were assessed immunocytochemically. Quantitative analysis was performed by double fluorescence video microscopy. Results were correlated with GSH levels and p53 status of the cell lines. This study showed that both JM216 and JM118 can partially circumvent intrinsic and acquired resistance to CDDP. Drug-induced cytotoxicity only correlated negatively with GSH levels for JM216 and CDDP in the tested unselected cell lines. At equimolar basis, JM216 induced lower levels of Pt-DNA adducts in the various cell lines than JM118 and CDDP, whereas the JM118-induced amount and pattern of Pt-DNA adducts was comparable to CDDP. No difference in initial Pt-DNA adducts levels was observed between cell lines sensitive, acquired or intrinsic resistant to CDDP suggesting a Pt-resistance mechanism based on tolerance or increased repair, rather than decreased initial Pt-DNA adduct formation.     

Induction of vascular endothelial growth factor by 4-hydroxynonenal and its prevention by glutathione precursors in retinal pigment epithelial cells

Although 4-hydroxynonenal, a highly reactive lipid peroxidation product, is implicated in several age-related disorders such as Alzheimer's and Parkinson's diseases, its role in age-related macular degeneration is not known. The purpose of this study was to determine whether 4-hydroxynonenal increases vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial cells (ARPE-19), a source of VEGF in choroidal neovascularization observed in age-related macular degeneration. In addition, it was the purpose of this study to assess whether glutathione (GSH) and GSH precursors can inhibit the effects of 4-hydroxynonenal. At 1 micro M, 4-hydroxynonenal did not alter cell viability, but elevated VEGF secretion and mRNA expression by 35% (p<0.05) and 1.9-fold (p<0.05), respectively. However, at concentrations 5 microM and above, 4-hydroxynonenal reduced VEGF secretion as well as cell viability. At 1 and 10 microM, 4-hydroxynonenal did not induce apoptosis in ARPE-19 cells. 4-Hydroxynonenal (1 microM) reduced intracellular GSH by 25% (p<0.05) and increased oxidative stress by 50% (p<0.05). GSH precursor pretreatment for 1 h, which increased intracellular GSH levels by 50% (p<0.05), as well as GSH co-treatment, inhibited the VEGF-inductive and cytotoxic effects of 4-hydroxynonenal. Thus, 4-hydroxynonenal (1 microM) induces VEGF expression and secretion in ARPE-19 cells. This effect is likely due to GSH depletion and an associated increase in intracellular oxidative stress, resulting in increased VEGF mRNA levels. 4-Hydroxynonenal-mediated VEGF secretion as well as cytotoxicity can be reversed with GSH precursor pretreatment or GSH co-treatment.     

BSO逆转人肺腺癌细胞株多药耐药性的实验研究

目的研究谷胱甘肽(GSH)合成酶抑制剂——BSO对人肺腺癌多药耐药细胞株A549DDP细胞内GSH含量影响;探讨BSO逆转多药耐药(MDR)作用机制及逆转效果。方法GSH还原酶循环法测定BSO对细胞内GSH含量的影响。MTT比色法测定经BSO预处理后顺氯氨铂(DDP)、阿霉素(ADM)对细胞50%抑制浓度(IC50)的影响。流式细胞仪检测BSO对MDR细胞内柔红霉素(DNR)荧光强度的影响。结果耐药细胞A549DDP细胞内GSH含量较人肺腺癌A549细胞内GSH含量明显增高。BSO在一定浓度范围内(50～200μmol·L-1)对A549细胞和耐药细胞A549DDP无明显细胞毒性作用(抑制率均小于10%)。BSO呈剂量依赖性非线性抑制细胞内GSH的合成,其对MDR细胞内GSH合成影响较为显著,而对A549细胞内GSH合成影响较小。BSO在一定浓度范围内能降低DDP、ADM对A549DDP细胞的IC50,而对A549细胞的IC50无明显影响。A549DDP细胞内DNR荧光强度较A549细胞显著降低,能不同程度提高A549DDP细胞内柔红霉素荧光强度,均较未处理组显著提高;与未经BSO处理的A549细胞比较,细胞内荧光强度轻度增高,但统计学上无显著性差异。结论BSO能有效逆转A549DDP细胞的MDR,其机制与降低MDR细胞内GSH含量有关。     

