Selective toxicity of Caspian cobra (Naja oxiana) venom on liver cancer cell mitochondria

Objective To explore the cytotoxicity effects of Caspian cobra (Naja oxiana or N. oxiana) venom on hepatocytes and mitochondria obtained from the liver of HCC rats. Methods In this study, HCC was induced by diethylnitrosamine (DEN), as an initiator, and 2-acetylaminofluorene (2-AAF), as a promoter. Rat liver hepatocytes and mitochondria for evaluation of the selective cytotoxic effect of N. oxiana venom were isolated and mitochondria and cellular parameters related to apoptosis signaling were then determined. Results Our results showed a raise in mitochondrial reactive oxygen species (ROS) level, swelling in mitochondria, mitochondrial membrane potential (Δψm) collapse and release of cytochrome c after exposure of mitochondria only isolated from the HCC group with the crude venom of the N. oxiana (12.5, 25, and 50 μg/mL). This crude venom also induced caspase-3 activation (P < 0.001) in the hepatocytes obtained only from the HCC rat liver. Conclusions Based on the over all results, we suggested that N. oxiana may be considered as a promising complementary therapeutic agent for the treatment of HCC.

comparison of hepatocyte cytotoxic mechanisms for docetaxel and PLGA-docetaxel nanoparticls

Docetaxel [DTX] is one of the most widely used drugs in oncology due to its high efficacy against several cancers. Though, its routine clinical administration, formulated in tween 80, causes serious side effects. Polylactide-co-glycolide [PLGA], biodegradable polyester synthesized and approved for human use, is employed to overcome these problems. In this investigation, we compare the cytotoxic mechanisms of DTX and PLGA-DTX in isolated rat hepatocytes. Cytotoxicity of DTX and PLGA-DTX were associated with reactive oxygen species formation, activation of caspases cascade, collapse of mitochondrial membrane potential [MMP], lysosomal membrane leakiness and ATP depletion. Our results also showed that CYP2E1 is involved in the oxidative stress cytotoxicity mechanism and both drugs are detoxified via phase II metabolic methylation. Furthermore, we concluded that PLGA-DTX is bioactivated by GSH. It could also potentiate hepatocyte toxicity through a mitochondrial/lysosomal toxic cross-talk. In addition to these observed differences, it is likely that mode of hepatocyte membrane penetration is different between these compounds

comparison of toxicity mechanisms of cigarette smoke on isolated mitochondria obtained from rat liver and skin

Previous studies demonstrated that CSE induces oxidative stress and its consequences on isolated mitochondria obtained from lung, heart and brain which may provide insight into the role of CSE in human health and disease. The present study was carried out to further characterize and compare toxic effect of CSE extract on isolated mitochondria obtained from either a directly contacting tissue [i.e. skin] or a vital visceral tissue [i.e. liver].We obtained Rat liver and skin mitochondria by differential ultracentrifugation and incubated the isolated mitochondria with different concentrations [1, 10 and 100%] ofstandardizedcigarette smoke extract [CSE]. Our results were similar to our previous study which discovered CSE toxicity mechanisms on isolated mitochondria obtained from lung, heart and brain with minor changes.CSE induced a significant rise in ROS formation, lipid peroxidation and mitochondrial membrane potential collapse and mitochondrial swelling on isolated mitochondria obtained from both liver and skin. CSE induced Decrease in ATP concentration on isolated mitochondria obtained from both liver and skin did not include CSE lowest concentration [1%]. Our findingsshowed that CSE-induced toxicity in liver and skin is due to disruptive effect on mitochondrial respiratory chain which canleads to cytochrome c release and apoptosis signaling

