Mechanism for the hepatotoxicity of the antiandrogen, nilutamide. Evidence suggesting that redox cycling of this nitroaromatic drug leads to oxidative stress in isolated hepatocytes.

The nitroaromatic drug nilutamide has been shown previously to undergo redox cycling in aerobic rat liver microsomes, being reduced by NADPH-cytochrome P-450 reductase to a nitro anion-free radical which reacts with oxygen, to regenerate the parent drug, and form a superoxide anion dismuted to hydrogen peroxide. In the present study, the effects of nilutamide on isolated rat hepatocytes have been determined. After 6 and 8 hr of incubation with 0.5 mM nilutamide, lactate dehydrogenase was released in the incubation medium, and cell viability was decreased markedly. Consistent with a redox cycle producing reactive oxygen species, nilutamide increased nonmitochondrial (cyanide-resistant) oxygen consumption; the toxicity of nilutamide occurred sooner and was more extensive in the presence of sodium azide (an inhibitor of catalase). Consistent with an oxidative stress, the toxicity of nilutamide was associated with depletion of reduced glutathione, increased levels of glutathione disulfide, increased Ca(++)-dependent phosphorylase a activity, oxidation and accumulation of cytoskeleton-associated proteins and formation of blebs; toxicity was prevented by glutathione precursors, thiol reductants and/or antioxidants, such as L-cystine, L-cysteine, N-acetyl-L-cysteine, dithiothreitol, N,N'-diphenyl-p-phenylene-diamine and alpha-tocopherol. Feeding the animals with a diet supplemented with 2% L-cystine increased the initial glutathione stores of hepatocytes and prevented nilutamide toxicity. It is concluded that nilutamide is toxic to isolated rat hepatocytes, as a probable consequence of an oxidative stress due to the redox cycling of this nitroaromatic compound.     

Mechanism of oxmetidine (SK&F 92994) cytotoxicity in isolated rat hepatocytes

Oxmetidine is an H2-receptor antagonist that has efficacy in the treatment of peptic ulcers. Isolated rat hepatocytes exposed to oxmetidine (0.5 mM) rapidly lost viability as estimated by increased leakage of lactate dehydrogenase, increased formation of plasma membrane surface blebs and decreased intracellular potassium concentration [K+]. Oxmetidine caused a reduction in hepatocyte reduced glutathione concentration that paralleled cell death; malondialdehyde formation was not observed. Hepatocyte respiration (O2 consumption) and intracellular ATP concentration were decreased markedly by oxmetidine in a concentration-related fashion. Oxmetidine (50 microM) blocked pyruvate/malate-supported state 3 (ADP-stimulated) respiration, caused a decrease in the ADP:0 ratio and a loss of respiratory control in isolated rat liver mitochondria. In contrast, oxmetidine did not block succinate-supported ADP-stimulated O2 consumption in isolated rat liver mitochondria. These data demonstrate that: 1) oxmetidine was cytotoxic to isolated rat hepatocytes in suspension and 2) the mechanism of oxmetidine-induced hepatocyte injury may be related to sustained inhibition of mitochondrial oxidative phosphorylation leading to decreased cellular ATP content and cell death. Although the exact site of action of oxmetidine within the mitochondrion has not been completely elucidated, it appears to reside in the inner mitochondrial membrane electron transport chain before ubiquinone oxidoreductase.     

The critical role of mitochondrial energetic impairment in the toxicity of nimesulide to hepatocytes

