Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N'-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.

Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.

No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca²⁺ concentration.

We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.     

Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes

Cultured hepatocytes were exposed to two chemicals, dinitrofluorobenzene (DNFB) and diethyl maleate (DEM), that abruptly deplete cellular stores of glutathione. Upon the loss of GSH, lipid peroxidation was evidenced by an accumulation of malondialdehyde in the cultures followed by the death of the hepatocytes. Pretreatment of the hepatocytes with a ferric iron chelator, deferoxamine, or the addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), to the culture medium prevented both the lipid peroxidation and the cell death produced by either DNFB or DEM. However, neither deferoxamine nor DPPD prevented the depletion of GSH caused by either agent. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or inhibition of catalase by aminotriazole sensitized the hepatocytes to the cytotoxicity of DNFB. In a similar manner, pretreatment with BCNU potentiated the cell killing by DEM. DPPD and deferoxamine protected hepatocytes pretreated with BCNU and then exposed to DNFB or DEM. These data indicate that an abrupt depletion of GSH leads to lipid peroxidation and cell death in cultured hepatocytes. It is proposed that GSH depletion sensitizes the hepatocyte to its constitutive flux of partially reduced oxygen species. Such an oxidative stress is normally detoxified by GSH-dependent mechanisms. However, with GSH depletion these activated oxygen species are toxic as a result of the iron-dependent formation of a potent oxidizing species.     

Oxidative cell injury in the killing of cultured hepatocytes by allyl alcohol

The killing of cultured hepatocytes by allyl alcohol depended on the metabolism of this hepatotoxin by alcohol dehydrogenase to the reactive electrophile, acrolein. An inhibitor of alcohol dehydrogenase, pyrazole, prevented both the toxicity of allyl alcohol and the rapid depletion of GSH. Treatment of the hepatocytes with a ferric iron chelator, deferoxamine, or an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), prevented the cell killing but not the metabolism of allyl alcohol and the resulting depletion of GSH. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitized the hepatocytes to allyl alcohol, an effect that was not attributable to the reduction in GSH with BCNU. The cell killing with allyl alcohol was preceded by the peroxidation of cellular lipids as evidence by an accumulation of malondialdehyde in the cultures. Deferoxamine and DPPD prevented the lipid peroxidation in parallel with their protection from the cell killing. These data indicate that acrolein produces an abrupt depletion of GSH that is followed by lipid peroxidation and cell death. Such oxidative cell injury is suggested to result from the inability to detoxify endogenous hydrogen peroxide and the ensuing iron-dependent formation of a potent oxidizing species. Oxidative cell injury more consistently accounts for the hepatotoxicity of allyl alcohol than does the covalent binding of acrolein to cellular macromolecules.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.     

Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP+) cytotoxicity

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.     

EGF mediates protection against Fas-induced apoptosis by depleting and oxidizing intracellular GSH stocks

Several pieces of evidence have demonstrated the importance of reduction/oxidation (redox) signaling in biological processes, including sensitivity toward apoptosis. In parallel, it was recently reported that growth factors induce the generation of reactive oxygen species (ROS). Therefore, we tested the hypothesis that the anti-apoptotic effect of epidermal growth factor (EGF) was mediated by changes in the redox state of hepatocytes through changes in GSH stocks. Isolated mouse hepatocytes were cultured and exposed to anti-Fas stimulation in order to induce apoptosis. Cell death by apoptosis was assessed by Hoechst 33258 staining and by measuring caspase-3 proteolysis activity. Cell treatment with EGF significantly decreased total (GSx) and reduced (GSH) glutathione levels in the presence and the absence of anti-Fas. Furthermore, glutathione reductase activity was lower in EGF-treated cultures (by 28%) as compared to untreated cultures which lead to a significant decline in GSH/GSx ratio. These effects were found to be EGF-receptor tyrosine kinase activity dependent. Co-stimulation of cells with anti-Fas and EGF attenuated caspase-3 activation and cell death by apoptosis by 70%. GSH monoethylester (GSHmee) significantly attenuated the effect of EGF on GSH and GSH/GSx ratio. It caused an increase in caspase-3 activation and in the percentage of apoptotic cells in anti-Fas + EGF-treated cells, thus resulting in a 53% decline in the protective effect of EGF. In conclusion, EGF induces a significant and specific depletion and oxidization of intracellular GSH, paralleled by a protection against Fas-induced apoptosis. GSH repenishment partly counteracted these effects suggesting that GSH depletion contributed to the protective effect of EGF against caspase-3 activation and cell death by apoptosis.     