Toxicity mechanisms of cigarette smoke on mouse fetus mitochondria

Maternal smoking has been recognized as a common cause of low birth weight, preterm birth and the decrease of gestational age period. Unfortunately, there is an increasing interest within public especially woman in Iran in the tobacco products consumption. On the other hand, the deleterious effect of maternal smoking on human fetus in pregnancy period especially in the first trimester encouraged us to investigate toxicity mechanisms of cigarette smoke on mouse fetus mitochondria. For this purpose different concentrations [1, 10 and 100%] of standardized cigarette smoke extract [CSE] were administrated on mitochondria isolated from fetus of NMRI mice on the 15 day of gestation. Our results showed a significant increase in ROS [Reactive oxygen species] formation, lipid peroxidation, mitochondrial membrane potential collapse, mitochondrial swelling and finally a decrease in ATP concentration in the CSE-treated isolated fetus mitochondria. Our results suggest that CSE-induced embryo toxicity is the result of disruptive effect on mitochondrial respiratory chain that leads to ROS formation, lipid peroxidation, mitochondrial MMP [mitochondrial membrane potential] decline and decrease of ATP level which starts apoptosis signaling

Embryo toxic effects of depleted uranium on the morphology of the mouse fetus

Although the biokinetics, metabolism, and chemical toxicity of uranium are well known, until recently little attention was paid to the potential toxic effects of uranium on reproduction and development in mammals. In recent years, it has been shown that uranium is a developmental toxicant when given orally or subcutaneously [SC] to mice. Decreased fertility, embryo/fetal toxicity including teratogenicity, and reduced growth of the offspring have been observed following uranium exposure at different gestation periods. For investigating the effects of DU on pregnant animals, three groups [control, sham and test] of NMRI mice were chosen. In test group 4mg/kg of DU were administered intraperitonealy at 11 day of gestation, in sham group only normal saline injected to interior peritoneum as indicated in the test group and in Control group which was considered as the comparison base line of our research, no injection was made. Caesarean sections were performed at 15 day of the gestation; and their placentas were examined externally. Base on our results DU caused significant external anomalies, and caused a significant decrease [p<0.05] in the weight and diameter of placentas, the number of the embryos, their body weight and crown-rump length of fetuses

Cytoprotective effects of hydrophilic and lipophilic extracts of pistacia vera against oxidative versus carbonyl stress in rat hepatocytes

This study was conducted to evaluate the cytoprotection of various extracts and bioactive compounds found in Pistacia vera againts cytotoxicity, ROS formation, lipid peroxidation, protein carbonylation, mitochondrial and lysosomal membrane damages in cell toxicity models of diabetes related carbonyl [glyoxal] and oxidative stress [hydroperoxide]. Methanol, water and ethyl acetate were used to prepare crude pistachios extracts, which were then used to screen for in-vitro cytoprotection of freshly isolated rat hepatocytes against these toxins. The order of protection by Pistacia vera extracts against both hydroperoxide induced oxidative stress [ROS formation] and glyoxal induced protein carbonylation was: pistachio methanolic extract > pistachio water extract, gallic acid, catechin > alpha-tochoferol and pistachio ethyl acetate extract. Finally due to higher protection achieved by methanolic extract even compared to sole pretreatment of gallic acid, catechin or alpha-tochoferol, we suggest that cytoprotection depends on the variety of polar and non-polar compounds found in methanolic extract, it is likely that multiple cytoprotective mechanisms are acting against oxidative and carbonyl induced cytotoxicity. To our knowledge, we are the first to report the cytoprotective activity of Pistacia vera extracts against oxidative and carbonyl stress seen in type 2 diabetes hepatocytes model

Schizophrenia induces oxidative stress and cytochrome C release in isolated rat brain mitochondria: a possible pathway for induction of apoptosis and neurodegeneration

