Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes

Microcystins are a group of highly liver-specific toxins, although their exact mechanisms of action remain unclear. We examined the effects of microcystic cyanobacteria extract (MCE) collected from a contaminated water source on the organization of cellular microtubules (MTs) and microfilaments (MFs) in hepatocytes. We also investigated the effects on lactate dehydrogenase (LDH) leakage and intracellular glutathione (GSH). Primary cultured rat hepatocytes exposed to MCE (equivalent to 125 microg/mL lyophilized algae cells) showed a characteristic disruption of MTs and MFs in a time-dependent manner. Under these conditions, MCE caused aggregation of MTs and MFs and a severe loss of MTs in some cells. Moreover, MCE-induced cytoskeletal alterations preceded the LDH leakage. On the other hand, the treatment of cells with MCE led to a dose-dependent increase of intracellular GSH. However, time-course study showed a biphasic change of intracellular GSH levels with a significant increase in the initial stage followed by a decrease after prolonged treatment. Furthermore, pretreatment with N-acetylcystein (NAC), a GSH precursor, significantly enhanced the intracellular GSH level and decreased the MCE-induced cytotoxicity as well as cytoskeleton changes. In contrast, buthionine-(S, R)-sulfoximine, a specific GSH synthesis inhibitor, increased the cell susceptibility to MCE-induced cytotoxicity by depleting the intracellular GSH level. These findings suggest that intracellular GSH plays an important role in MCE-induced cytotoxicity and cytoskeleton changes in primary cultured rat hepatocytes. Increasing intracellular GSH levels protect cells from MCE-induced cytotoxicity and cytoskeleton changes.     

稽留流产绒毛中线粒体膜电位和活性氧的变化

目的:通过对稽留流产患者绒毛滋养叶细胞中线粒体膜电位(mitochondrial membrane potential MMP)、活性氧物质(reactive oxygen species ROS)的测定,探讨MMP、ROS与稽留流产的关系。方法:稽留流产患者20例作为研究组,正常早孕要求流产妇女20例作为对照组。研究组20例胚胎稽留宫内时间<4周。应用荧光分光光度计和流式细胞仪检测研究组及对照组绒毛滋养叶细胞中线粒体膜电位(MMP)、活性氧物质(ROS)的水平。结果:与正常早孕妇女相比较,稽留流产患者绒毛滋养叶细胞中MMP水平显著降低(P<0.05),ROS水平显著增高(P<0.05)。结论:绒毛滋养叶细胞中线粒体膜电位下降、活性氧物质增多可能是稽留流产发生的信号。     

Folate deprivation and copper exposure potentiate reactive oxygen species generation and chromosomal DNA loss but not mitochondrial DNA deletions in rat hepatocytes

Both increased copper and reduced folate levels are commonly found in patients with liver diseases. To better understand the mechanisms by which folate deprivation interacts with copper to contribute to hepatocellular toxicity, rat primary hepatocytes were isolated, cultured in folate-deprived (FD) RPMI medium, and assayed for cytotoxicity after copper sulfate (CuSO4) exposure. MTT measurement and trypan blue assay showed that elevated CuSO4 levels aggravated cell death of folate-deprived but not folate-sufficient hepatocytes. CuSO4 treatment increased the levels of intracellular reactive oxygen species (ROS) by 3 times in FD hepatocytes and tripled the proportion of FD hepatocytes with hypodiploid DNA contents. Measurement of membrane phosphatidylserine exposure indicated that the CuSO4-mediated toxicity in FD hepatocytes was not mediated by the apoptotic pathway. Real-time polymerase chain reaction (PCR) analysis revealed that CuSO4 treatment did not increase the occurrence of a 4834-bp mtDNA (mtDNA4834) deletion in FD hepatocytes. Preincubation of FD hepatocytes with various concentrations of folate prior to CuSO4 treatment did not modulate the mtDNA4834 deletion. Taken together, the data suggest that elevated copper levels potentiate cell death of folate-deprived hepatocytes, which is primarily associated with increased ROS generation and chromosomal DNA loss. The cytotoxicity exerted by folate depletion and elevated copper levels, however, is not due to apoptosis or accumulated mtDNA4834 deletions in primary hepatocytes.     

Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes.

Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation.     