We described the effects of nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) and its reduced metabolite in isolated rat hepatocytes. Nimesulide stimulated the succinate-supported state 4 respiration of mitochondria, indicating an uncoupling effect of the drug. Incubation of hepatocytes with nimesulide (0.1-1 mM) elicited a concentration- and time-dependent decrease in cell viability as assessed by lactate dehydrogenase leakage, a decrease of mitochondrial membrane potential as assessed by rhodamine 123 retention, and cell ATP depression. Nimesulide also decreased the levels of NAD(P)H and glutathione in hepatocytes, but the extent of the effects was less pronounced in relation to the energetic parameters; in addition, these effects did not imply the peroxidation of membrane lipids. The decrease in the viability of hepatocytes was prevented by fructose and, to a larger extent, by fructose plus oligomycin; it was stimulated by proadifen, a cytochrome P450 inhibitor. In contrast, the reduced metabolite of nimesulide did not present any of the effects observed for the parent drug. These results indicate that: 1) nimesulide causes injury to the isolated rat liver cells, 2) this effect is mainly mediated by impairment of ATP production by mitochondria due to uncoupling, and 3) on account of the activity of its nitro group, the parent drug by itself is the main factor responsible for its toxicity to the hepatocytes.     

Lysophosphatidic acid induces rapid and sustained decreases in epidermal growth factor receptor binding via different signaling pathways in BEAS-2B airway epithelial cells

L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.     

Mechanism of toxicity of an experimental bidentate phosphine gold complexed antineoplastic agent in isolated rat hepatocytes

SK&F 104524 (bis-[1,2 bis(diphenylphosphino)-ethane]gold(l) lactate) [( Au(dppe)2]+) is an experimental antineoplastic agent that is hepatotoxic in vivo in the dog as well as highly cytotoxic to isolated canine hepatocytes in vitro. Preliminary studies in isolated dog hepatocytes have indicated that [Au(dppe)2]+ causes an increase in hepatocyte respiration and a decrease in cellular ATP. The purpose of the present investigation was to characterize [Au(dppe)2]+-induced cytotoxicity and biochemical lesions in the intact cell and to correlate these changes with mitochondrial function. The uptake of [14C][Au(dppe)2]+ by rat hepatocytes was rapid, reaching a maximum by 30 min. [Au(dppe)2]+ was distributed throughout the hepatocyte and associated rapidly with mitochondria, nuclei, cytosol and cellular membranes. [Au(dppe)2]+ caused cell lethality in a concentration-dependent fashion; although 5 microM did not cause any changes in lactic dehydrogenase leakage, 20 microM produced 100% cell death by 120 min. [Au(dppe)2]+ also caused concentration-dependent bleb formation of the hepatocyte plasma membrane, increased oxygen consumption and loss of ATP within 30 min. ATP loss was associated with transient increases in AMP and ADP and a profound drop in the ATP/ADP ratio and energy charge. Total nucleotides (adenine and xanthine nucleotides) remained constant. The pattern of glutathione depletion coincided with that of lactic dehydrogenase leakage. Electron microscopy of hepatocytes exposed to [Au(dppe)2]+ for 30 min revealed depletion of glycogen granules and marked swelling of mitochondria. In isolated rat liver mitochondria, [Au(dppe)2]+ caused a stimulation of state 4 respiration and loss of the respiratory control ratio. [Au(dppe)2]+ also relieved the oligomycin-induced inhibition of state 3 (ADP-stimulated) respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-associated enhancement of diquat-induced lipid peroxidation and cytotoxicity in isolated rat hepatocytes.

The effect of age on the toxicity of diquat, a redox cycling compound, was investigated in hepatocytes isolated from mature (6 months) and old (24-29 months) male Fischer 344 rats. Hepatocytes of old rats were more sensitive than those of mature rats to diquat-induced cytotoxicity (lactate dehydrogenase release into the medium). Cell death was preceded by glutathione disappearance, and rates of glutathione depletion were similar in mature and old hepatocytes. In contrast, diquat-induced formation of thiobarbituric acid-reactive substances was much greater in the hepatocytes from old rats, suggesting that increased lipid peroxidation caused the enhanced cytotoxicity. Further experiments revealed that: 1) hepatocytes of mature and old rats were equally sensitive to iron-induced lipid peroxidation; 2) diquat-stimulated production of superoxide anion radical in liver microsomes did not increase with age, but decreased 43%; 3) superoxide dismutase activity was similar in hepatocytes of mature and old rats; 4) inhibition of catalase activity (which diminishes with age in male rats) did not increase diquat toxicity; and 5) malondialdehyde disappearance in intact hepatocytes decreased (33%) with age, but the toxicological significance of the decline in metabolism was uncertain. Thus, the results demonstrated that diquat-induced lipid peroxidation and cytotoxicity increase with age in male rat hepatocytes, but the enhanced sensitivity to diquat poisoning remains unexplained.     

Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation

Whereas alpidem is hepatotoxic, zolpidem is not. Despite closely related chemical structures, alpidem, but not zolpidem, is a peripheral benzodiazepine receptor (PBR) ligand, and is also more lipophilic than zolpidem. We compared their effects in isolated rat liver mitochondria and rat hepatocytes. Zolpidem did not affect calcium-induced mitochondrial permeability transition (MPT) in mitochondria, caused little glutathione depletion in hepatocytes, and was not toxic, even at 500 microM. At 250 to 500 microM, alpidem prevented calcium-induced MPT in isolated mitochondria, but caused severe glutathione depletion in hepatocytes that was increased by 3-methylcholanthrene, a cytochrome P4501A inducer, and decreased by cystine, a glutathione precursor. Although cell calcium increased, mitochondrial cytochrome c did not translocate to the cytosol and cells died of necrosis. Cell death was prevented by cystine, but not cyclosporin A, an MPT inhibitor. At low concentrations (25-50 microM), in contrast, alpidem accelerated calcium-induced MPT in mitochondria. It did not deplete glutathione in hepatocytes, but nevertheless caused some cell death that was prevented by cyclosporin A, but not by cystine. Alpidem (10 microM) also increased the toxicity of tumor necrosis factor-alpha (1 ng/ml) in hepatocytes. In conclusion, low concentrations of alpidem increase both calcium-induced MPT in mitochondria, and TNF-alpha toxicity in cells, like other PBR ligands. Like other lipophilic protonatable amines, however, alpidem inhibits calcium-induced MPT at high concentrations. At these high concentrations, toxicity involves cytochrome P4501A-mediated metabolic activation, glutathione depletion, and increased cell calcium, without MPT involvement. In contrast, zolpidem has no mitochondrial effects, causes little glutathione depletion, and is not toxic.     

Mechanism of hepatotoxicity in periportal regions of the liver lobule due to allyl alcohol: studies on thiols and energy status

The possible involvement of thiols and adenine nucleotides in the selective toxicity to periportal regions by allyl alcohol was evaluated in isolated perfused rat livers. Infusion of allyl alcohol (350 microM) for 20 min depleted hepatic glutathione content by 95% in both regions of the liver lobule yet damage was undetectable as indexed by release of lactate dehydrogenase or uptake of trypan blue. Perfusion for an additional 40 min in the absence of allyl alcohol resulted in lactate dehydrogenase release (2400 U/l) and uptake of trypan blue by 75% of hepatocytes in periportal regions of the liver lobule; however, dye was not taken up by cells in pericentral areas. Because thiol content was depleted in the undamaged pericentral area, it was concluded that thiol depletion alone cannot explain local toxicity to periportal regions by allyl alcohol. Perfusion with dithioerythritol (1.5 mM) prevented damage due to allyl alcohol totally. In contrast, addition of dithioerythritol 20 min after allyl alcohol did not prevent allyl alcohol-induced damage to periportal regions indicating that irreversible changes occur during the first 20 min which ultimately lead to damage. Fasting or pretreatment of rats with diethylmaleate (0.7 g/kg; 1 hr) to deplete glutathione decreased the T1/2 required for release of lactate dehydrogenase from 45 to 35 and 22 min, respectively. When methionine was infused into livers from diethylmaleate-treated rats, the T1/2 for release of lactate dehydrogenase by allyl alcohol was increased to 45 min. Infusion of allyl alcohol for 60 min also produced a significant decrease in ATP content and in the ATP/ADP ratio in periportal but not pericentral regions of the liver lobule.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of isolated enterocytes to study Phase I intestinal drug metabolism: validation with rat and pig intestine

