Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

谷胱甘肽治疗帕金森病30例临床观察

目的 探讨还原型谷胱甘肽(古拉定，GLT)对帕金森病人治疗效果，观察GLT治疗后病人血超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH—Px)、丙二醛(MDA)水平的变化。方法 选择30例临床未治疗或服用美多巴因副反应被迫减药病人，应用GLT进行治疗，治疗前后行血SOD、GSH—PX、MDA测定，并设立对照组、正常人组比较；治疗、Px对照组进行了治疗前后H—Y分级及Webster评分。结果 治疗组和正常人组比较，血SOD、GSH—Px显减低(P＜o．01)，MDA显增高(P＜o．01)；GLT治疗后，SOD、GSH—Px值较治疗前明显升高(P＜o．05、P＜o．01)，MDA值下降(P＜o．05)，而对照组改变不明显。治疗前后评分，治疗后临床症状有显改善(P＜o．01)，与对照组比较，两组有显差异(P＜o．01)。结论 GLT对PD病人有较显治疗作用。     

古拉定在肝硬化亚临床肝性脑病治疗中的临床意义

目的　探讨古拉定 (GLT)在肝硬化亚临床性肝性脑病 (SHE)治疗中的临床意义。方法　以古拉定联合乳果糖治疗 4 7例SHE ,在 1mon治疗结束后随访 6mon ,并以单用乳果糖治疗的 5 0例SHE作对照 ,观察智力测验好转情况及肝功能改变情况。结果　数字连接试验 (NCT)及数字符合试验 (DST)在治疗结束及随访 6mon时的好转率分别为 69 0 %、5 2 6%和 4 7 6%、5 0 % ,而对照则分别为 4 0 8%、4 5 0 %和 17 4 % ,除DST治疗 1mon时两组差异无显著性外 ,两组其余各项比较差异均有显著性 (P <0 0 5或P <0 0 1) ;治疗组肝功能Child Pugh计分在治疗前为 8 92± 1 16,治疗 1mon时降为 7 73± 1 3 1(P <0 0 1) ,随访 6mon时为 8 0 2± 1 17(P <0 0 5 ) ,而对照组在治疗前为 8 65± 2 4 1,1mon时为 7 86± 1 4 6(P <0 0 5 ) ,至 6mon时为 8 4 4± 1 87,与治疗前差异无显著性。结论　古拉定能提高乳果糖对肝硬化SHE治疗的近期疗效及远期疗效 ,机制可能是通过改善肝功能状态而起作用     

谷胱甘肽治疗急性酒精中毒

目的 :探讨谷胱甘肽注射液治疗急性酒精中毒的疗效及安全性。方法 :5 0例急性酒精中毒昏睡期病人随机分为 2组 :谷胱甘肽组 2 8例 [男性 2 3例 ,女性 5例 ,年龄 (33±s 11)a],给予谷胱甘肽注射液 1.8g加入 5 %葡萄糖注射液 2 5 0mL中静脉滴注。促进乙醇氧化组 2 2例 [男性 18例 ,女性 4例 ,年龄 (35± 9)a],给予 5 %葡萄糖注射液 2 5 0mL +2 0U正规胰岛素注射液静脉滴注 ,同时给予维生素C、维生素B6 静脉注射。 2组病人均给予温水洗胃 ,并给予静脉注射呋塞米、西咪替丁及补液治疗。结果 :在清醒时间及给药后 3h清醒例数方面 ,治疗组明显优于对照组 ,治疗组病人苏醒时间为 (76± 4 4 )min ;对照组为 (133± 5 9)min(P <0 .0 1)。给药后 3h治疗组清醒 2 5例 (89% ) ,对照组清醒 7例(32 % ) (P <0 .0 1)。结论 :谷胱甘肽注射液治疗急性酒精中毒昏睡期病人的苏醒时间明显短于传统的促进乙醇氧化疗法 ,对急性酒精中毒具有可靠的治疗效果     

Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

Objective To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13μg/mL, 10.95μg/mL and 16.52μg/mE respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34μg/mL, 7.48μg/mL and 13.70μg/mE respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.     

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR.

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km2 and located in the South-Western of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 microg microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes.     

The use of Lepidium sativum in a plant bioassay system for the detection of microcystin-LR.

