Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutathione and the modulation of glutathione S-transferase A4 expression by butyrate.

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.     

Liv.52 attenuate copper induced toxicity by inhibiting glutathione depletion and increased antioxidant enzyme activity in HepG2 cells

Altered copper metabolism plays a pivotal role in the onset of several hepatic disorders and glutathione (GSH) plays an important role in its homeostasis. Hepatic diseases are often implicated with decreased content of intracellular GSH. GSH depleted cells are prone to increased oxidative damage eventually leading to its death. Liv.52 is used to treat hepatic ailments since long time. Hence, in the present study the potential cytoprotective effect of Liv.52 against toxicity induced by copper (Cu²⁺) was evaluated in HepG2 cells. Cu²⁺ at 750 μM induced cytotoxicity to HepG2 cells as determined by MTT assay. The toxicity was brought about by increased lipid peroxidation, DNA fragmentation and decreased GSH content. But, upon treatment with Liv.52 cell death induced by Cu²⁺ was significantly abrogated by inhibition of lipid peroxidation by 58% and DNA fragmentation by 37%. Liv.52 increased the GSH content by 74%. Activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase were increased by 46%, 22% and 81% respectively in Liv.52 treated cells. Thus, it is apparent from these results that Liv.52 abrogates Cu²⁺ induced cytotoxicity in HepG2 cells by inhibiting lipid peroxidation and increased GSH content and antioxidant enzyme activity.     

Comparison of inhibitory effects between acetaminophen–glutathione conjugate and reduced glutathione in human glutathione reductase

Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen–glutathione conjugate (APAP‐SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP‐SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP‐SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP‐SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP‐SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP‐SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP‐SG, which might enhance the pro‐oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen. Copyright © 2013 John Wiley & Sons, Ltd.     

The relationship between the anti-inflammatory effects of curcumin and cellular glutathione content in myelomonocytic cells

Oxidative stress plays an important role during inflammatory diseases and recent therapies have focused on antioxidant administration to diminish oxidative stress and to arrest inflammatory processes. In this study, we investigated the impact of the GSH modulating effects of curcumin, a naturally derived polyphenol, on inflammatory processes in myelomonocytic U937 cells. One hour after administration of 10 micromol/l curcumin reactive oxygen species (ROS) production was significantly increased in undifferentiated U937 cells (+43%). Twenty-four hour after addition of curcumin, a significantly decreased ROS concentration was found (-32%), whereas GSH (+110%) and GSSG (+88%) content increased. A higher concentration of curcumin (25 micromol/l) caused an even stronger increase of GSH (+145%) and GSSG (+101%), but significantly decreased percentage of living cells to 84%. The increased GSH content of differentiated U937 cells after pre-incubation with curcumin was associated with lowered ROS production, nuclear factor kappa B (NFkappaB) activation (-34%) and tumor necrosis factor alpha (TNF-alpha) secretion (-51%) after LPS exposure. Curcumin inhibited TNF-alpha formation was also seen after GSH depletion by buthionine sulfoximine (BSO). This study shows that the antioxidative effects of curcumin are preceded by an oxidative stimulus, which is time and dose-dependent. Excessive concentrations of curcumin may even harm cells, as cell viability was decreased, in spite of elevated GSH contents. There was no clear relationship between intracellular GSH concentrations and the anti-inflammatory effects of curcumin.     

The effect of estrous cycle and buthionine sulfoximine on glutathione release from the in vitro perfused rat ovary

There is little known regarding the intracellular mechanisms of modification of damage in the ovary. Ovarian perfusion of en block dissections of the rat right ovary with aorta and vena cava were done to determine (a) if glutathione (GSH) is released by the ovary, (b) if the release is cycle dependent, and (c) if GSH released is the product of de novo ovarian synthesis. All perfused ovaries released GSH and the release was maximal at estrus and least at metestrus. Perfusion with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase, resulted in a dose-dependent reduction in GSH released, indicating inhibition of de novo synthesis during perfusion.     