丹那唑抑制大鼠线粒体呼吸链复合酶I 导致原代肝细胞坏死

摘要:目的　研究丹那唑对大鼠原代肝细胞的损伤作用与机制。方法　原代大鼠肝细胞贴壁培养4 h,加入丹那唑0,50,100,200 μmol/L,4 h和24 h后采用乳酸脱氢酶法测定细胞活力并检测三磷酸腺苷（ATP）水平,同时提取线粒体,分别测5个呼吸链复合酶（complex I～V）活性。培养液中加入20 mmol/L果糖促进糖酵解,观察果糖对丹那唑细胞毒性的影响。结果　丹那唑50,100 μmol/L处理4 h和24 h对细胞几乎没有杀伤作用,但可致ATP显著降低;200 μmol/L处理4 h和24 h均可导致细胞坏死。果糖可对抗丹那唑4 h具杀伤作用,但对24 h杀伤作用无效。丹那唑（50～200 μmol/L）显著抑制线粒体呼吸链复合酶I活性,但不影响complex Ⅱ～V活性。结论　丹那唑选择性抑制线粒体呼吸链复合酶I活性而导致ATP生成减少并诱发细胞坏死。     

氟化钠对原代培养大鼠肝细胞的毒性作用

目的　探讨氟化钠 (NaF)对原代培养大鼠肝细胞的毒性作用。方法　采用原代培养的方法 ,观察氟化钠对原代培养大鼠肝细胞存活率和肝细胞培养液中谷草转氨酶 (AST)和谷丙转氨酶 (ALT)活性的影响。结果　氟化钠可使原代培养大鼠肝细胞存活率下降 ,且呈现明显的剂量 -效应关系 ,其IC50 为 3 5 8mmol/L ;肝细胞培养液中ALT和AST的活性随染毒剂量的增加而逐渐升高 ,2和 4mmol/L染氟组肝细胞培养液中ALT和AST的活性与对照组相比显著升高 (P <0 0 5 )。结论　氟化钠对原代培养大鼠肝细胞有明显的毒作用。     

A对大鼠海马细胞钙浓度和线粒体膜电位影响

目的 探讨β-淀粉样蛋白(Aβ_25~35)对原代培养大鼠脑海马细胞内游离钙离子浓度和线粒体膜电位影响。方法 原代培养8 d的SD大鼠脑海马神经细胞,利用老化的Aβ_25~35 1、10和20 μmol/L对其染毒,分别观察染毒后24、48和72 h时海马细胞形态改变、细胞内游离钙离子浓度和线粒体膜电位。结果 随着染毒剂量增加,海马神经细胞开始变形、萎缩、神经元胞体模糊,突起变细、变短、分支减少,边缘模糊,神经网络破裂中断;电镜结果显示,随着染毒剂量增加和染毒时间延长,海马细胞凋亡比例增加;20 μmol/L Aβ_25~35染毒24、48和72 h后,细胞内Ca²⁺浓度分别为(30.79±1.28)、(38.19±2.13)和(41.65±3.60),细胞内线粒体膜电位分别为(46.94±9.55)、(39.98±6.51)和(34.52±5.67),与对照组比较,Ca²⁺浓度均升高,膜电位均降低(P＜0.01)。结论 Aβ_25~35可以通过破坏细胞内钙稳态和线粒体膜电位而发挥神经毒性作用。     

The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells

Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.     

Stimulation of NADPH-dependent reactive oxygen species formation and DNA damage by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat peritoneal lavage cells

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] and its congeners involves binding to a specific TCDD [Ah] receptor, interaction of this complex with chromatin, and the ultimate production of pleiotropic responses. The mechanism whereby these effects are produced following interaction of TCDD with the receptor complex is not known. Oxidative stress following the production of reactive oxygen species (ROS) may play an important role in the toxic manifestations of TCDD. Thus, the dose and time-dependent effects of TCDD on the production of superoxide anion by peritoneal lavage cells (primarily macrophages) from rats were examined. A maximum increase in superoxide anion production occurred on day 1 after treatment in rats with 50 and 125 g TCDD/kg. At 6 h after a single dose of 125 g TCDD/kg, a 2.4-fold increase in superoxide anion production was observed in peritoneal lavage cells from rats. A single dose of 5 g TCDD/kg had no effect on superoxide anion production by peritoneal lavage cells. A significant increase in DNA single strand breaks within peritoneal lavage cells occurred at 12 h after the oral administration of 50 g TCDD/kg, and a maximum increase in DNA single strand breaks was observed on days 3–5 after treatment. No DNA damage was detected at a dose of 5 g TCDD/kg. No difference was observed with respect to dose and time in the composition of the peritoneal lavage cells. The results clearly indicate that the oral administration of TCDD activates peritoneal lavage cells in rats, and that the activation precedes the formation of DNA single strand breaks. The results support the hypothesis that the tissue damage by TCDD may be due, at least in part, to the formation of reactive oxygen species, and macrophages may serve as one source of reactive oxygen species in response to TCDD.     

