曲格列酮对大鼠肝细胞氧化性损伤的研究

目的:研究曲格列酮的肝细胞毒性及其可能的机制.方法:在原代大鼠肝细胞模型和其他3种不同来源非肝细胞系,用WST-1法、乳酸脱氢酶释放法、荧光检测法、生物发光法等方法,观察曲格列酮对肝细胞的损伤作用.结果:不同剂量曲格列酮作用后,其在原代培养大鼠肝细胞的IC50值最低,即其对原代肝细胞毒性最大.曲格列酮诱导活性氧(ROS)生成,并引起还原型谷胱甘肽(GSH)和ATP水平比对照细胞显著降低.不同剂量的曲格列酮作用5h后诱发肝细胞凋亡、坏死.结论:曲格列酮对大鼠肝细胞的毒性作用强于对其他非肝细胞来源细胞的毒性作用.曲格列酮可以引起肝细胞氧化性损伤,肝细胞凋亡和坏死.     

海兔素对大鼠原代肝细胞酒精性氧化损伤的保护作用

目的探讨海兔素对大鼠原代肝细胞酒精性氧化损伤保护作用。方法门静脉胶原酶Ⅳ原位灌注及密度梯度离心获得大鼠原代肝细胞。MTT实验检测乙醇和海兔素最佳作用剂量及肝细胞活力。酶学实验检测细胞AST、LDH、SOD、MDA、GSH水平;流式细胞术检测细胞凋亡情况;单细胞凝胶电泳观察细胞DNA损伤状况;JC-1荧光探针检测细胞线粒体膜电位水平;比色法及Western blot检测细胞CYP2E1活性及蛋白表达。结果经30 mg·L~(-1)。海兔素预作用2 h,再与300 mmol·L~(-1)。乙醇共作用8 h后,肝细胞活力较酒精模型组明显上升,AST和LDH释放也得到明显抑制;同时,肝细胞SOD和GSH水平明显升高,MDA含量则明显降低,差异均具有显著性(P<0.05)。海兔素干预后,肝细胞凋亡率明显降低,DNA损伤及线粒体膜电位水平明显得到改善。海兔素干预后,肝细胞CYP2E1活性及蛋白表达水平明显受到抑制(P<0.05)。结论海兔素对大鼠原代肝细胞酒精性氧化损伤具有保护作用,其作用机制可能与海兔素抑制酒精对CYP2E1的活化,缓解氧化应激,提高机体抗氧化能力有关。     

Inhibition of rat liver catechol-O-methyltranferase by lanthanum,neodymium and europium

Partially purified rat liver catechol-O-methyltransferase (COMT) is inhibited by lanthanum, and by the lanthanides, neodymium and europium. The 50 per cent inhibitory concentrations are 3 × 10^?6, 1.2 × 10^?6, and 8.5 × 10^?7 M for La³⁺, Nd³⁺ and Eu³⁺ respectively. The inhibition of COMT by these ions is reversible. Lineweaver-Burk plots of the results of experiments in which the enzyme activity was measured in the presence of varying concentrations of magnesium, an activator of COMT, and different concentrations of La³⁺, Nd³⁺ and Eu³⁺ were compatible with non-competitive or “mixed” inhibition. Double reciprocal plots of the results of experiments in which COMT activity was determined in the presence of different concentrations of La³⁺ and varying quantities of the two co-substrates for the reaction, S-adenosyl-1-methionine and 3,4-dihydroxybenzoic acid, were also compatible with non-competitive or “mixed inhibition”. The characteristics of the inhibition of rat liver COMT by La³⁺, Nd³⁺ and Eu³⁺ are similar to those of the inhibition of COMT by Ca²⁺. However, lanthanum, neodymium and europium are two to three orders of magnitude more potent inhibitors of COMT than is calcium.     