An important step in the development of new drugs is to evaluate the extent of their metabolism during absorption in the small intestine. Reliable in vitro systems to do this can expediate the development process, but the current systems are often unsuitable because they lack the appropriate metabolic enzymes (e.g. Caco-2 cell monolayers) or are not representative of the physiological conditions present in the intact intestinal cells (e.g. isolated microsomes). The aim of this study was to validate the use of isolated intestinal epithelial cells (enterocytes), equivalent to hepatocytes, to evaluate Phase I drug metabolism. A method was developed to prepare enterocytes from rat and pig (as metabolically closer to man) that maintained good viability and activity for up to 90 min as judged by trypan blue exclusion and the release of the cytosolic enzyme lactate dehydrogenase. The Phase I metabolism of the established marker drugs: midazolam, bupropion and dextromethorphan were measured by LC-MS and confirmed the activities of the 3A, 2B and 2D families of CYP isoforms, respectively. The kinetic parameters, K(m) and V(max), were compared between isolated cells and isolated intestinal microsomes from the rat. The use of isolated intestinal cells is a simple and practical method to study the Phase I metabolism of drugs during their absorption and the potential for drug-drug interactions. The method could eventually be modified and usefully applied to human studies.     

Protein kinase C-epsilon is a trigger of delayed cardioprotection against myocardial ischemia of kappa-opioid receptor stimulation in rat ventricular myocytes.

Kappa-opioid receptor (OR) stimulation with a selective agonist, U50,488H (U50), known to mediate the delayed cardioprotection of metabolic inhibition preconditioning (MIP) against cell injury/death in rat ventricular myocytes, has been shown to act via protein kinase C (PKC). We attempted to identify the PKC isoform(s) that is activated, thus triggering delayed cardioprotection of MIP and pretreatment with 10 microM U50 (U50 pretreatment, UP). Release of lactate dehydrogenase and exclusion of trypan blue by isolated rat ventricular myocytes were used as indices of cell injury and death, respectively. Both MIP and UP induced translocation of PKC-epsilon, but not other PKC isoforms, -alpha and -delta, from cytosolic to membrane fractions. This was accompanied by reductions in cell injury/death induced by lethal simulated ischemia. The effects of MIP and UP were attenuated and abolished by 1 microM nor-binaltorphimine, a selective kappa-OR antagonist, administered before and during preconditioning/pretreatment, respectively. The effects were mimicked by 10 nM phorbol-12-myristate-13-acetate, a PKC activator, but attenuated by 5 microM chelerythrine, a PKC inhibitor. More importantly, 0.1 microM epsilonV1-2, a selective PKC-epsilon inhibitor administered before and during MIP/UP, also attenuated the effects of both treatments on cell injury/death and translocation of PKC-epsilon. On the other hand, 5 microM rottlerin, a selective PKC-delta inhibitor, did not alter the effects of either treatment on injury/death. The results indicate that both MIP and UP activate PKC-epsilon, leading to delayed cardioprotection in rat ventricular myocytes.     

Effect of extracellular Ca++ omission on isolated hepatocytes. II. Loss of mitochondrial membrane potential and protection by inhibitors of uniport Ca++ transduction

Incubation of isolated rat hepatocytes in Ca++-free medium generates an oxidative stress which causes significant cell injury. Ruthenium red and La , which block Ca++ uptake through the mitochondrial uniport, totally prevented malondialdehyde formation, glutathione and protein thiol oxidation and vitamin E loss induced by Ca++ omission. Accordingly, these agents also prevented leakage of intracellular K+ and lactate dehydrogenase. Similar protective effects were provided by the Ca++ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. The absence of extracellular Ca++ resulted in a marked decline of the mitochondrial transmembrane potential which could be prevented by ruthenium red, ethylene glycol bis(beta-aminoethyl ether)-N,N'tetraacetic acid, the antioxidant vitamin E and the iron chelator, desferrioxamine. In contrast, oxidative stress induced by treatment with the redox active agent paraquat and 1,3-bis(2-chloroethyl)-1-nitrosourea had little effect on mitochondrial transmembrane potential and malondialdehyde formation and lactate dehydrogenase leakage were not affected by ruthenium red or La . These results indicate that the incubation of rat hepatocytes in the absence of extracellular Ca++ creates an unusual oxidative stress which markedly affects mitochondrial function. The ability of vitamin E and desferrioxamine to inhibit the loss of mitochondrial transmembrane potential indicates that oxidative damage is involved in producing mitochondrial dysfunction. Furthermore, the potent inhibitory effects of ruthenium red and La suggest that Ca++ movement through the uniport, perhaps indicative of mitochondrial Ca++ cycling, plays a major role in generating this oxidative stress and promoting cell injury.     