氟化物对原代培养大鼠肝细胞酶活力及超微结构的影响

目的研究不同剂量的氟化物对原代培养大鼠肝细胞存活率、酶活力及超微结构的影响。方法采用半原位胶原酶消化法分离大鼠肝细胞;MTT法检测细胞存活率;赖氏法检测培养液中谷草转氨酶(AST)和谷丙转氨酶(ALT)活性;透射电镜观察细胞超微结构改变。结果氟化钠染毒24小时后肝细胞存活率下降,且呈现明显的剂量-效应关系;2mmolL和4mmolL染氟组肝细胞培养液中ALT和AST的活性显著升高(P<005);透射电镜下染氟组肝细胞线粒体肿胀,内质网排列紊乱或断裂。结论过量氟化物对原代培养大鼠肝细胞有明显的毒作用,其主要作用方式是引起细胞膜和细胞器质膜损伤。     

新生SD大鼠缺氧缺血性脑损伤早期线粒体内膜跨膜电势的变化

【目的】　了解缺氧缺血性脑损伤 (HIBD)早期脑细胞△Ψm的变化。　【方法】　 7日龄新生大鼠随机分为正常对照组 (n =6) ,HIBD组 (n =3 6,分为HIBD后 0、1、2、3、4h时间点组 )。予以右颈总动脉分离结扎后再置8%O2 低氧舱 2 .5h ;断头处死后分离左右大脑半球制成单细胞悬液 ,加入终浓度为 1μmol的罗丹明 12 3于 3 7℃避光孵育 45min后 ,以流式细胞仪测定△Ψm并计算左右大脑半球△Ψm比值。　【结果】　正常P7SD大鼠左、右侧大脑半球脑细胞△Ψm分别为 ( 18.2 1± 1.2 6)MFL和 ( 18.93± 0 .74)MFL ,右∶左比值为 1.0 6。HIBD损伤使双侧大脑半球脑细胞△Ψm降低程度更明显 ,其中HIBD后 0h时△Ψm右∶左比值比正常时降低了 2 4.5 %(P <0 .0 5 )。HIBD后 1～ 3h时间内 ,△Ψm稍有回升。HIBD后 4h时HI损伤侧△Ψm值及右∶左比值均出现第二次降低现象 ,且程度更重 ,其中右 :左比值比正常时降低了 3 4%(P <0 .0 5 )。　【结论】　HIBD早期脑细胞△Ψm出现两次降低 (初次及二次降低 ) ,分别发生在HI损伤 0h和 4h时 ,提示有关改善线粒体功能的治疗尽量在△Ψm出现第二次降低前应用     

Magnesium inhibits nickel-induced genotoxicity and formation of reactive oxygen.

Nickel compounds are recognized to cause nasal and lung cancers. Magnesium is an effective protector against nickel-induced carcinogenesis in vivo, although its mechanisms of protection remain elusive. The effects of magnesium carbonate on the cytotoxicity and genotoxicity induced by nickel subsulfide were examined with respect to the inhibition of cell proliferation, micronuclei formation, DNA-protein cross-link formation, and intranuclear nickel concentration. The generation of reactive oxygen by nickel chloride was also analyzed by observing 8-hydroxy-deoxyguanosine formation from deoxyguanosine in the presence and absence of magnesium chloride. The suppression of up to 64% of the proliferation of BALB/3T3 fibroblasts by nickel subsulfide (1 microgram/ml) was reversed by magnesium. The nickel compound increased not only the number of micronuclei but also the amount of DNA-protein cross-links examined with CHO and BALB/3T3 cells, respectively. These genotoxic effects of nickel were again lessened by magnesium carbonate. In addition, the cellular accumulation of nickel increased 80-fold with nickel subsulfide treatment and decreased with magnesium carbonate treatment. Nickel also enhanced 8-hydroxy-deoxyguanosine formation in the presence of H2O2 and ascorbic acid, where magnesium played another suppressive role. In fact, inhibition by magnesium was still observed even in the absence of nickel treatment. These results suggest that the protective role of magnesium in nickel-induced cytotoxicity and genotoxicity can be attributed to its ability to reduce either the intracellular nickel concentration or reactive oxygen formation.     