钩吻生物碱化合物体外抗消化系统肿瘤的活性

目的探讨钩吻生物碱化合物抗消化系统肿瘤的活性,并初步探索其构效关系。方法采用四甲基偶氮唑盐（MTT法）比色法,观察钩吻生物碱化合物对人肝肿瘤细胞HepG2、人胃癌细胞MGC80-3、人食管癌细胞TE-11和人结肠癌细胞SW480增殖的抑制作用。结果钩吻素子、钩吻素甲、钩吻素己和1-甲氧基钩吻碱对SW480细胞和MGC80-3细胞增殖具有显著的抑制作用,作用呈剂量依赖性,对SW480细胞半数抑制浓度（IC50）分别为0.45±0.10,0.76±0.28,0.52±0.22和1.41±0.06mmol·L^-1。对MGC80-3细胞IC50分别为0.82±0.19,1.20±0.33,1.14±0.23和1.22±0.11mmol·L^-1。钩吻素子、钩吻素甲和钩吻素己对TE-11细胞和HepG2细胞的增殖也有一定的抑制作用,作用呈剂量依赖性,对TE-11细胞半数抑制浓度（IC50）分别为0.74±0.05,1.94±0.30和1.73±0.35mmol·L^-1。对细胞HepG2的IC50分别为1.26±0.32,1.82±0.35和1.79±0.54mmol·L^-1。结论钩吻生物碱化合物具有抗消化系统肿瘤活性,并存在一定的构效关系。     

In vivo effects of fenthion on oxidative processes by the modulation of glutathione metabolism in the brain of Oreochromis niloticus

The present study was designed to understand the oxidative stress potential of fenthion, an organophosphate (OP) pesticide and its involvement in glutathione metabolism modulated buthionine sulfoximine (BSO, 50 mg/kg) and N-acetylcysteine (NAC, 100 mg/kg) in the brain of fish, Oreochromis niloticus. A sublethal fenthion concentration (0.45 mg/L) was applied for 24, 48, and 96 h together with injection with BSO or NAC; following treatment, recovery periods for 24, 48, and 96 h were allowed. Total glutathione (tGSH), oxidized glutathione (GSSG), lipid peroxidation, protein level, and GSH-related enzyme activities were analyzed by using spectrophotometric methods. Fenthion in applied concentration did not change GSH levels, but increased GSSG levels. BSO application in fenthion exposure caused a depletion in GSH, while increasing the GSSG levels. Glutathione peroxidase (GPx; EC 1.11.1.9) specific activity increased in fenthion-applied groups at 24-h treatment. gamma-Glutamylcysteinyl synthetase (gamma-GCS; EC 6.3.2.2) was not detected in the brain. NAC injection in fenthion treatment decreased GSH and increased GSSG levels and GST activity. In conclusion, fenthion in sublethal concentration induced an oxidative stress processes in brain. BSO application provided an evidence for the involvement of fenthion in GSH metabolism. NAC elevated the fenthion-induced effects in spite of its antioxidant properties. Recovery period for 96 h was not adequate to eliminate the fenthion-induced changes.     

谷胱甘肽合成抑制对人胚肾细胞的毒作用

目的：研究细胞谷胱甘肽（GSH）合成抑制对人胚肾细胞的影响。方法：用GSH合成特异抑制剂（BSO）抑制GSH合成，从而降低胞内GSH的水平。测定BSO处理不同时间人胚肾细胞内GSH的水平、细胞周期的变化、活性氧水平、细胞凋亡率和细胞存活率。结果：细胞内总GSH水平随BSO处理时间的延长而下降。处理24时，总GSH水平下降至对照的10％，分析发现细胞周期G2－M期增多（P＜0．01），S期减少（P＜0．05）；但活性氧水平、细胞凋亡率及细胞存活率没有明显改变。处理48h时，总GSH水平下降至对照的4％，细胞存活率明显降低（P＜0．01），活性氧水平明显升高（P＜0．01）。细胞凋亡率直到处理72h时升高才能显著性（P＜0．01，n=3），此时细胞内总GSH已基本耗竭。结论：由于GSH水平下降而引起的氧化还原状态失衡可能是凋亡发生的普遍诱导因素。     

The effect of buthionine sulfoximine, an inhibitor of glutathione synthesis, on hepatic drug metabolism in the male mouse

Buthionine sulfoximine significantly reduced the hepatic non-protein sulfhydryl (NPSH) content of male B6C3F1 mice within 2 h after intraperitoneal (i.p.) injection. This treatment did not affect the activity of several hepatic microsomal and cytosolic enzymes responsible for xenobiotic metabolism. Pretreatment of mice with buthionine sulfoximine (2 mmol/kg) increased the hepatotoxicity of chloroform, but did not affect the hepatotoxicity of carbon tetrachloride. These findings suggest that buthionine sulfoximine can be a useful agent for studying the role of glutathione (GSH) in hepatic biotransformation of xenobiotics.     