Antinociceptive effects of lanthanum,neodymium and europium following intrathecal administration

The trivalent cations lanthanum, neodymium and europium were found to have a weak dose-dependent antinociceptive effect as measured on hot plate and tail nick following administration into the lumbar subarachnoid space of rats implanted with chronic intrathecal catheters. The effects were limited to the hindlimbs and tail. The lanthanum effect was reversible by intrathecal administration of calcium, but was not altered by intraperitoneal administration of naloxone. In equimolar doses, neodymium and europium were found to be significantly more potent than lanthanum. Doses of lanthanum higher than 1 μmol and of neodymium and europium greater than 0.3 μmol resulted in significant motor weakness of the lower extremities.     

Mechanisms participating in oxidative damage of isolated rat hepatocytes

The aim of the study was to evaluate time course and dose dependence of peroxidative damage induced by tert-butyl hydroperoxide (tBHP) in rat hepatocytes cultured in suspension and in monolayer. At the lowest (0.1 mM) concentration, decrease of cytosolic glutathione and discharge of mitochondrial membrane potential (MMP) could be detected. Significant increases in leakage of lactate dehydrogenase and in malondialdehyde concentrations together with decrease of pyruvate-dependent respiration were detected at higher tBHP concentrations (above 0.5 mM) and after longer periods of incubation. Changes in plasma membrane integrity were observed at 1 mM concentration of tBHP. Succinate-dependent oxidation was most resistant to peroxidative damages. Opening of the mitochondrial permeability transition pore was responsible for the discharge of mitochondria membrane potential. In the presence of cyclosporine A and succinate, the membrane potential could be restored. Our data showed that the most sensitive indicators of the peroxidative damage are changes of cytosolic glutathione concentration and MMP.     

Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes

Capillarisin (Cap), a main constituent of Artemisia capillaris (Compositae), was studied for its antioxidant bioactivity. In the preliminary study, Cap expressed a antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). This antioxidant bioactivity of Cap was investigated further using a model of t-butylhydroperoxide (t-BHP)-induced cytotoxicity and genotoxicity in rat primary hepatocytes. Results presented here demonstrate that Cap, at concentrations of 0.01–1.00 mg/ml, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Cap also attenuated the t-BHP-induced diminution of glutathione (GSH) and high level of DNA repaired synthesis. These results lead to speculation that Cap presents inhibitory effects against t-BHP-caused cytotoxicity and genotoxicity in rat primary hepatocyte cultures at least via two distinct pathways, stabilizing the GSH system and quenching free radicals. Received: 4 November 1998 / Accepted: 18 February 1999     

Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes.

Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.     

Effects of monochlorobimane on cerebral ischemia-induced damage to mitochondria

A possible involvement of inhibitory effects of monochlorobimane (MCB) on the opening of mitochondrial permeability transition (MPT) pore in the cerebroprotection against the ischemic brain injury was examined. MCB (1 mM) inhibited the opening of MPT pore in vitro. Sustained cerebral ischemia was induced by injecting 900 microspheres (48 microm in diameter) into the right hemisphere of rats. At 12 to 72 h after microsphere embolism (ME), the mitochondrial activity was determined histochemically by staining cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) of the brain sections. The COX and SDH stainings in the hippocampus were observed intensively in the pyramidal neurons in the CA2-3 and dentate gyrus rather than those in the CA-1 region. The staining was decreased with time after the embolism. Pretreatment with 10 microg/animal MCB 30 min prior to the embolism significantly attenuated the ME-induced reduction in the staining of COX and SDH in the hippocampus, but not in the pariatal cortex. The results suggest that prevention of the opening of MPT pore by MCB may play an important role in the cerebroprotection against cerebral ischemic injury.     

Early oxidative damage in primary cultured trout hepatocytes: a time course study

The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.     

