Subchronic microcystin-LR exposure increased hepatic apoptosis and induced compensatory mechanisms in mice

Acute lethal cytotoxicity of microcystin-LR (MC-LR), a toxin produced by fresh-water cyanobacteria, has been attributed to protein phosphatases type 1 and type 2A (PP1/PP2A) inhibition and reactive oxygen species (ROS) generation. However, the effects and molecular mechanisms of prolonged, sublethal MC-LR exposure are less known. We studied mice intraperitonealy injected with saline or 25 μg MC-LR/kg for 28 days (every 2 days). MC-LR induced apoptosis in liver and not in kidneys or heart of treated animals. Liver also showed decreased α-tubulin levels (45.56% ± 7.65% of controls) and activation of p38-MAPK and CaMKII pathways (137.93% ± 11.64% and 419.35% ± 67.83% of the control group, respectively). PP1/PP2A activity decreased from 1.82 ± 0.23 (controls) to 0.91 ± 0.98 mU/mg (MC-LR-treated mice); however, no difference in total Ser/Thr phosphatase activity was found between both the groups. The results demonstrated that apoptosis and cytoskeleton disruption contributed to the hepatic cytotoxic effects of subchronic MC-LR administration. These effects occurred in association with sustained activation of signaling cascades and development of compensatory mechanisms to maintain total Ser/Thr phosphatase activity.     

Bioaccumulation, oxidative stress and HSP70 expression in Cyprinus carpio L. exposed to microcystin-LR under laboratory conditions

Microcystin-LR (MC-LR) produced by cyanobacteria are potent specific hepatotoxins. So far the pathogenesis of environmental MC-LR toxicity to aquatic organisms has not been fully elucidated. In the present study the accumulation of MC-LR was investigated in various organs/tissues of Cyprinus carpio L. (C. carpio) following exposure to MC-LR for 14 d at environmentally relevant concentrations (0.1 to 10 μg L(-1)). Results showed that the presence of MC-LR enhanced toxin accumulation in all investigated organs and the highest accumulation was found in the liver of fish exposed to 5.0 μg L(-1) of MC-LR. An EPR analysis indicated ·OH intensity in liver was significantly induced at 0.1 μg L(-1) of MC-LR and then restored when the MC-LR concentration was greater than 0.1 μg L(-1). After 14-day exposure, MC-LR (1.0-10.0 μg L(-1) of MC-LR) caused a pronounced promotion of glutathione S-transferase (GST) activity and a depletion of reduced glutathione (GSH) content in fish liver, which indicated that GSH was involved in detoxification of MC-LR and the conjugation reaction of MC-LR and GSH occurred. A mild oxidative damage was evidenced by the accumulation of malondialdehyde (MDA) level at 5.0 μg L(-1) of MC-LR exposure, but which was restored when the MC-LR concentration was increased to 10.0 μg L(-1). The responses of antioxidant enzymes and the induction of HSP70 expression might contribute to MC-LR tolerance of C. carpio. However, the protein phosphatase (PP) activities were strikingly inhibited in all treated groups. Thus, the overall toxicity of environmental MC-LR on C. carpio seems to be initiated in the liver via both the ROS pathway and the PP inhibition pathway, and the latter might be more important when ambient MC-LR concentration is greater than 0.1 μg L(-1). More importantly, these results can help to support the evaluation on the potential effects of MC-LR under common environmental concentrations.     

Involvement of JNK regulation in oxidative stress-mediated murine liver injury by microcystin-LR

Microcystin-LR (MC-LR) produced by cyanobacteria in diverse water systems is a potent specific hepatotoxin and has been documented to induce various liver diseases via oxidative stress. However, the underlying mechanisms are largely unknown. In the current study, we investigated the molecular events involved in the oxidative liver injury by MC-LR. Our results demonstrated that MC-LR induced liver injury in mice through a series of steps that began with the production of reactive oxygen species (ROS), which stimulated the sustained activation of JNK and its downstream targets, AP-1 and Bid. Furthermore, the mitochondrial proteomic analysis indicated that JNK activation affected some crucial enzymes of energy metabolism, led to mitochondria dysfunction, which contributed to hepatocyte apoptosis and oxidative liver injury by MC-LR. Our results reveal significant insights into the mechanisms of liver injury induced by microcystins, and serve as a framework for deciphering the role of JNK in oxidative stress-associated liver diseases. Yinna Wei and Dan Weng equally contributed to this work. D. Owen Young—An international exchange student from USA.     