Modification of butylated hydroxytoluene-induced pulmonary toxicity in mice by diethyl maleate, buthionine sulfoximine, and cysteine

Treatment of mice with diethyl maleate (DEM) or buthionine sulfoximine (BSO) significantly enhanced the lung injury caused by butylated hydroxytoluene (BHT). Conversely, cysteine protected mice from the lung toxicity of BHT. BHT administration to mice produced a time-dependent reduction of glutathione (GSH) content in the lung, but not in the liver. These results support the concept that conjugation of 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide), a proposed reactive metabolite of BHT, with GSH is involved in the detoxification of BHT in mice.     

谷胱甘肽的耗竭与肿瘤细胞凋亡

细胞凋亡是由一系列基因控制的细胞主动死亡过程，凋亡的通路受到抑制是肿瘤产生的重要原因。凋亡机制的最新研究发现细胞内谷胱甘肽(GSH)的水平与细胞凋亡密切相关。通过GSH的耗竭可以启动肿瘤细胞凋亡。本文综述了GSH的耗竭诱导肿瘤细胞凋亡的分子生物学机制、GSH耗竭剂——GSH合成抑制剂与GSH结合剂的研究进展以及GSH耗竭剂作为肿瘤细胞凋亡诱导剂的应用前景。     

Plasmodium berghei: dehydroepiandrosterone sulfate reverses chloroquino-resistance in experimental malaria infection; correlation with glucose 6-phosphate dehydrogenase and glutathione synthesis pathway

In Plasmodium falciparum-infected cells or in P. berghei infected mice, increase of reduced glutathione (GSH) levels confers resistance to chloroquine (CQ). GSH is synthesized within the cells through a complex biochemical pathway composed of several well known enzymes, in which glucose-6-phosphate dehydrogenase (G6PD) plays an important role. The physiological hormone dehydroepiandrosterone sulfate (DHEAS) is a potent inhibitor of G6PD activity, and G6PD deficiency is known to exert antimalaria protection. This study aimed to investigate the ability of DHEAS to enhance the antimalarial activity of CQ, via an inhibition of G6PD activity and GSH synthesis. Two P. berghei CQ resistant strains (CQR6 and CQR30) were selected in vivo from the sensitive strain NK65. Drug effects were checked both by monitoring the evolution of parasitaemia and by the survival of infected mice. In addition, intra-parasite levels of GSH and G6PD activity were measured before and after the treatment. Results demonstrate that acquisition of CQ resistance in P. berghei is associated with a significant increase in parasite G6PD activity and GSH level. Combination of CQ with DHEAS or buthionin sulfoximin (BSO, a specific inhibitor of GSH synthesis) significantly increased sensitivity of resistant parasites to CQ and increased the survival period of the infected mice. This reduction of parasitaemia and improvement of the survival of infected mice were associated with intra-parasite depletion of GSH and inhibition of G6PD activity due to DHEAS action. This experimental study suggests that DHEAS could be used to potentiate antimalarial action of CQ, particularly on CQ resistant strains.     

Tissue-specific effects of fenthion on glutathione metabolism modulated by NAC and BSO in Oreochromis niloticus

Introduction:?The present study was designed to understand the effects of organophosphate (OP) insecticide and avicide fenthion on cellular redox status and the role of reduced glutathione (GSH) on fenthion toxicity in the liver and kidney of Oreochromis niloticus as a model organism. N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were injected intraperitoneally to fenthion-exposed fish as modulators of GSH metabolism. GSH redox status, GSH-related enzyme activities, and thiobarbituric acid reactive substances (TBARS) contents were then measured spectrophotometrically at 24, 48, and 96 hours. To assess recovery from fenthion exposure, similar analyses were performed on fish transferred to non-treated water for 24, 48, and 96 hours.