加味小柴胡汤对顺铂诱导大鼠肝BRL细胞过氧化损伤的保护作用

目的:探讨加味小柴胡汤对顺铂诱导大鼠肝BRL细胞氧化损伤的保护作用。方法:采用体外细胞培养方法,观察加味小柴胡汤对顺铂诱导的BRL细胞生长、SOD、GSH-px、NOS活性、MDA、GSH、T-AOC含量的影响。结果:顺铂各剂量处理细胞OD值皆显著下降,且随剂量而加重;加味小柴胡汤大小剂量细胞OD值皆明显升高;顺铂组SOD、GSH-px、T-AOC活性、GSH含量均明显降低;MDA含量及NOS活性明显升高;两实验组SOD、GSH-px、T-AOC及GSH均较顺铂组升高,MDA及NOS降低,而以1组效果更显著。结论:顺铂可明显抑制肝细胞生长,诱致细胞过氧化损伤,加味小柴胡汤可提高细胞生长率,其作用与其明显的抗氧化能力有关,且有一定的量效关系。     

束缚应激反应对小鼠晶状体抗氧化能力指数的影响

目的探讨束缚应激反应对小鼠晶状体抗氧化能力指数的影响。方法建立小鼠束缚应激模型,采用荧光酶标仪测定晶状体的抗氧化能力指数,用高效液相-电化学检测器(HPLC-ECD)测定小鼠晶状体中谷胱甘肽含量。结果随着应激时间的延长,小鼠晶状体的抗氧化能力指数和谷胱甘肽含量与正常组相比均有不同程度下降。结论束缚应激状态下,晶状体的抗氧化能力指数和谷胱甘肽水平均相应降低,其机制可能与晶状体内的内源性抗氧化能力降低及氧自由基产生过多和蓄积有关。     

复方参果液对大鼠亚硒酸钠性白内障晶状体中巯基和谷胱甘肽含量的影响

目的 观察复方参果液对亚硒酸钠性白内障形成的抑制作用及其机理。方法 选用10日龄Wistar大鼠40只随机分为正常对照组、亚硒酸钠性白内障大剂量复方参果液治疗组，亚硒酸性钠白内障小剂量复方参果液治疗组和亚硒酸钠性白内障未治疗对照组，每组均为10只。在4组中分别用不同剂量的复方参果液和等量清水灌胃给药30天。灌胃结束后测定晶状体上清液中非蛋白巯基（nonprotein sulfhydryl,NP-SH)，沉淀部分蛋白巯基（protein sulfhydryl,P-SH)和晶状体匀浆液中谷胱甘肽（glutathione,GSH)等含量。结果 白内障未治疗组中NP－SH，P－SH和GSH含量均明显降低，大剂量和小剂量复方参果液组中NP－SH，P－SH，GSH含量均明显增加（P＜0．05，P＜0．001），接近正常对照组水平。结论 复方参果液能提高大鼠亚硒酸钠性白内障晶状体中NP－SH，P－SH和GSH的含量，这可能与复方参果液抑制亚硒酸钠性白内障形成有关。     

Reduced glutathione, dithiothreitol and cytochrome P-450 inhibitors do not influence hypoxic chemosensory responses in the rat carotid body

Glomus cells and carotid sinus afferents are anatomically connected, and the chemical events in the glomus cells are expected to be conveyed reflexly as afferent signals. Accordingly, K(+) channel inhibition of the glomus cell membrane is expected to be followed by excitation of the afferents. In order to test the redox inhibition of K(+) channels of glomus cells by reduced glutathione (GSH), dithiothreitol (DTT) and by cytochrome P-450 inhibitors (clotrimazole and miconazole), we measured the carotid sinus nerve (CSN) discharge using an in vitro perfused adult rat carotid body (CB) in the presence and absence of these chemicals which are expected to excite the afferents. Our findings were that these agents did not stimulate the CSN activities during normoxia and kept the hypoxic responses intact. These results led us to conclude that the redox modulation of glomus cells was not conveyed to the afferents, and this functional disconnection did not support the redox hypothesis of O(2) chemoreception in the whole carotid body.