微囊藻毒素与细菌内毒素对草鱼淋巴细胞凋亡的联合毒效应

采用体外暴露方式,以草鱼淋巴细胞为试验对象,研究了水体中微囊藻毒素(MC-LR)与细菌内毒素(LPS)复合作用对鱼免疫系统的影响.结果表明： MC-LR和LPS在单一与复合暴露下都能够诱导草鱼淋巴细胞发生凋亡,呈现细胞凋亡典型的阶梯状DNA电泳特征.但对比复合暴露与单一暴露的凋亡率可以发现,MC-LR与LPS复合暴露会发生协同作用,并呈显著剂量-效应关系,其中复合暴露Ⅳ组凋亡率为单一暴露Ⅰ(MC-LR)和Ⅱ(LPS)组的2-1和3.3倍.MC-LR可协同LPS抑制谷胱甘肽硫转移酶(GST)活性,引发细胞内活性氧(ROS)和丙二醛(MDA)水平上升,导致DNA损伤,致使淋巴细胞阻滞于G0期,增殖受到明显抑制,加速鱼淋巴细胞的凋亡,提高细胞凋亡率.表明MC-LR能够协同LPS加剧其对鱼体免疫细胞毒性,对水产养殖业产生严重不利影响.     

Protein phosphatase 5 protects neurons against amyloid-β toxicity

Amyloid-β (Aβ) is thought to promote neuronal cell loss in Alzheimer's disease, in part through the generation of reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Protein phosphatase 5 (PP5) is a ubiquitously expressed serine/threonine phosphatase which has been implicated in several cell stress response pathways and shown to inactivate MAPK pathways through key dephosphorylation events. Therefore, we examined whether PP5 protects dissociated embryonic rat cortical neurons in vitro from cell death evoked by Aβ. As predicted, neurons in which PP5 expression was decreased by small-interfering RNA treatment were more susceptible to Aβ toxicity. In contrast, over-expression of PP5, but not the inactive mutant, PP5(H304Q), prevented MAPK phosphorylation and neurotoxicity induced by Aβ. PP5 also prevented cell death caused by direct treatment with H₂O₂, but did not prevent Aβ-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Aβ-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Aβ and oxidative stress. Consequently, PP5 might be an effective therapeutic target in Alzheimer's disease and other neurodegenerative disorders in which oxidative stress is implicated.     

In vivo DNA damage induced by the cyanotoxin microcystin-LR: comparison of intra-peritoneal and oral administrations by use of the comet assay

Microcystin-LR (MC-LR), involved in human and animal poisonings by cyanobacteria, has been shown to be both a potent tumour promoter in rat liver and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of MC-LR counts only few in vivo studies. In order to determine the target organs for DNA-damage induction by MC-LR, the single-cell gel electrophoresis (SCGE) or comet assay was performed in mice. Following a single oral administration of 2 and 4mg/kg bw of MC-LR, a statistically significant induction of DNA damage in blood cells was obtained after 3h. However, after an intra-peritoneal injection (ip), DNA lesions were mainly induced in the liver, but were also reported in the kidney, the intestine and the colon. The sensitivity of the ip route compared to the oral route suggested a difference in the bio-disponibility of the toxin. In any case, DNA damage was induced by MC-LR irrespective of the administration route. Among the target organs, the DNA damage induced in the intestinal tissues (ileum and colon) may contribute to an increased cancer risk.     

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.     