微波对骨细胞活力和生物力学的影响

目的研究微波辐射对大鼠股骨细胞活力和生物力学的影响,以探求杀灭所有骨、软骨细胞时骨组织生物力学的变化。方法32只大鼠处死后,将其双侧股骨完整取出,左侧股骨为实验组,右侧股骨为对照组。应用微波对实验组股骨分别加热3、5、10、15min后,用特殊的乳酸脱氢酶活性染色,判断骨、软骨细胞的活力;用Instron万能材料试验机测定股骨的生物力学并同步记录;用原子吸收分光光度仪测定股骨钙的含量。结果实验组与对照组的生物力学及钙含量差异无显著性（P〉0.05）,股骨经微波加热10min后,温度可达62℃,能够杀灭骨、软骨所有的活细胞。结论微波产生的高温,在较短的时间内能杀灭骨、软骨活细胞,而对骨的生物力学无显著损害;通过组织化学观察乳酸脱氢酶活性判断骨、软骨细胞活力的方法是特异和敏感的,体外微波加热可应用于骨肿瘤的保肢手术。     

乳鼠心肌细胞缺血预适应模型的建立及电镜下超微结构的改变

目的探讨培养乳鼠心肌细胞建立缺血预适应(IPC)模型的方法及对细胞超微结构的影响。方法出生1～2d的SD大鼠用改良法分离培养心肌细胞,检测模拟缺血各时间点乳酸脱氢酶(LDH)漏出率、细胞死亡率,据此建立预适应模型并与单纯缺血/再灌注组比较其LDH、细胞死亡率和超微结构的改变。结果乳鼠心肌细胞在模拟缺血4h以后LDH、细胞死亡率与对照组相比有明显差异(P <0 0 0 1) ;以模拟缺血90min ,再灌60min诱导的IPC模型与单纯缺血/再灌注(I/R)组相比明显降低LDH漏出率、细胞死亡率(P <0 0 0 5 ,P <0 0 0 1) ,I/R组心肌细胞超微结构破坏严重;IPC组心肌细胞超微结构基本完整清晰。结论乳鼠心肌细胞对缺血有较强耐受力,模拟缺血4h以上才有明显损伤。模拟缺血90min ,再灌60min能成功建立IPC模型,且维持超微结构完整性。     

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.     

C3A细胞与L-02永生化肝细胞低温保存条件下的生物学特性比较

背景：获得大量功能良好的肝细胞是生物人工肝的核心。探索出一种可靠的肝细胞低温保存方法进而构建一个肝细胞库是目前生物人工肝研究的热点。目的：比较用UW液在4℃条件下保存已经进入Ⅲ期临床试验的C3A细胞与国内构建的永生化肝细胞株L-02细胞的生物学特性。方法：贴壁培养C3A与L-02细胞,胰酶消化,制备成细胞悬液,UW液保存。4℃低温保存0,24,48及72h后,采用流式细胞术分别测定细胞存活率与凋亡率,测定谷草转氨酶与乳酸脱氢酶释放、尿素合成功能及白蛋白分泌功能。结果与结论：随低温保存时间延长,C3A与L-02细胞存活率呈下降的趋势,但C3A细胞的存活率明显高于L-02细胞（P〈0.01）;细胞凋亡率呈上升趋势,但48h后C3A细胞同L-02细胞无差异（P〉0.05）。谷草转氨酶及乳酸脱氢酶释放呈现上升的趋势,但C3A细胞明显低于L-02细胞（P〈0.01）。白蛋白分泌功能呈下降的趋势,但C3A细胞明显优于L-02细胞（P〈0.01）。尿素合成功能呈下降的趋势,但是L-02细胞明显优于C3A细胞（P〈0.01）。结果提示,UW液4℃保存C3A细胞与L-02细胞时间不易超过48h。以C3A细胞为材料的人工肝可能更适用于肝功能衰竭合并低白蛋白血症,以L-02细胞为材料的人工肝更适用于肝功能衰竭合并肝性脑病。     