汞对大鼠肾皮质线粒体ATP酶和膜电位的影响

目的 通过线粒体孵育液染汞并用N-乙酰半胱氨酸(NAC)干预研究汞对大鼠肾皮质线粒体损伤的影响.方法 提取Wistar大鼠肾皮质线粒体液并分设为阴性对照组,5、50、500 μmol/L氯化汞(HgCl2)直接染毒组及线粒体液NAC预处理后50 μmol/L HgCl2染毒组5组.各组于培养箱内37℃孵育1 h后,测定Na+-K+-三磷酸腺苷(ATP)酶活力、Ca2+-ATP酶活力、线粒体膜电位及孵育液中细胞色素C含量.结果 与阴性对照组比较,50、500 μmol/L HgCl2染毒组Na+-K+-ATP酶、Ca2+-ATP酶活力及线粒体膜电位显著降低(P<0.05),细胞色素C含量显著升高(P<0.05);与50 μmol/L HgCl2染毒组比较,NAC预处理后Na+-K+-ATP酶活力及线粒体膜电位显著升高,细胞色素C含量显著降低(P<0.05).结论 汞可以剂量依赖性地诱导大鼠肾皮质线粒体损伤,NAC预处理在某种程度上能有效地预防汞所致大鼠肾皮质线粒体损伤.     

Effects of active oxygen species on damage to and prostaglandin synthesis in cultured rat gastric cells.

Active oxygen species cause gastric mucosal damage in vivo. However, it is not known if these species are directly cytotoxic toward gastric cells. Prostaglandins have important physiological roles in the gastric mucosa, including direct cell protection against damaging factors. So, to find if active oxygen species affect prostaglandin synthesis in gastric mucosal cells is important, but this also is not known. This study was done to investigate the effects of such species on damage to and prostaglandin synthesis in cultured mucus-producing cells from rat gastric mucosa. Active oxygen species were produced by the addition of xanthine and xanthine oxidase to the culture medium. Cytotoxicity was assayed by 51Cr release. Xanthine (1 mM) and xanthine oxidase (100 mU/ml) increased specific 51Cr release as the thiobarbituric acid reactants increased. This increase in 51Cr release was inhibited by catalase, a scavenger of hydrogen peroxide, or dimethyl sulfoxide, a scavenger of hydroxyl radicals, but not by superoxide dismutase, a scavenger of superoxide, nor deferoxamine, an inhibitor of hydroxyl radical generation. Catalase, dimethyl sulfoxide, and superoxide dismutase each had no effect on prostaglandin E2 synthesis when xanthine and xanthine oxidase were not added. In the presence of xanthine and xanthine oxidase, catalase and dimethyl sulfoxide stimulated the synthesis of prostaglandin E2 and superoxide dismutase inhibited it. Indomethacin, a prostaglandin synthetase inhibitor, did not affect the decrease in 51Cr release caused by catalase in the presence of xanthine and xanthine oxidase, but it abolished the decrease caused by dimethyl sulfoxide. These results suggest that hydrogen peroxide, but not superoxide nor hydroxyl radicals, is involved in damage to cultured rat gastric cells, and that superoxide stimulates prostaglandin E2 synthesis, but that hydrogen peroxide inhibits it. Protection of the cells by dimethyl sulfoxide may be related to stimulation of prostaglandin E2 synthesis in the cells, but not via scavenging hydroxyl radicals.     

Formation of copper oxychloride and reactive oxygen species as causes of uterine injury during copper oxidation of Cu-IUD

Magainins are antimicrobial peptides with known spermicidal activity. Their activity is inhibited by cholesterol present in eukaryotic cell membranes. Pretreatment of spermatozoa with methyl-beta-cyclodextrin, which extracts cholesterol from cell membranes and induces capacitation, sensitizes them to magainin-2-amide as shown by a decrease in human sperm motility determined by computer-assisted sperm analysis and a concomitant decrease in sperm viability, as measured by MitoTracker(R) Red CMXRos labeling. Magainin-2-amide affects mainly the fast progressive spermatozoa inducing them directly into an immotile state, without an increase in motile non-progressive and slow progressive spermatozoa. We conclude that methyl-beta-cyclodextrin highly potentiates the deleterious effect of magainin-2-amide on human spermatozoa. Most probably, this effect can be explained by cholesterol extraction from sperm cell membranes.     