Metformin in contrast to berberine reversed arsenic‐induced oxidative stress in mitochondria from rat pancreas probably via Sirt3‐dependent pathway

Exposure to arsenic has been linked to the development of type 2 diabetes though its mechanism of toxicity remains unresolved. In this study berberine (BBR) effects on arsenic‐induced sirtuin 3 (Sirt3) modifications in isolated mitochondria from rat pancreas were evaluated and compared with metformin (MET). With arsenic, mitochondrial reactive oxygen species (ROS), oxidative stress, and insulin resistance were obtained higher than control. From our results and in the presence of arsenic trioxide, insulin resistance and Sirt3 levels were found to be predominantly elevated that could be the result of compensating mechanisms. Apparently, BBR and MET recruit both direct (as an antioxidant) and indirect mechanisms (Sirt3 content) to deal with arsenic trioxide toxicity. Metformin compared with BBR exhibited a less significant effect on ROS levels and since its direct antioxidant property is minor, depressed the ROS level mainly through the Sirt3 modification.     

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H-R)

Hypoxia and hypoxia-reoxygenation (H-R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H-R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H-R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies.     

Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.     

Distinct mechanisms account for β-amyloid toxicity in PC12 and differentiated PC12 neuronal cells

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph(+) and Ph(-) ALL cell lines and patient samples with IC 50 values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph⁺ and Ph^- ALL cell lines and patient samples with IC₅₀ values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

草鱼TP53INP1基因的克隆及微囊藻毒素对其表达的影响

为深入研究肿瘤蛋白p53诱导核蛋白1(Tumor protein 53-induced nuclear protein 1, TP53INP1)的结构及其在微囊藻毒素-LR(MC-LR)胁迫下的表达变化,以MC-LR诱导的草鱼(Ctenopharygodon idella)肝脏转录组测序获得的unigenes序列为基础,扩增获得了TP53INP1基因的cDNA序列(GenBank登录号:MG797689),其中开放阅读框(Open reading frame, ORF)为759 bp,编码252个氨基酸,属于β类型,具有4个PEST结构。氨基酸同源性分析结果表明, TP53INP1具有较高的保守性,其中与鱇浪白鱼(Anabarilius grahami)相似性最高。系统进化分析结果表明其与鱇浪白鱼(Anabarilius grahami)、斑马鱼(Danio rerio)等鱼类聚为一大支。采用荧光定量PCR分析发现TP53INP1基因在草鱼各组织中广泛分布,其中,在肝脏和血液等组织中表达丰富,显著高于在头肾组织中的表达(P<0.05)。采用Western blot检测分析不同剂量...     

Intraperitoneal gardiquimod protects against hepatotoxicity through inhibition of oxidative stress and inflammation in mice with sepsis

Many reports recapitulate the contribution of reactive oxygen species (ROS) over‐accumulation to the organ damage; it is of significance to strictly target ROS production. In this study, we evaluated the potential role of TLR7 agonist gardiquimod (GDQ) in oxidative stress (OS) in liver injury induced by sepsis. Here, we observed that intraperitoneal pretreatment with GDQ dramatically elevated the septic survival rate and effectively attenuated the septic liver injury. Interestingly, the increased ROS and inflammatory factor IL‐6 levels were reversed after GDQ intervention. Subsequently, Western blot was employed to determine the definite mechanism. As expected, it was showed that the upregulation of c‐Jun N‐terminal kinase (JNK)/c‐Jun pathway in liver of septic animals was considerably suppressed by GDQ pre‐exposure. Our current result highlight that pre‐administration of GDQ ameliorated sepsis induced hepatotoxicity and reduced the generation of IL‐6 and OS responses, which was associated with downregulation of JNK/c‐Jun pathway. Our strategies might be ultimately beneficial in mitigating liver injury symptom.     