C3A细胞与L-02永生化肝细胞低温保存条件下的生物学特性比较

背景:获得大量功能良好的肝细胞是生物人工肝的核心。探索出一种可靠的肝细胞低温保存方法进而构建一个肝细胞库是目前生物人工肝研究的热点。目的:比较用UW液在4℃条件下保存已经进入Ⅲ期临床试验的C3A细胞与国内构建的永生化肝细胞株L-02细胞的生物学特性。方法:贴壁培养C3A与L-02细胞,胰酶消化,制备成细胞悬液,UW液保存。4℃低温保存0,24,48及72h后,采用流式细胞术分别测定细胞存活率与凋亡率,测定谷草转氨酶与乳酸脱氢酶释放、尿素合成功能及白蛋白分泌功能。结果与结论:随低温保存时间延长,C3A与L-02细胞存活率呈下降的趋势,但C3A细胞的存活率明显高于L-02细胞(P<0.01);细胞凋亡率呈上升趋势,但48h后C3A细胞同L-02细胞无差异(P>0.05)。谷草转氨酶及乳酸脱氢酶释放呈现上升的趋势,但C3A细胞明显低于L-02细胞(P<0.01)。白蛋白分泌功能呈下降的趋势,但C3A细胞明显优于L-02细胞(P<0.01)。尿素合成功能呈下降的趋势,但是L-02细胞明显优于C3A细胞(P<0.01)。结果提示,UW液4℃保存C3A细胞与L-02细胞时间不易超过48h。以C3A细胞为材料的人工肝可能更适用于肝功能衰竭合并低白蛋白血症,以L-02细胞为材料的人工肝更适用于肝功能衰竭合并肝性脑病。     

抗氧化剂预防低血糖所致肌肉损伤的研究

目的 探讨低血糖状态下血浆和组织中谷胱甘肽含量的变化以及还原型谷胱甘肽（GSH）对血清酶活性的抑制作用。方法 静脉注射胰岛素诱发低血糖，并持续60min，然后输注葡萄糖解除低血糖。结果发现诱发低血糖后6h，血浆GTSH，氧化型谷胱甘肽（GSSG），谷胱甘肽总量（TGSH）及GSSG/TGSH比值均明显升高，肝脏GSH和TGSH明显减少，心肌和骨骼肌CSSG和GSSG/TGSH比值明显增高，同时伴有血清酶（ALT，AST，LDH，CK）活性升高，预先注射CSH后诱发低血糖组，血浆GSSG和GSSG/TGSH比值明显下降，并抑制了血清酶的活性。结论 低血糖所致的血清酶活性的升高与体内谷胱甘肽氧化还原状态的改变有关。补充抗氧化剂GSH可以预防低血糖所致的肌肉损伤。     

Hepatotoxicity of tacrine: occurrence of membrane fluidity alterations without involvement of lipid peroxidation

Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells using a spin-trapping technique coupled to electron paramagnetic resonance (EPR) spectroscopy. No increase of the EPR signal was observed over the period of 30 min to 24 h. In contrast, treatment of cells with the spin label 12-doxyl stearic acid followed by EPR spectroscopy showed that THA (0.05 and 0.25 mM) rapidly increased hepatocyte membrane fluidity. Extracellular application of GM1 ganglioside (60 microM) both reversed this increase in fluidity and partially reduced lactate dehydrogenase release on THA exposure. In conclusion, this work indicates that early alterations of membrane fluidity, not resulting from lipid peroxidation, are likely to play an important role in the development of THA toxicity.     