硫丹对成年大鼠生精功能的影响和氧化损伤

目的　研究硫丹对大鼠生精功能的影响是否与氧化损伤有关。方法　成年雄性SPFWistar大鼠随机分为 6组 ,每组 6只。 1～ 4组ig硫丹 0 ,2 .5 ,5 .0 ,7.5mg·kg- 1,每天 1次 ,每周 6次 ,持续 10周。5～ 6组在给硫丹 7.5mg·kg- 1的同时 ,ip维生素C(VitC) 2 0 ,4 0mg·kg- 1。给药结束时检查各组动物的每日精子生成量 (DSP)、附睾精子数和形态 ,并检测血清和睾丸、肝组织中的过氧化脂质 (LPO)和 8 羟基脱氧鸟苷 (8 OHdG)的含量。结果　给硫丹的5 .0和 7.5mg·kg- 1组出现短暂的中枢神经系统中毒症状。给药结束时 3个单给硫丹组DSP和附睾精子计数都低于对照组 ,精子畸形率则高于对照组。同时ipVitC两组的DSP和精子计数虽然仍低于对照组 ,但都比单给硫丹组有所改善。给硫丹组血清、肝脏和睾丸组织中的LPO和 8 OHdG都高于对照组。同时注射VitC组血清和上述组织中的LPO和8 OHdG都比单给硫丹 7.5mg·kg- 1组低。结论　大鼠长期大剂量接触硫丹能引起精子生成减少 ,异常精子比例增多 ,并能引起肝脏和睾丸组织脂质过氧化和DNA氧化损伤 ,而这些改变都能被抗氧化剂VitC部分改善 ,提示氧化损伤可能是硫丹生殖毒性的作用机理之一。     

Effect of iron and ascorbate on cyclosporine-induced oxidative damage of kidney mitochondria and microsomes.

The stimulatory effect of iron and ascorbate on the damaging action of cyclosporine in kidney mitochondria, microsomes and epithelial cells was examined. Cyclosporine induced malondialdehyde formation and hydrogen peroxide production in mitochondria and attenuated the activity of MnSOD and glutathione peroxidase. The damaging effect of cyclosporine (50 microM) plus Fe2+(20 microM) on mitochondrial and microsomal lipids and proteins as well as mitochondrial thiols was greater than the summation of the oxidizing action of cyclosporine alone and Fe2+ alone. As for tissue components, iron enhanced cyclosporine-induced viability loss in kidney epithelial cells. Fe2+, EDTA and H2O2- induced 2-alpha deoxyribose degradation was attenuated by 10 mM DMSO and 200 microM DTPA but not affected by 200 microM cyclosporine. The addition of Fe2+ caused a change in the absorbance spectrum of cyclosporine in the wavelength range 230-350 nm. The simultaneous addition of cyclosporine (50 microM) and ascorbate (100 microM) showed the enhanced peroxidative effect on mitochondrial and microsomal lipids, which was inhibited by DTPA and EDTA (1 mM). Similar to iron, ascorbate enhanced cyclosporine-induced cell viability loss. The results show that iron and ascorbate promote the damaging action of cyclosporine in kidney cortex mitochondria and microsomes and in kidney epithelial cells, which may contribute to the enhancement of cyclosporine-induced nephrotoxicity.     

Attenuation of brain mitochondria oxidative damage by Albizia julibrissin Durazz: neuroprotective and antiemetic effects

Medicinal plants, as new drugs, are considered for treatment of insomnia, anxiety, depression, confusion, nausea, and vomiting symptoms. The current study aimed to evaluate the neuroprotective and antiemetic effects of Albizia. julibrissin Durazz. flower extract in the chickens. Emesis was induced by copper sulfate and ipecac (60 and 600?mg/kg, orally, respectively) and the methanolic extract (50, 100, and 200?mg/kg) were injected intraperitoneally (i.p.). Mitochondrial function, lipid peroxidation (LPO), protein carbonyl (PC) content, and catalase activity as biomarkers of oxidative damage were evaluated in the brain mitochondria. All doses of extract showed significant (p?<?0.001) antiemetic activity against induced emesis by copper sulfate and ipecac. Brain mitochondria function (by 50, 100, and 200?mg/kg of extract) were increased 48%, 85%, and 90% against emesis induced by ipecac and 32%, 18%, and 24% against emesis induced by copper sulfate, respectively. LPO and PC contents were significantly decreased after the administration of extract in emesis induced by copper sulfate and ipecac. A significant decrease (p?<?0.01) of CAT activity was observed in the extract (200?mg/kg) group in emesis induced by copper sulfate in chickens brain mitochondria. The present study suggests that the extract had antiemetic effects against emesis induced by copper sulfate and ipecac in young chickens via peripheral and central mechanisms. Neuroprotective effect of the extract could be due to the increase in bioactive compounds, plasma antioxidants, or direct free radical scavenging that could prevent lipid and protein alteration and impede the formation of oxidative damage.     

Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons

AIM: To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. METHODS: Primary cultured rat cortical neurons wereperformed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cellapoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Aψm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer.‘ Bcl-2family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected byWestern blot. RESULTS: Exposure of tBHP 100 μmol/L to neurons for 60 rain resulted in △ψm loss and cyto-chrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis.After removal of tBHP and then further treatment with curcumin (2.5-20 μmol/L) for 18 h, curcumin abrogated △ψm loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family.Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly.Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. CONCLUSION: Curcumin mayattenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mito-chondria from oxidative damage.     

菟丝子醇提液对衰老模型小鼠肝线粒体氧化损伤和复合体酶活性的影响

目的:研究菟丝子醇提液对D-半乳糖致衰小鼠抗氧化作用和线粒体呼吸链复合体和活性的作用。方法:纯系昆明种小鼠50只,随机分为青年组10只,衰老模型组10只,菟丝子醇提液组30只(15d组、30d组和45d组)。D-半乳糖注射法制作衰老模型。给药组灌服菟丝子醇提液。测定各组小鼠肝线粒体Mn-SOD活性、呼吸链复合体、呼吸链复合体活性及MDA含量的变化,同时观察给药15d、30d和45d时上述指标的变化。结果:D-半乳糖可诱导小鼠产生拟衰老变化,与青年对照组比较,表现出肝组织线粒体SOD活性下降(P<0.05),MDA含量升高(P<0.05),呼吸链复合体、呼吸链复合体活性降低(P<0.05)。菟丝子醇提液可提高衰老模型小鼠Mn-SOD、呼吸链复合体和呼吸链复合体活性。结论:菟丝子醇提液可通过提高衰老小鼠肝线粒体MnSOD活性和清除自由基能力,降低自由基代谢产物的含量,提高线粒体酶复合物活性,其作用随给药时间的延长而更显著。     

Molecular mechanism of oxidative damage of lung in mice following exposure to lanthanum chloride

Exposure to lanthanoids (Ln) elicits an adverse response such as oxidative injury of lung in animals and human. The molecular targets of Ln remain unclear. In the present study, the function and signal pathway of nuclear factor erythroid 2 related factor 2 (Nrf2) in LaCl₃‐induced oxidative stress in mouse lung were investigated. Mice were exposed to 2, 5, and 10 mg/kg body weight by nasal administration for 6 consecutive months. With increased doses, La was markedly accumulated and promoted the reactive oxygen species (ROS) production in the lung, which in turn resulted in peroxidation of lipids, proteins and DNA, and severe pulmonary damages. Furthermore, LaCl₃ exposure could significantly increase levels of Nrf2, heme oxygenase 1 (HO‐1) and glutamate‐cysteine ligase catalytic subunit (GCLC) expressions in the LaCl₃‐exposed lung. These findings imply that the induction of Nrf2 expression is an adaptive intracellular response to LaCl₃‐induced oxidative stress in mouse lung, and that Nrf2 may regulate the LaCl₃‐induced pulmonary damages. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 357–365, 2015.     

Comparative effects of sulfhydryl compounds on target organellae,nuclei and mitochondria,of hydroxylated fullerene‐induced cytotoxicity in isolated rat hepatocytes

