Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes

The influence of oxidative stress by hydrogen peroxide (H2O2) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30 min in saline A containing H2O2. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H2O2. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addition of antioxidants (N-acetylcysteine, L-ascorbic acid), a metal-chelator (1,10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H2O2-induced cell viability reduction suggested that oxidative stress by H2O2 itself or H2O2-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.     

过氧化氢对原代培养大鼠肝细胞的毒性作用及其机理

本文报道了过氧化氢诱发原代培养大鼠肝细胞毒性作用的可能机理．过氧化氢（０．２～１．０ｍｍｏｌ·Ｌ－１）温育６ｈ可以引起大鼠肝细胞坏死性损伤，导致谷丙转氨酶释放增加及细胞存活率下降，加入过氧化氢酶（２５０～１５００Ｕ·ｍＬ－１）及抗氧化剂五味子乙素（１０～１００μｍｏｌ·Ｌ－１）均可降低过氧化氢的毒性作用．加入过氧化氢（０．６和１．０ｍｍｏｌ·Ｌ－１）可在６ｍｉｎ内使大鼠肝细胞内钙从１８０ｎｍｏｌ·Ｌ－１明显持续升高至７００ｎｍｏｌ·Ｌ－１以上（约３．５倍）．过氧化氢与肝细胞作用３０ｍｉｎ至１ｈ既可导致细胞膜脂质过氧化，表现为丙二醛蓄积及膜流动性下降，明显早于肝细胞发生坏死性损伤的时间．肝细胞胞浆中还原型谷胱甘肽（ＧＳＨ）含量在加入过氧化氢３０ｍｉｎ后明显降低，可推测肝细胞在后面的温育中对过氧化氢毒性的敏感性增加．以上结果证实过氧化氢诱发的原代培养大鼠肝细胞致死性损伤可能与细胞内钙迅速持续增高，细胞膜脂质过氧化及ＧＳＨ含量下降有关．     

High glucose induces cell death of cultured human aortic smooth muscle cells through the formation of hydrogen peroxide

Alterations of the vessel structure, which is mainly determined by smooth muscle cells through cell growth and/or cell death mechanisms, are characteristic of diabetes complications. We analysed the influence of high glucose (22 mM) on cultured human aortic smooth muscle cell growth and death, as hyperglycaemia is considered one of the main factors involved in diabetic vasculopathy. Growth curves were performed over 96 h in medium containing 0.5% foetal calf serum. Cell number increased by 2 - 4 fold over the culture period in the presence of 5.5 mM (low) glucose, while a 20% reduction in final cell number was observed with high glucose. Under serum-free conditions, cell number remained constant in low glucose cultures, but a 40% decrease was observed in high glucose cultures, suggesting that high glucose may induce increased cell death rather than reduced proliferation. Reduced final cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum. The possible participation of oxidative stress was investigated by co-incubating high glucose with different reactive oxygen species scavengers. Only catalase reversed the effect of high glucose. Intracellular H(2)O(2) content, visualized with 2',7'-dichlorofluorescein and quantified by flow cytometry, was increased after high glucose treatment. To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quantified. No differences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures. In conclusion, high glucose promotes necrotic cell death through H(2)O(2) formation, which may participate in the development of diabetic vasculopathy.     

Protein thiol depletion and the killing of cultured hepatocytes by hydrogen peroxide

The H2O2 generated by menadione kills cultured hepatocytes by a mechanism that depends in large part on a cellular source of ferric iron. Chelation of this iron by deferoxamine reduced by two-thirds the number of dead cells without any effect on the loss of 30% of total protein thiols, the formation of protein mixed disulfides, or the accumulation of oxidized glutathione (GSSG). The loss of protein thiols was accounted for by the formation of glutathione mixed disulfides from GSSG and the arylation of protein nucleophiles by menadione. Nevertheless, such a loss occurred despite the chelation of cellular iron and a substantial reduction in the extent of cell killing. With the H2O2 generated by glucose oxidase, lipid peroxidation and a loss of 40% of the total protein thiols accompanied the cell killing within 1 hr. Deferoxamine, superoxide dismutase and the antioxidant N,N'-diphenyl phenylenediamine (DPPD) prevented the cell killing and two-thirds of the loss of protein thiols. Peroxidation of liver microsomes in vitro with ADP:Fe3+ similarly depleted protein thiols, an effect that was prevented by DPPD. The supernatant fraction from the peroxidation assay depleted the protein thiols of cultured hepatocytes without an effect on viability. Thus, lipid peroxidation accounted for the major part of the loss of protein thiols with glucose oxidase. The 10-15% decrement in protein thiols after 1 hr that occurred in the absence of cell killing reflected the formation of glutathione mixed disulfides. Finally, in the presence of DPPD, glucose oxidase killed 75% of the cells between 1 and 3 hr without any further change in protein thiols. Thus, under the conditions studied, the depletion of protein thiols by the three mechanisms, namely lipid peroxidation, formation of glutathione mixed disulfides, and arylation, does not necessarily have a causal relationship to the killing of cultured hepatocytes.     