Results:?Fenthion increased glutathione S-transferase (GST; EC 2.5.1.18) activity and caused changes in total GSH (tGSH), GSH and oxidized glutathione (GSSG) contents and glutathione peroxidase (GPx; EC 1.11.1.9) specific activity in the liver tissue over time. Increases observed in tGSH and GSSG contents at 24 hours were decreased by fenthion treatment at 96 hours. BSO caused a sharp decline in liver tGSH, GSH, and GSSG contents and an elevation in GST and γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) enzyme activities. A significant decrease was observed in tGSH and GSH contents and, also, GST enzyme activities in the kidney at 48-hour fenthion treatment. On the contrary to the liver, a significant increase was observed in tGSH and GSH contents in the kidney by BSO injection. NAC application eliminated the decreasing effects of fenthion on GST activity in this tissue. NAC injection caused decreases in lipid peroxidation (LPO) levels. Decline in tGSH and GSH contents were maintained in the liver during the recovery period, and elevations in LPO levels in the kidney were observed during the same period.

Conclusions:?In conclusion, tissue-specific and time-dependent GSH redox status disturbance of fenthion were observed. BSO revealed the significance of GST-mediated GSH conjugation on the detoxification process of fenthion. NAC seemed useful to avoid the fenthion-related oxidative toxicity.     

Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)

Glutathione is a tripeptide that plays an important role in ameliorating metal toxicity. Depletion of glutathione has been associated with an increased risk of metal toxicity in mammals. An understanding of the toxicological significance of glutathione depletion in oysters would be of considerable importance given the widespread use of bivalves in biological monitoring. Laboratory studies were conducted by using an inhibitor of glutathione synthesis (buthionine sulfoximine) to investigate the effects of glutathione depletion on metallothionein expression, lysosomal membrane destabilization, and lipid peroxidation in Cu-exposed oysters. In oysters exposed to Cu (20 and 80 μg/l) and buthionine sulfoximine (20 mg/l), metallothionein induction was suppressed and cellular stress responses were frequently higher than those observed in oysters exposed singly to Cu. Together, these results suggest that environmental conditions that cause glutathione depletion may increase the potential for adverse effects to pollutants during in situ exposures.     

三萜皂苷混合物ardisiacrispin(A+B)对人白血病HL-60细胞的增殖抑制作用及机制探讨

目的观察三萜皂苷混合物ardisiacrispin(A+B)对人白血病HL-60细胞增殖的抑制作用,并对其可能的作用机制进行探讨。方法应用MTT法测定增殖抑制作用,采用流式细胞术(FACS)分析阻断细胞周期及诱导凋亡作用,采用PI荧光染色观察凋亡小体的存在,以Western blot分析凋亡相关蛋白表达变化。结果ardisiacrispin(A+B)可明显抑制HL-60细胞的增殖,具有浓度依赖性,作用48h的IC50值为4.2mg·L-1。ardisiacrispin(A+B)可阻断HL-60细胞于S期,在1.0～5.0mg·L-1范围内具有浓度依赖性,3.5mg.L-1ardisiacrispin(A+B)作用6、24和48h时,给药组中S期细胞比例分别为对照组的1.26、1.46和1.71倍。ardisia-crispin(A+B)可诱导HL-60细胞凋亡,在1.0～7.5mg.L-1浓度范围内具有浓度依赖性;此外,ardisiacrispin(A+B)可浓度依赖性地诱导PARP蛋白的裂解,2.5mg.L-1ardisi-acrispin(A+B)作用24h使裂解比例达到27.1%。结论Ardisiacrispin(A+B)可通过阻断细胞于S期、诱导凋亡来抑制HL-60细胞的增殖。     

Different effects of nine clausenamide ennatiomers on liver glutathione biosynthesis and glutathione S-transferase activity in mice

AIM: To study the effects of nine synthetic clausenamide with different stereo structures on liver glutathione (GSH) biosynthesis and glutathione S-transferase (GST) activity in mice. METHODS: The nine test compounds were racemic mixtures and their ennatiomers of clausenamide, neoclausenamide and epineoclausenamide. Mice were administered clausenamide 250 mg/kg once daily for 3 consecutive days, ig, and were killed 24 h after the last dosing. The mouse liver cytosol GSH and GST were determined with related biochemical methods. RESULTS: Nine clausenamides exhibited different effects on liver GSH and GST. Of nine clausenamides, only (+) and (+/-)clausenamide markedly increased liver cytosol GSH content. The mechanism of increasing liver GSH content of (+)clausenamide is mainly due to stimulating the key limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS) activity for GSH biosynthesis. The other test clausenamides had no such effect on liver GSH. All of the nine clausenamides induced a significant increase of GST activity. CONCLUSION: The effects of clausenamide ennatiomers on liver GST and GSH varied with the alterations of their spatial structures. (+)Clausenamide stimulated liver GSH biosynthesis through enhancing gamma-GCS activity.     

Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinate in cultured human cells and the role of glutathione

Thio-dimethylarsinate (thio-DMA), a recently discovered urine metabolite in humans, was investigated for its cytotoxic, genotoxic and cell-cycle disruptive effects in the cultured human hepatocarcinoma cell line, HepG2, and Syrian hamster embryo cells. In addition, the role of glutathione (GSH) on the cytotoxic effects of thio-DMA was investigated in terms of the effects of GSH depletion and the effects of exogenously added GSH. LC50 values of arsenicals for cells incubated for 48 h were 0.026 mM for thio-DMA, 0.343 mM for DMA and 3.66 mM for dithio-DMA. Depletion of cell GSH reduced the cytotoxic effects of thio-DMA. The cytotoxic effects of 0.02 mM and 0.05 mM thio-DMA were enhanced markedly when used in combination with 1 to 3 mM GSH, but decreased again when combined with 5 mM GSH. These results suggested that cytotoxic intermediates were generated by the interaction of thio-DMA with GSH, while an excessive amount of GSH suppressed the generation of these intermediates. Flow-cytometry showed that thio-DMA was an inducer of cells with 4N DNA and hypo 2N DNA. The results also demonstrated that cells arrested in the mitotic phase had abnormalities in their spindle organization and centrosome integrity. In addition, cells arrested in mitosis by thio-DMA had chromosome structural aberrations, such as chromatid gaps, chromatid breaks and chromatid exchanges. Moreover, the cytotoxic effects of thio-DMA may in part be associated with an apoptotic mode of cell death that was evaluated by the appearance of nucleosome level DNA fragmentations and an 85-kDa cleavage fragment of poly (ADP-ribose) polymerase. These findings suggest that the presence of thio-DMA in human urine has implications for human health in terms of arsenic metabolism and toxicity.     

Rimonabant (SR141716) exerts anti-proliferative and immunomodulatory effects in human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE: Rimonabant (SR141716) is the first selective cannabinoid receptor CB(1) antagonist described. Along with its anti-obesity action, emerging findings show potential anti-proliferative and anti-inflammatory action of SR141716 in several in vitro and in vivo models. In this study we have investigated the anti-proliferative and immunomodulatory effects of SR141716 in human peripheral blood mononuclear cells (PBMCs). EXPERIMENTAL APPROACH: We have evaluated in vitro the effect of SR141716 in human PBMCs stimulated with different mitogens. Cell proliferation was assessed by (3)H-thymidine incorporation. Cell cycle, cell death and apoptosis were analysed by flow cytometry. Protein expression was investigated by Western blot. KEY RESULTS: SR141716 significantly inhibited the proliferative response of PBMCs and this effect was accompanied by block of G(1)/S phase of the cell cycle without induction of apoptosis and cell death. SR141716 used in combination with 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), a stable analogue of the endogenous cannabinoid anandamide, showed synergism rather than antagonism of the inhibition of cell proliferation. The immunomodulatory effects of SR141716 were associated with increased expression of IkappaB, phosphorylated AKT (p-AKT) and decreased expression of NF-kappaB, p-IkappaB, p-ERK, COX-2 and iNOS. CONCLUSIONS AND IMPLICATIONS: Our findings suggest SR141716 is a novel immunomodulatory drug with anti-inflammatory properties.     

Cytotoxicity of rhenium(I) alkoxo and hydroxo carbonyl complexes in murine and human tumor cells

The rhenium(I) alkoxo/hydroxo carbonyl complexes were shown to be very potent in suspended tumor cell lines in suppressing growth but were more selective in inhibiting the growth of cultures from solid tumors. Their mode of action in L1210 lymphoid leukemia cells indicated that they were not alkylating agents but interfered with nucleic acid metabolism at multiple enzyme sites, e.g. dihydrofolate reductase, PRPP-amido transferase, thymidine kinase, with DNA strand scission after 60 min incubation. These compounds did not function mechanistically exclusively as cisplatin derivatives causing intrastrand linkages of DNA but rather they mimicked the metal complexes of aminecarboxyboranes, furan oximes, N-substituted thiosemicarbazones, trifluoromethyl borons and ferratricarbadecarbanyl complexes acting as antimetabolites.