DNA damage and cytotoxicity induced by a hydroxylated fullerene [C₆₀(OH)₂₄], which is a spherical nanomaterial and/or a water‐soluble fullerene derivative, and their protection by sulfhydryl compounds were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ at a concentration of 50 μM caused time (0 to 3 h)‐dependent cell death accompanied by the formation of cell surface blebs, the loss of cellular levels of ATP and reduced glutathione, accumulation of glutathione disulfide, and induction of DNA fragmentation assayed using alkali single‐cell agarose‐gel electrophoresis. C₆₀(OH)₂₄‐induced cytotoxicity was effectively prevented by pretreatment with sulfhydryl compounds. N‐acetyl‐L‐cysteine (NAC), L‐cysteine and L‐methionine, at a concentration of 2.5 mM, ameliorated cell death, accompanied by a decrease in cellular ATP levels, formation of cell surface blebs, induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential caused by C₆₀(OH)₂₄. In addition, DNA fragmentation caused by C₆₀(OH)₂₄ was also inhibited by NAC, whereas an antioxidant ascorbic acid did not affect C₆₀(OH)₂₄‐induced cell death and DNA damage in rat hepatocytes. Taken collectively, these results indicate that incubation of rat hepatocytes with C₆₀(OH)₂₄ elicits DNA damage, suggesting that nuclei as well as mitochondria are target sites of the hydroxylated fullerene; and induction of DNA damage and oxidative stress is ameliorated by an increase in cellular GSH levels, suggesting that the onset of toxic effects may be partially attributable to a thiol redox‐state imbalance caused by C₆₀(OH)₂₄. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of pH on nicotine-induced DNA damage and oxidative stress

Epidemiological evidence suggests that chewing betel quid and smoking have synergistic potential in the development of oral squamous-cell carcinoma in Taiwan. Chewing betel quid produces alkalization of saliva. This study investigated the response of human oral cancer OEC-M1 cells to nicotine in different pH environments (6.5 and 8) by examining its effects on DNA damage as evidenced by single-cell gel electrophoresis. Nicotine (1 and 10 muM) significantly induced DNA strand breakage when cultured at pH 8 for 6 h but did not induce DNA damage at pH 6.5. Nicotine-induced DNA damage was also time dependent. When cells were pretreated with catalase or N-acetylcysteine, a significant reduction in nicotine-induced DNA damage was observed. Flow cytometric analyses showed that the production of 8-oxoguanine was significantly increased following nicotine (10 muM) treatment. Posttreatment of nicotine-damaged DNA by endonuclease III and formamidopyrimidine-DNA glycosylase, recognizing oxidized DNA bases, increased the extent of DNA damage. These results suggest that nicotine-induced DNA strand breakage is pH dependent, and oxidative stress might be involved in nicotine-induced DNA damage. Finally, cigarette smoke condensate (equivalent to 8 muM nicotine) induced significant DNA strand breaks in OEC-M1 cells at pH 8 and correlated with the generation of oxidative DNA damage. Thus, alkaline saliva generated by chewing betel quid plays an important role in cigarette-related nicotine-induced DNA damage, and reactive oxygen species may be involved in generating this DNA damage.     

Effects of Epigallocatechin-3-gallate on lead-induced oxidative damage

Recent studies have shown that lead (Pb) could disrupt the prooxidant/antioxidant balance of tissue which leads to biochemical and physiological dysfunction. Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is found to be an effective antioxidant. The present study investigated whether EGCG administration could reverse the changes on redox states in rat hippocampus caused by lead exposure. The association between redox status changes and long-term potentiation (LTP) in CA1 area of hippocampus were also examined. Wistar rats exposed to lead from postnatal day 1 were followed by 10 days of EGCG (10, 25 and 50 mg/kg) administration through intraperitoneally (ip), and the rats were sacrificed for experiments at the age of 21–23 days. The experimental results showed that glutathione (GSH) and superoxide dismutase (SOD) activity decreased accompanied with LTP amplitude decrease in CA1 area of hippocampus in the lead-exposed group. EGCG supplementation following lead intoxication resulted in increases in the GSH and SOD levels and increases in the LTP amplitude. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following lead exposure and decreased following EGCG treatment. In hippocampal neuron culture model, lead exposure (20 μM) significantly inhibited the viability of neurons which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (ΔΨ_m). Treatment by EGCG (10–50 μM) effectively increased cell viability, decreased ROS formation and improved ΔΨ_m in hippocampal neurons exposed to lead. These observations suggest that EGCG is a potential complementary agent in the treatment of chronic lead intoxication through its antioxidative character.