Schizophrenia is a chronic and often debilitating illness which affects about 1% of the world population. Some reagents have been used to simulate schizophrenic disorders in laboratory animals, such as amphetamine and ketamine. Previous studies have suggested that reactive oxygen species [ROS] production, reduced levels of ATP, mitochondrial dysfunction and apoptosis are involved in the pathophysiology and etiology of schizophrenia. In this study we divided Wistar rats in to 2 groups; control group received normal saline and test group received ketamine 30 mg/Kg daily for five consecutive days. Then, locomotor activity including side to side head rocking and arcing of neck, proved schizophrenia in the test group rats. Rats in both control and test groups were then decapitated and brain mitochondria were isolated. Our results showed increased ROS formation, mitochondrial membrane potential collapse, mitochondrial swelling and cytochrome c release in mitochondria of schizophrenic test group. Our findings suggested that mitochondrial ROS formation and apoptosis signaling are likely involved in cellular pathology of Schizophrenia. To our knowledge this is the first report that provides a mechanistic justification between mitochondrial events and neuodegeneration in the Schizophrenia

Ichthyotoxic cochlodinium polykrikoides induces mitochondrial mediated oxidative stress and apoptosis in rat liver hepatocytes

In this research, we investigated the cytotoxic mechanisms of CochlodiniumpolykrikoidescQ/ lysate on isolated rat liver hepatocytes.This micro algae is responsible for a severe and widespread harmful algal bloom in the Persian Gulf and Gulf of Oman [2008-2009]. Isolated hepatocytes were obtained by collagenase perfiision of Sprague-Dawley rat liver.According to our results, incubation of algal lysate with isolated rat hepatocytescaused hepatocyte membrane lysis, reactive oxygen species [ROS] formation, glutathione depletion, collapse of mitochondrial membrane potential,ATP depletion and increase in ADP/ATP ratio, cytochrome c release in to the hepatocyte cytosol,activation of caspase-3 [final mediator of apoptosis] and appearance of apoptosis phenotype. On the other hand, pre-treatment of antioxidants [a-tocopherol succinate and BHT], radical scavengers [mannitol and DMSO], mitochondrial permeability transition [MPT] pore sealing agents [cyclosporine A, carnitine and trifluoperazine], NADPH P450 reductase inhibitor [Diphenyliodonium chloride], CYP2E1 inhibitors [Phenylimidazole and 4-Methylpyrazole] and ATP generators [L-glutamine, Fructose and Xylitol]inhibitedcaspase-3 activation and cell death in algal lysate treated hepatocytes.Our data also confirmed that algal lysate activates apoptosis signaling via oxidative stress and mitochondrial pathway. Thus, ROS formation caused by the lysate exposure could directly be involved in mitochondrial MPT pore opening and activation of caspase-3 leading to C.polykrikoides lysateinduced apoptosis on rat hepatocytes. These findings contribute to a better understanding QfC.polykrikoides-toxic effects on mammalian liver cells

Mitochondrial toxicity of depleted uranium: protection by beta-glucan

Considerable evidence suggests that mitochondrial dysfunction contributes to the toxicity of uranyl acetate [UA], a soluble salt of depleted uranium [DU]. We examined the ability of the two antioxidants, beta-glucan and butylated hydroxyl toluene [BHT], to prevent UA-induced mitochondrial dysfunction using rat-isolated kidney mitochondria. Beta-glucan [150 nM] and BHT [20 nM] attenuated UA-induced mitochondrial reactive oxygen species [ROS] formation, lipid peroxidation and glutathione oxidation. Beta-glucan and BHT also prevented the loss of mitochondrial membrane potential [MMP] and mitochondrial swelling following the UA treatment in isolated mitochondria. Our results show that beta-glucan and BHT prevented UA-induced mitochondrial outer membrane damage as well as release of cytochrome c from mitochondria. UA also decreased the ATP production in isolated mitochondria significantly inhibited with beta-glucan and BHT pre-treatment. Our results showed that beta-glucan may be mitochondria-targeted antioxidant and suggested this compound as a possible drug candidate for prophylaxis and treatment against DU-induced nephrotoxicity

Toxicity of arsenic [III] on isolated liver mitochondria: a new mechanistic approach

