Approach to the abdominal retroperitoneum in patients with gynecologic malignancies

The treatment of patients with gynecologic malignancies is still widely dependent on clinical staging. The introduction of the concept of surgical staging has significantly altered treatment plans. Better staging allows better treatment and more accurate comparison of survival and cure rates. We describe a surgical technique to expose the abdominal retroperitoneum. Sixteen patients have been explored by this technique. There has been no apparent difficulty in this procedure, even when performed on obese patients.     

Chemosensitization: do thiols matter?

It is well known that endogenous sulfhydryls are radioprotective in mammalian cells. Their comparable role in chemotherapeutic drug toxicity has been known for almost as long but less well defined. Thiol depletion as a mechanism responsible for enhanced cytotoxicity of melphalan was assayed by pretreatment of cells in vitro with misonidazole and buthionine sulfoximine (BSO). Hypoxic cell sensitizers, such as MISO, deplete endogenous thiols by metabolic activation under hypoxic conditions to thiol reactive intermediates, whereas BSO specifically inhibits a key enzyme in the synthesis of glutathione. For a given level of thiol reduction, sensitization to melphalan was far greater by preincubation with MISO than it was for BSO. This indicated that thiol reduction itself was not the sole factor involved in chemosensitization by MISO. As evidence that the method of thiol depletion predisposes to the expression of biological damage, it was shown that cells preincubated with MISO were appreciably more vulnerable to oxidative stress than those exposed to BSO. BSO was shown to totally inhibit the repair of damage from a preincubation treatment with MISO, demonstrating that recovery is dependent upon thiol regeneration. Thiol depletion "per se" is a good qualitative but not necessarily a quantitative indicator of chemosensitization--the biological and biochemical function of the thiol depleting agents used influences further drug interactions. The results of the study with these two agents suggest that thiols may play a potentially more critical role in the repair rather than the initiation of drug-induced damage.     

The accumulation and efflux of lead partly depend on ATP-dependent efflux pump–multidrug resistance protein 1 and glutathione in testis Sertoli cells

Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump–multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10–40 μM for 12 h or 24 h, the differences were about 2–8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9 h time points (P < 0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.     

Acetaminophen alters microsomal ryanodine Ca2+ channel in HepG2 cells overexpressing CYP2E1

Acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding of drug metabolites to liver proteins. Acetaminophen metabolites have been shown to affect rat liver microsomal Ca2+ stores, but the mechanism is not well understood. The aim of the current work was to find out if the metabolism of acetaminophen by CYP2E1 affects ryanodine-sensitive Ca2+ stores in the endoplasmic reticulum of transduced HepG2 cells. Five millimoles acetaminophen decreased proliferation of CYP2E1-overexpressing HepG2 cells, increased cytosolic Ca2+ levels and produced significant cytotoxicity, while only little, mostly anti-proliferative effects were found in HepG2 cells lacking CYP2E1. CYP2E1 inhibitor-4-methylpyrazole decreased drug cytotoxicity in transduced cells and normalized elevated Ca2+ levels. Acetaminophen cytotoxicity was significantly higher in CYP2E1 expressing cells with depleted glutathione. In the cells engineered to overexpress CYP2E1, an increased [3H]ryanodine affinity (by 45%) and increased ligand maximal binding to ryanodine receptors (by 64%) was observed, most probably due to increased association rate of [3H]ryanodine. Ca2+ loading was decreased by about 53% in microsomal fractions isolated from transduced cells treated with acetaminophen and by 92% in glutathione depleted transfected cells treated with the drug. Ca2+/Mg2+-ATPase activity was unchanged in all microsomal fractions. Such effects were not observed in cells lacking CYP2E1. Our results confirm significant role of CYP2E1 in metabolic activation of acetaminophen and indicate that ryanodine receptors located in the liver endoplasmic reticulum are sensitive targets for acetaminophen metabolites.     

Evaluation of glutathione deficiency in rat livers by microarray analysis

Hepatic glutathione content was measured and gene expression data were obtained using an Affymetrix RG U34 array after treatment with tap water containing 20mM l-buthionine (S, R)-sulfoximine (BSO) to male F344 rats for four consecutive days. Both Spearman's and Pearson's correlation coefficients were calculated between the glutathione content and the mRNA content level obtained from the microarray analysis individually. Sixty-nine gene probes, which were statistically significant (Spearman's correlation, P < 0.05) and showed a Pearson's correlation coefficients (Pearson's r) less than -0.8 between mRNA content and hepatic glutathione content, were identified as glutathione deficiency-correlated probes. By comparing the hepatic gene expression profiles between BSO- and butylated hydroxyanisole (BHA)-treated rats, 14 probes of genes that showed an increase in the corresponding gene mRNA levels only after the BSO treatment were thought to be good indicators of glutathione deficiency. A principal component analysis successfully illustrated the time-course of hepatic gene expression after the treatment with acetaminophen, phenobarbital and clofibrate, and the expression profiles were thought to reflect the changes in hepatic glutathione levels. The identified gene probes in the present study would be useful as markers for assessing hepatocellular glutathione deficiency, or oxidative stress level, based on microarray data.     

谷胱甘肽对顺铂所致肾小管上皮细胞毒性的保护作用

目的 探讨谷胱甘肽（GSH）对顺铂所致不同性别大鼠肾小管上皮细胞毒性的影响。方法 从不同性别的大鼠分离的肾小管上皮细胞接种于96孔培养液，培养24h后加入一系列浓度的顺铂，或在加入顺铂前的16和4h，分别加入GSH合成抑制剂BSO和GSH合成前体物半胱氨酸，再培养24h后用MTT方法检测细胞存活率。结果：顺铂对雌雄大鼠肾小管上皮细胞具有形状相似的剂量－反应关系曲线，半数抑制浓度（IC50）分别为0.178mmol/L和0.182mmol/L；BSO能使2组IC50均2降低为0.001mmol/L，可使剂量－反应曲线左移，而半胱氨酸则可使2组IC50均升高，均大于5mmol/L，使剂量－反应曲线下沉。结论 顺铂对雌雄大鼠肾小管上皮细胞同样具有明显的毒性；BSO和半胱氨酸可分别增强和降低顺铂的毒性，间接证明细胞内GSH对顺铂所致大鼠肾小管上皮细胞毒性有保护作用，且与性别无前。     

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.     

丁胱亚磺酰亚胺对脑胶质细胞瘤GSH作用的研究

目的研究巯基修饰剂了胱亚磺酰亚胺（BSO）对脑胶质细胞瘤GSH含量的修饰作用。方法利用Tietze还原酶法观察BSO对体外培养的脑胶质细胞瘤细胞株及裸小鼠移植瘤模型脑胶质细胞瘤细胞GSH的作用。结果经BSO作用后脑胶质细胞瘤细胞株GSH含量最低为对照4．50％；实体瘤最低为6．74％。结论无论是离体还是整体用药，BSO均能降低脑胶质细胞瘤细胞的GSH含量。