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Detection of microcystins in water generally relies on specialised equipment and a delay of several days for transport and analysis. Little work has, however, been done on establishing a simple, cost-effective and sensitive plant bioassay for the detection of microcystin-LR (MCLR) in water at the WHO Tolerable Daily Intake guideline level of 1 microg/l. We investigated the effect of a MCLR extract at 1 and 10 microg/l on the growth of Lepidium sativum over 6 days. Exposure to 10 microg/l MCLR resulted in a significant decrease in root and leaf lengths and fresh weights of seedlings when compared to the controls. These results were consistent with seedlings exposed to pure MCLR at 10 microg/l. Seedlings exposed to 1 microg/l MCLR showed a significant decrease in root development from day 2 to day 6. Glutathione S-transferase and glutathione peroxidase activities were also significantly raised in plants from days 5 and 4, respectively, at both toxin levels investigated.     

亚硝基谷胱甘肽对大鼠肝微粒体谷胱甘肽转移酶的激活及机制

目的　探索一氧化氮供体亚硝基谷胱甘肽(GSNO)能否在体外通过S 亚硝酰化机制激活大鼠肝微粒体谷胱甘肽转移酶 (mGST)。方法　微粒体粗提物与GSNO体外共孵育 ,测定mGST催化动力学改变 ,结合N 乙基马来酰亚胺 (NEM )再激活实验和二巯基苏醇 (DTT)逆转实验 ,以及酶蛋白游离巯基和酶S 亚硝酰化蛋白的改变 ,研究酶的激活机制。结果　GSNO在 0 .12 5～ 2mmol·L- 1浓度范围内呈浓度和时间 (3～ 15min)依赖性地激活mGST ,NEM对酶的再激活效应消失 ,DTT可以逆转上述激活作用 ,同时酶蛋白游离巯基浓度依赖性减少 ,而S 亚硝酰化蛋白浓度依赖性增多。结论　GSNO体外可激活大鼠肝mGST ,激活机制可能与mGST第 4 9位半胱氨酸 (Cys4 9)的巯基被亚硝酰化形成S 亚硝基硫醇结构有关。     

GENETIC DELETION OF DETOXIFIC ENZYME GSTM1 AND GSTTI AS A HOST SUSCEPTIBLE FACTOR FOR NASOPHARYNGEAL CARCINOMA

Objective: To study the gene polymorphisms of GSTT1 and GSTM1 in nasopharyngeal carcinoma (NPC) patients and controls in an incidental area to evaluate the relationship between specific genotype and genotype combinations of these polymorphisms with the risk of NPC. Methods: Cases and controls all came from the Southwestern Guangxi. DNAs were extracted from their WBC. PCR technique was used to calculate the deletion rate of the two detoxific enzyme genes. Results: In this high risk area of NPC, the residents had high level deletion rates of 47.4% (64/135) Ml and T1 40.7% (55/135). The deletion rates were even higher in NPC patients, 61.5% (56/91) for Ml and 59.3% (54/91) for T1 respectively. There were statistical significances compared with control,P<0.05 andP<0.01 for Ml and T1 respectively. The difference was more significant in terms of combined Ml and T1 deletion between patients and controlsx ²=12.533,P=0.002. Conclusion: The combined deletion of detoxific enzyme genes GSTM1 and GSTT1 may be an important genetic susceptible factor for NPC in Guangxi. Biography: DENG Zhuo-lin (1929-), male, professor of pathology, Guangxi Medical University, majors in tumor pathology. E-mail :zhuolin@hotmail.com     

三种耐药蛋白在胃癌中的表达及意义

目的：探讨多药耐药蛋白(MRP)、谷胱甘肽转移酶Pi(GST—π)和肺耐药蛋白(LRP)在胃癌中的表达及意义。方法：应用免疫组化S—P法，检测MRP、GST—π和LRP在90例胃癌及30例正常胃黏膜中的表达。结果：MRP、GST—π及LRP在胃癌中的阳性表达率分别为92．2％、99．0％和93．3％，均显著高于其在正常胃黏膜中的表达(P＜0．05)，且MRP、GST—π和LRP在高、中分化腺癌中的表达显著高于在低分化腺癌和黏液癌中的表达(P＜0．05)。但它们的表达与胃癌浸润深度、淋巴结转移无关。结论：MRP、GST—π和LRP在胃癌中高表达，在胃癌的原发性耐药中起重要作用。这可能是胃癌化疗效果差、死亡率高的重要原因之一。