Diclofenac induces apoptosis in hepatocytes

Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 μM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3–5-fold control) was maximal after 12 h of exposure to 350 μM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug.     

Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.     

Combination acetaminophen and doxapram potentiated hepatotoxicity in mouse primary cultured hepatocytes

Doxapram-induced potentiation of acetaminophen-induced reductions in cell viability and apoptosis was examined in mouse primary cultured hepatocytes. Loss of viability following exposure of acetaminophen and/or doxapram in cultured hepatocytes was assessed by monitoring [3H]-thymidine incorporation and mitochondrial activity, and apoptosis of hepatocytes was determined by nuclear microscopic observation and from detection of a ladder-like DNA fragmentation pattern in agarose gel electrophoresis. The combination of acetaminophen (5 mM) and doxapram (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased lipid peroxide levels of hepatocytes. Hepatocytes exposed for 24 h to acetaminophen (5 mM) plus doxapram (100 microM) showed atrophy of nuclei including chromatin condensation and a ladder-like DNA fragmentation pattern characteristic of apoptosis. Antioxidant (N-acetylcysteine), iron-chelator (deferoxamine), intracellular calcium ion chelator (quin 2-AM), endonuclease inhibitor (aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitor (3-aminobenzamide) all improved the viability of cells and eliminated the ladder-like DNA fragmentation in cells exposed to acetaminophen plus doxapram. In conclusion, the combination acetaminophen and doxapram potentiated the reduction in cell viability and apoptosis in mouse primary cultured hepatocytes. We suggest that careful observation for hepatotoxicity is recommended when acetaminophen and doxapram are prescribed simultaneously.     

Potentiation of acetaminophen hepatotoxicity by doxapram in mouse primary cultured hepatocytes

The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 microM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH2OC (O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB, 50 microM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 microM) alone or AA ( 5 mM) plus DOP (100 microM). However, loss of viability on exposure to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH2-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.     

Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.     

Deltamethrin induces apoptotic cell death in cultured cerebral cortical neurons

In this study we investigated the induction of apoptotic cell death and its potential mechanisms in cultured cortical neurons in response to deltamethrin exposure. The cultured cortical neurons were treated at 7 days with deltamethrin at concentrations of 10, 100, and 1000 nM, respectively. MTT assay showed that higher concentrations of deltamethrin (100 and 1000 nM) decreased neuronal viability in a time- and dose-dependent way. TUNEL staining revealed that numerous apoptotic cells appeared in the treated cultures compared to controls at 24, 48, and 72 h after treatment of 100 nM deltamethrin. Western blot analysis demonstrated that p53 and Bax expression were dramatically increased at the same time points, whereas Bcl-2 expression was significantly reduced at all time points after deltamethrin treatment. Further, we found that nitric oxide synthase inhibitor N(G)-nitro-L-arginine prevented deltamethrin-induced neuronal apoptosis and altered expression of p53, Bax, and Bcl-2. These results suggest that nitric oxide synthase might mediate deltamethrin-elicited neuronal apoptosis through modulating the expression of apoptosis-related genes.     

T-2 toxin induces apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells

To explore the toxic effect of T-2 toxin on mouse Leydig cells and its underlying molecular mechanisms, we isolated Leydig cells from mature mice, set-up Leydig cells culture, treated cells with T-2 toxin, evaluated cell proliferation, detected the caspase-3 activity, mitochondrial activity and apoptosis rate, and measured the mRNA levels of Bcl-2, Bax, PARP and caspase-3. T-2 toxin inhibited cell proliferation at concentrations higher than 10^?9 M or time more than 12?h, T-2 toxin also decreased Bcl-2 expression at the mRNA levels and mitochondrial activity at concentrations higher than 10^?9 M. While, T-2 toxin increased the mRNA expressions of Bax and PARP at concentrations higher than 10^?8 M and 10^?9 M, respectively, triggered mitochondria-mediated apoptosis, activated downstream caspase-3, and then increased caspase-3 at the activity and mRNA levels at concentrations higher than 10^?9 M. These data showed that T-2 toxin appears to activate specific intracellular death-related pathways leading to Bax-dependent caspase-3 activation and the induction of apoptosis in Leydig cells.     