The role of the glyoxalase pathway in reducing mesothelial toxicity of glucose degradation products.

BACKGROUND: The glucose degradation products (GDP) presentin conventional peritoneal dialysis fluids (PDF) may exert adverse effects toward human peritoneal mesothelial cells (HPMC). Some GDP can be detoxified by the glyoxalase/ glutathione pathway. It has been shown that the addition of glyoxalase I (GLO-I) and reduced glutathione (GSH) to PDF effectively eliminates GDP. We have therefore examined the GLO-I/GSH system in HPMC and assessed the impact of GLO-I/ GSH-treated PDF on the viability and function of HPMC. METHODS: Heat-sterilized PDF (H-PDF) was incubated in the presence or absence of GLO-I and GSH for 1 hour at 37 degrees C, and then mixed with an equal volume of serum-free M199 medium and applied to HPMCin culture. After 24 hours, HPMC were assessed for viability, the release of interleukin-6, GLO-I activity, and cellular glutathione. The effects were compared to those exerted by filter-sterilized PDF (F-PDF), which was devoid of GDP. RESULTS: Exposure of HPMC to H-PDF resulted in reduced GLO-I activity, GSH depletion, and a decrease in cell viability. Pretreatment of H-PDF with either a combination of GLO-I and GSH or GSH alone markedly reduced inhibitory effects of H-PDF toward HPMC, as measured by cell viability and inter-Leukin-6 generation. Exposure of HPMC to the GSH precursor L-2-oxothiazolidine-carboxylic acid increased cellular GSH and prevented the loss of GLO-I activity in response to H-PDF. CONCLUSIONS: Exposure to conventional GDP-rich PDF impairs the activity of the glyoxalase/glutathione system in HPMC. Pretreatment of PDF with GSH or replenishment of cellular GSH protects HPMC against GDP-mediated toxicity.     

Inhibition of endogenous oxalate production: biochemical considerations of the roles of glycollate oxidase and lactate dehydrogenase

1. Both the peroxisomal, flavin-linked glycollate oxidase [(S)-2-hydroxy-acid oxidase; EC 1.1.3.15] and the cytosolic, nicotinamide-adenine dinucleotide (NAD)-linked lactate dehydrogenase (L-lactate dehydrogenase; EC 1.1.1.27) are thought to contribute to the formation of oxalate from its immediate precursors, glycollate and glyoxylate, but the relative contributions of each enzyme to endogenous oxalate production is not known. 2. In rat liver homogenates, [14C]oxalate production from labelled glycollate is halved and that from labelled glyoxylate is increased fourfold by the addition of either NAD or NADH. 3. In isolated rat hepatocytes, the 3-hydroxy-1H-pyrrole-2,5-dione derivatives of glycollate, which are specific inhibitors of glycollate oxidase, have a greater effect on glycollate metabolism than on glyoxylate metabolism. 4. These findings are consistent with an important role for lactate dehydrogenase in oxalate formation from glyoxylate. 5. With human and rat liver homogenates and with purified human liver glycollate oxidase and rabbit muscle lactate dehydrogenase, DL-phenyl-lactate (2 mmol/l) completely inhibits glycollate oxidase but has not effect on lactate dehydrogenase. On the other hand, the reduced form of a chemically synthesized, NAD-pyruvate adduct (1 mmol/l) almost completely inhibited lactate dehydrogenase but had no effect on glycollate oxidase. 6. Either alone or in combination, DL-phenyl-lactate and reduced NAD-pyruvate adduct reduce oxalate production from glycollate and glyoxylate in isolated rat hepatocytes, but do not abolish it completely. 7. These findings support a role for another enzyme, probably glycollate dehydrogenase (EC 1.1.99.14), in oxalate production in integrated cell metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)