Effects of sesamin and curcumin on delta 5-desaturation and chain elongation of polyunsaturated fatty acid metabolism in primary cultured rat hepatocytes.

Effects of sesamin and curcumin on delta 5-desaturation and chain elongation of polyunsaturated fatty acid (PUFA) were studied in rat primary cultured hepatocytes. When sesamin was added to culture medium containing 20:4 (n-3), rat hepatocytes after 24 h of incubation produced 20:5 (n-3) from 20:4 (n-3), whereas when incubated with 20:3 (n-6), the metabolite by delta 5-desaturation did not accumulate, and consequently, the ratio of 20:3 (n-6)/20:4 (n-6) increased with the amount of sesamin added. Curcumin was more effective than sesamin in this respect. Both sesamin and curcumin interfered with chain elongation of PUFAs. An addition of 18:3 (n-6) or 18:4 (n-3) increased the cellular concentrations of 20:3 (n-6) or 20:4 (n-3), respectively, but the simultaneous addition of sesamin or curcumin inhibited the chain elongation of C18 acids (the fatty acids with 18 carbons) into corresponding C20 and C18 acids. Similarly, the elongation from C20 of n-3 and n-6 families to C22 was also inhibited with sesamin and curcumin. These results suggested that: 1) sesamin and curcumin inhibited delta 5-desaturation of n-6 fatty acid, but not n-3 fatty acid in rat hepatocytes; 2) curcumin was more effective than sesamin; 3) chain elongation was also inhibited by sesamin and curcumin.     

Lycopene I--effect on osteoclasts: lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in rat bone marrow cultures

Osteoclasts have been shown to produce reactive oxygen species (ROS) that can stimulate bone resorption. We explored the hypothesis that lycopene, the antioxidant carotenoid from tomatoes, can inhibit mineral resorption by inhibiting osteoclast formation and the production of ROS. Cells from bone marrow prepared from rat femur were plated into 16-well calcium phosphate-coated Osteologic Multi-test Slides and cultured in alpha-minimal essential medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid. The cells were treated with varying doses of lycopene in the absence or presence of parathyroid hormone (PTH) at the start of culture and at each medium change (i.e., every 48 hours). On day 8, mineral resorption pits were quantitated. Similar, parallel experiments were carried out in 12-well plastic dishes to assess tartrate-resistant acid phosphatase (TRAP) activity. Results showed that lycopene inhibited TRAP + formation of multinucleated cells in both vehicle- and PTH-treated cultures. Osteoclasts reduced nitroblue tetrazolium (NBT) to purple-colored formazan, indicating the presence of ROS in these cells. The formazan-staining cells were decreased by treatment with 10(-5) M lycopene, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibits basal and PTH-stimulated osteoclastic mineral resorption and formation of TRAP + multinucleated osteoclasts, as well as the ROS produced by osteoclasts. These findings are novel and may be important in the pathogenesis, treatment, and prevention of osteoporosis.     

Glutamine attenuates nitric oxide synthase expression and mitochondria membrane potential decrease in interleukin-1β-activated rat hepatocytes

Background  

Mitochondrial dysfunction induced by nitric oxide (NO) overproduction is involved in the pathogenesis of organ failure during many severe diseases. Recently, several experiments have reported that glutamine (Gln) modifies inducible nitric oxide synthase (iNOS) gene expression which is thought to be responsible for its beneficial effects in critical illnesses.     

Contrasting reactive oxygen species and transition metal concentrations in combustion aerosols

The presence of reactive oxygen species (ROS) and 10 transition metals (Ag, Cd, Co, Cu, Fe, Mn, Ni, Ti, V and Zn) in both the acid-soluble and water-soluble fractions of fine particles of combustion origin were determined. ROS was analyzed using the dichlorofluorescin fluorescence technique. Particles emitted from on-road vehicles, gas cooking, incense burning, and cigarette smoke were characterized along with those in the background air of outdoor and indoor environments. In addition, this study evaluated the possible relationships between ROS and individual transition metals. It is found that cigarette smoke which had the highest concentration of metals also contained the highest concentration of ROS. Regression analysis performed showed that water-soluble metals including Cd, Co, Cu, Fe, Mn, and Ni showed better correlation with ROS concentration as compared to acid-soluble (total) metals. The findings demonstrated that water-soluble metals could be one of the species influencing ROS formation in ambient air.