Depletion of S-adenosyl-l-methionine with cycloleucine potentiates cytochrome P450 2E1 toxicity in primary rat hepatocytes

S-Adenosyl-l-methionine (SAM) is the principal biological methyl donor. Methionine adenosyltransferase (MAT) catalyzes the only reaction that generates SAM. Hepatocytes were treated with cycloleucine, an inhibitor of MAT, to evaluate whether hepatocytes enriched in cytochrome P450 2E1 (CYP2E1) were more sensitive to a decline in SAM. Cycloleucine decreased SAM and glutathione (GSH) levels and induced cytotoxicity in hepatocytes from pyrazole-treated rats (with an increased content of CYP2E1) to a greater extent as compared to hepatocytes from saline-treated rats. Apoptosis caused by cycloleucine in pyrazole hepatocytes appeared earlier and was more pronounced than control hepatocytes and could be prevented by incubation with SAM, glutathione reduced ethyl ester and antioxidants. The cytotoxicity was prevented by treating rats with chlormethiazole, a specific inhibitor of CYP2E1. Cycloleucine induced greater production of reactive oxygen species (ROS) in pyrazole hepatocytes than in control hepatocytes, and treatment with SAM, Trolox, and chlormethiazole lowered ROS formation. In conclusion, lowering of hepatic SAM levels produced greater toxicity and apoptosis in hepatocytes enriched in CYP2E1. This is due to elevated ROS production by CYP2E1 coupled to lower levels of hepatoprotective SAM and GSH. We speculate that such interactions e.g. induction of CYP2E1, decline in SAM and GSH may contribute to alcohol liver toxicity.     

草鱼自噬相关基因Beclin1的克隆及其在MC-LR胁迫下的表达特征

为评估微囊藻毒素-LR (MC-LR)对鱼类自噬的影响, 根据转录组测序结果得到的EST序列, 采用RACE技术获得了草鱼(Ctenopharygodon idella)自噬基因Beclin1 (CiBeclin1)的cDNA全长序列。该基因序列全长1590 bp, 包括1344 bp的开放阅读框, 编码447个氨基酸, 分子量为51.2 kD, 理论等电点为4.88。CiBeclin1包含1个BH3和1个ECD结构域。CiBeclin1氨基酸序列与其他物种的相似性为88%—98%, 构建的进化树显示CiBeclin1与稀有鲫(Gobiocypris rarus)的Beclin1亲缘关系最近。实时荧光定量PCR (qRT-PCR)检测结果表明, CiBeclin1在肝、肾、脾等10种不同组织中均广泛表达, 但在肝脏组织中的相对表达量最丰富, 显著高于相对表达量最低的头肾组织(P<0.05)。在不同剂量(25和100 μg MC-LR/kg BW) MC-LR胁迫24h、48h、72h和96h后, 草鱼肝脏中CiBeclin1表达量均显著低于对照组的表达水平, 研究结果表明, MC-LR能抑制草鱼CiBeclin1的表达, 但MC-LR是否在该剂量下诱导了草鱼的自噬还有待于进一步研究。     

Protective effects of green tea polyphenols on human HepG2 cells against oxidative damage of fenofibrate

The aim of this work was to investigate the protective effects of green tea polyphenols on the cytotoxic effects of hypolipidemic agent fenofibrate (FF), a peroxisome proliferator (PP), in human HepG2 cells. The results showed that high concentrations of FF induced human HepG2 cell death through a mechanism involving an increase of reactive oxygen species (ROS) and intracellular reduced glutathione (GSH) depletion. These effects were partially prevented by antioxidant green tea polyphenols. The elevated expression of PP-activated receptors alpha (PPARalpha) in HepG2 cells induced by FF was also decreased by treatment with green tea polyphenols. In conclusion, this result demonstrates that oxidative stress and PPARalpha are involved in FF cytotoxicity and green tea polyphenols have a protective effect against FF-induced cellular injury. It may be beneficial for the hyperlipidemic patients who were administered the hypolipidemic drug fenofibrate to drink tea or use green tea polyphenols synchronously during their treatment.