Cocaine-induced biochemical changes and cytotoxicity in hepatocytes isolated from both mice and rats

The mechanism of cocaine-induced cytotoxicity was investigated in hepatocytes isolated from both male C3H mice and male Sprague-Dawley rats. Cocaine was more cytotoxic to mouse hepatocytes than rat and induced reduced glutathione (GSH) depletion prior to marked increases in cytotoxicity in both systems. In both mouse and rat cells, GSH depletion was accompanied by GSSG production, but in rat cells, quantitative measures suggested that other mechanisms contributed to GSH depletion. No cocaine-induced depletion of protein-thiol groups or generation of protein-glutathione mixed disulfides could be detected in rat cells. Cocaine induced lipid peroxidation, using malondialdehyde (MDA) production as an index of the peroxidation process, in both mouse and rat hepatocytes. Inhibition of MDA production to below control levels using the antioxidant N,N'-diphenyl-phenylene diamine (DPPD) however, had no inhibitory effect on cocaine-induced cytotoxicity in either mouse or rat cells. These data suggest that neither generalized protein thiol depletion nor lipid peroxidation are critical determinants of cocaine-induced cytotoxicity in cellular systems.     

Danthron Induced Apoptosis Through Mitochondria- and Caspase-3-Dependent Pathways in Human Brain Glioblastoma Multiforms GBM 8401 Cells

Danthron (1,8-dihydroxyanthraquinone), is one of component from Rheum palmatum L. (Polygonaceae), has been shown several biological activities but did not show to induce apoptosis in human brain tumor cells. The aim of this study is to investigate the mechanisms by danthron for the induction of apoptotic potential on human brain glioblastoma multiforms GBM 8401 cell line. Danthron showed a marked concentration- and time-dependent inhibition of GBM 8401 cell viability and induced apoptosis in a dose-and time-dependent manner. There was an attenuation of mitochondrial membrane potential (ΔΨ _m ) with the alterations of Bcl-2/Bax protein ratio in GBM 8401 cells, indicating the participation of a mitochondria-related mechanism. Pretreatment of a caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVE-FMK) significantly increased the viable of GBM 8401 cells implied that the participations of caspases. Western blotting analysis also showed the activation of initiator caspase-8 and caspase-9, and executor caspase-3 in GBM 8401 cells. Meanwhile, danthron also promoted the generation of reactive oxygen species (ROS) and cytosolic Ca²⁺ in GBM 8401 cells. Taken together, our data showed that danthron induced apoptosis in GBM 8401 cells through mitochondria-related and caspase-related pathways, and it may be further evaluated as a chemotherapeutic agent for human brain cancer.     

Antigenotoxic properties of Cassia tea (Cassia tora L.): mechanism of action and the influence of roasting process

Antigenotoxic properties and the possible mechanisms of water extracts from Cassia tora L. (WECT) treated with different degrees of roasting (unroasted and roasted at 150 and 250 degrees C) were evaluated by the Ames Salmonella/microsome test and the Comet assay. Results indicated that WECT, especially unroasted C. tora (WEUCT), markedly suppressed the mutagenicity of 2-amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole (Glu-P-1) and 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1). In the Comet assay performed on human lymphocytes, WECT exhibited significant protective effect on Trp-P-1-mediated DNA damage followed the order of unroasted (55%) > roasted at 150 degrees C (42% ) > roasted at 250 degrees C (29%). Pre-treatment of the lymphocytes with WEUCT resulted in 30% repression of DNA damage. However, no significant effect on excision-repair system was found during DNA damage expression time in post-treatment scheme (p>0.05). WEUCT showed 84% scavenging effect on oxygen free radicals generated in the activation process of mutagen detected by electron paramagentic resonance system. Two possible mechanisms were considered: (1) neutralization the reactive intermediate of Trp-P-1; and (2) protecting cells directly as an antioxidant that scavenge the oxygen radicals from the activation process of mutagen. The individual anthraquinone content in extracts of C. tora was measured by HPLC. Three anthraquinones, chrysophanol, emodin and rhein, have been detected under experimental conditions. The anthraquinone content decreased with increased roasting temperature. Each of these anthraquinones demonstrated significant antigenotoxicity against Trp-P-1 in the Comet assay. In conclusion, our data suggest that the decrease in antigenotoxic potency of roasted C. tora was related to the reduction in their anthraquinones.     

Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes

Zn(2+) has multiple implications in cellular metabolism, including free radicals metabolism and cell death by apoptosis. In the present study, we examined the role of Zn(2+) in the regulation of apoptosis in cultured rat hepatocytes. The chelation of Zn(2+) by a membrane permeable metal ion chelator, N, N, N', N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), induced apoptosis. Addition of ZnSO(4) prevented TPEN-induced apoptosis. Unlike the effect of TPEN, a membrane impermeable metal ion chelator, diethylenetriamine pentaacetic acid (DTPA), did not induce apoptosis, indicating that chelation of intracellular Zn(2+) was required to trigger apoptosis. Caspase-3-like proteolytic activity, a general biochemical mediator of apoptosis in a variety of cells and tissues, was also activated with the treatment of TPEN but not DTPA. TPEN treatment, but not DTPA, also resulted in the depletion of intracellular reduced glutathione (GSH) but addition of Zn(2+) recovered the GSH level. N-acetyl-L-cysteine (NAC), a thiol antioxidant, prevented TPEN-induced apoptosis. These results taken together suggest that intracellular Zn(2+) interfere with the apoptosis process, possibly through the regulation of cellular redox potential involving GSH.     

粗柄独尾草不同器官蒽醌类成分的消长规律

采用高效液相色谱法对沙生类短命植物粗柄独尾草苗期、营养生长期、初花期、盛花期、果期各器官中大黄素、大黄酚、大黄酸、芦荟大黄素含量的消长规律进行了研究。结果表明:叶中,芦荟大黄素的含量在苗期和初花期都较高,在盛花期时最低;大黄酸的含量在苗期最高,盛花期时最低;大黄素的含量在苗期达到最高,初花期和盛花期最低;大黄酚的含量也以苗期最高,盛花期和果期最低。且在初花期时,4种蒽醌类物质含量均呈现明显的叶先端>叶中部>叶基部的空间差异性。根中,芦荟大黄素的含量在苗期和营养生长期较高,而以盛花期和果期较低;大黄酸的含量在果期最高,其余时期差异不显著;大黄素的含量以苗期和初花期较高;大黄酚的含量在果期达最高,而盛花期时最低。同时期的根叶蒽醌含量相比,叶中的芦荟大黄素要高于根,而根中大黄酚含量要高于叶。同时期各器官蒽醌总量相比:叶>根>花>花葶。故若选取粗柄独尾草作为蒽醌类药材利用,建议最佳采集方式为采集初花期的叶先端部分。     

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Anthracen-derivate in kalluskulturen aus Cassia angustifolia

Callus cultures from cotyledons of Cassia angustifolia were shown to contain dianthrones. Proof of their structures was obtained by co-chromatography (TLC), oxidative cleavage and UV analysis. The basic components chrysophanol, physcion, rheum emodin, aloe emodin and rhein have been isolated and identified by UV and IR analysis.     

高速逆流色谱对大黄蒽醌类成分的分离研究

利用高速逆流色谱对大黄中的5个蒽醌活性成分进行了分离,当两相溶剂系统的组成是石油醚∶乙酸乙酯∶甲醇∶水=8∶2∶8∶1时,分离出大黄素;当两相溶剂比为3∶4∶3∶2时,分离出大黄酸和芦荟大黄素;当溶剂比为12∶2∶12∶1时,分离出大黄酚和大黄素甲醚;经高压液相色谱检测大黄素、大黄酸和芦荟大黄素、大黄酚和大黄素甲醚的含量分别为98.81%9、9.15%、98.51%9、8.89%和98.16%。     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

大黄属3种大黄植物不同部分蒽醌含量的测定与比较

采用C18反相柱高效液相色谱方法分离、外标法定量对大黄属掌叶组唐古特大黄、波叶组波叶大黄、穗序组穗序大黄的根（及根茎）、叶片、叶柄、主茎四部分的芦荟大黄素、大黄酸、大黄素、大黄酚4种游离和结合蒽醌的含量进行了测定和比较。结果表明：唐古特大黄中,叶片中的游离蒽醌含量高于其它部分,游离蒽醌总量地上部分远高于地下部分;结合蒽醌则根中最高,蒽醌总量地下部分远高于地上部分。波叶大黄和穗序大黄中,游离和结合蒽醌均为根中最高,穗序大黄蒽醌总量地下部分远高于地上部分,而波叶大黄中,游离蒽醌总量地上部分高于地下部分,结合蒽醌总量地上部分与地下部分相差不大,地上部分略高于地下部分。     

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.