Drug-induced hydrogen peroxide production in isolated rat hepatocytes

A method for measuring drug-induced hydrogen peroxide production in freshly isolated rat hepatocytes is described. 3-Amino-1,2,4-triazole, an irreversible inhibitor of the enzyme catalase markedly reduces the capacity of isolated hepatocytes to metabolize hydrogen peroxide, with maximum inhibition (80%) being observed after 40 min of co-incubation. The present method is based on the observation that this inhibition of catalase by 3-amino-1,2,4-triazole is prevented by methanol and that the effect of methanol is reversed in the presence of hydrogen peroxide. Using this assay we could demonstrate increased hydrogen peroxide production during the metabolism of diquat, paraquat, xanthine, benzylamine and glycolate by hepatocytes. Inhibition of the hydrogen peroxide metabolic capacity was greatest with glycolate and diquat, whereas paraquat and benzylamine only had a minor effect.     

Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibition of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death.     

普罗布考对过氧化氢诱导心肌细胞凋亡的影响

目的探讨普罗布考对过氧化氢(H2O2)诱导的心肌细胞凋亡的影响,探讨其可能的机制。方法培养的新生鼠心肌细胞随机分组并给予0.2mmol.L-1H2O2或0.2mmol.L-1 H2O2及不同浓度的普罗布考(100μmol.L-1、10μmol.L-1和1μmol.L-1)。采用琼脂糖凝聚电泳检测细胞凋亡;逆转录聚合酶链式反应(RT-PCR)法检测细胞Bcl-2 mRNA及Bax mRNA表达;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性,硫代巴比妥酸法检测细胞内丙二醛(MDA)含量。结果普罗布考明显抑制H2O2诱导的心肌细胞凋亡,降低心肌细胞BaxmRNA表达,升高Bcl-2mRNA表达。经不同浓度普罗布考干预后,心肌细胞SOD活性显著升高,MDA含量显著降低。以上这些效应均呈剂量依赖性。结论普罗布考抑制H2O2诱导的心肌细胞凋亡,其作用机制与改善心肌细胞氧化应激水平有关。     

Tri-n-butyltin delays the cell death induced by hydrogen peroxide in rat thymocytes

Tri-n-butyltin (TBT), one of environmental pollutants accumulated in mollusks, at nanomolar concentrations decreases cellular content of glutathione (GSH), suggesting that TBT increases cell vulnerability to oxidative stress because GSH has a role in catabolizing hydrogen peroxide (H(2)O(2)). In order to examine this possibility, the effect of tri-n-butyltin chloride (TBTCl) on rat thymocytes suffering from oxidative stress induced by H(2)O(2) was examined using a flow cytometer with four fluorescent probes; ethidium bromide, 2',7'-dichlorofluorescin diacetate, 5-chloromethylfluorescein diacetate and annexin-V-FITC. TBTCl at concentrations ranging from 100 nM to 1 μM attenuated H(2)O(2)-induced decrease in cell viability in a dose-dependent manner. It was unlikely that TBTCl reduced H(2)O(2)-induced oxidative stress because TBTCl failed to affect H(2)O(2)-induced oxidation of intracellular molecule (2',7'-dichlorofluorescin) and H(2)O(2)-induced decrease in cellular content of GSH. Results suggest that TBTCl may inhibit the pathway of cell death induced by H(2)O(2) or that TBTCl may induce a protective substance against the oxidative stress produced by H(2)O(2).     

Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.     

Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes

Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl₂), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl₂ significantly attenuated the CoCl₂-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl₂-induced decrease of Bcl-2 expression and attenuated the CoCl₂-induced increase in the expression of Bax and caspase-3. Treatment with CoCl₂ resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl₂-induced decrease of Bcl-2 expression and decreased the CoCl₂-induced increase of Bax and caspase-3 expressions. CoCl₂-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl₂-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl₂-induced COX-2 expression. Additionally, CoCl₂-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl₂-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.     

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.     

Hepatoprotective effects of reynosin against thioacetamide-induced apoptosis in primary hepatocytes and mouse liver

The aim of this study was to identify the hepatoprotective effects of reynosin, sesquiterpenes from the leaves of Laurus nobilis, against thioacetamide (TAA)-induced apoptosis in primary hepatocyte cultures and an in vivo mouse model. Rat hepatocytes were isolated and pretreated with 0.13, 0.64, or 3.22 μM reynosin and then exposed to 100 mM TAA. Reynosin treatment significantly inhibited TAA-induced apoptosis and hepatocellular DNA damage in primary rat hepatocytes. We observed an increase in levels of antiapoptotic Bcl-2, Bcl-XL mRNA and a decrease in levels of proapoptotic Bax mRNA following reynosin treatment of hepatocytes. Apoptosis in BALB/c mice was induced with intra-peritoneal injection of 200 mg/kg TAA for 2 weeks every other day. Then reynosin (5 mg/kg) and TAA were intragastrically given for 3 weeks every other day. Aspartate aminotransferase and alanine aminotransferase levels in the blood of mice were decreased in the reynosin administration group. Bcl-2 and Bcl-XL mRNA levels were increased, and the Bax mRNA level was decreased in reynosin-treated mice. Thus, reynosin inhibited TAA-induced apoptosis in primary hepatocytes and an in vivo mouse model.