Arsenic exposure mainly through food and water has been shown to be associated with increased incidence of numerous cancers and non-cancer harmful health. It is also used in cancer chemotherapy and treatment of several cancer types due to its apoptogenic effects in the various cancer and normal cell lines. We have already reported that liver is the storage site and important target organ in As [III] toxicity and recently, it has been suggested that hepatic toxicity of arsenic could be resulted from impairment of the liver mitochondria. In this study, interaction of As [III] with freshly isolated rat mitochondria was investigated. We determined different mitochondrial toxicity factors as well as mitochondrial sources of ROS formation using specific substrates and inhibitors following addition of As [III] to the mitochondria. Our results showed that arsenic [III] increased mitochondrial ROS formation, lipid peroxidation and mitochondrial membrane potential collapse, cytochrome c release and mitochondrial swelling in a concentration dependent manner. Addition of As [III] in to the isolated mitochondria, inhibited complexes I and II leading to disruption of mitochondrial electron transfer chain, decreased mitochondrial ATP content and ROS formation

Synthesis and molecular-cellular mechanistic study of pyridine derivative of dacarbazine

Dacarbazine is an antitumor prodrug which is used for the treatment of malignant metastatic melanoma and Hodgkin's disease. It requires initial activation in liver through an N-demethylationreaction. The active metabolite prevents the progress of disease via alkylation of guanine bases in DNA strands. In order to investigate the importance of imidazole ring and its dynamictautomerization in anticancer activity of dacarbazine, a pyridine analog of this drug was synthesized and the cytotoxic activity and cellular-molecular mechanisms of action for this compound were compared with those of dacarbazine. EC50 values for dacarbazine and the pyridine analog were found to be 56 micro M and 33 micro M respectively. Both dacarbazine and the pyridine analog resulted in formation of reactive oxygen species [ROS] upon their addition to the isolated rat hepatocytes. They also decreased the mitochondrial membrane potential and caused lysosomal membrane rupture. Cytotoxicity was prevented by ROS scavengers and antioxidants. Cytotoxicity was also prevented by CYP[450] inhibitors, lysosomalinactivators and MPT [Mitochondrial Permeability Transition Pore] blockers

Involvement of four different intracellular sites in chloroacetaldehyde- induced oxidative stress cytotoxicity

Chloroacetaldehyde [CAA] is a chlorination by-product in finished drinking water and a toxic metabolite of a wide variety of industrial chemicals [e.g. vinyl chloride] and chemotherapeutic agents [e.g. cyclophosphamide and ifosfamide]. In this research, the cytotoxic mechanisms of CAA in freshly isolated rat hepatocytes were investigated.CAA cytotoxicity was associated with reactive oxygen species [ROS] formation and glutathione depletion suggesting that oxidative stress contributed to the CAA cytotoxic mechanism. CAA-induced oxidative stress cytotoxicity markers were significantly prevented by antioxidants, ROS scavengers, mitochondrial permeability transition [MPT] pore sealing agents, endocytosis inhibitors, ATP generators and xanthine oxidase inhibitor. In our study the hepatocyte mitochondrial membrane potential was rapidly decreased by CAA which was prevented by antioxidants and ROS scavenger indicating that mitochondrial membrane damage was a consequence of ROS formation. CAA cytotoxicity was also associated with lysosomal membrane rupture. Our findings showed that at least four different intracellular sources including: metabolic enzymes cytochrome P[450] and xanthine oxidase, mitochondrial respiratory chain disruption and lysosomal Haber-weiss reaction, were involved in CAA induced ROS formation and other subsequent cytotoxic events. Our other interesting finding was that the lysosomotropic agents prevented CAA induced mitochondrial membrane potential collapse and mitochondrial MPT pore sealing agents inhibited lysosomal membrane damage caused by CAA. It can therefore be suggested that there is probably a toxic interaction [cross-talk] between mitochondrial and lysosomal oxidative stress generating systems, which potentiates each organelle damage and ROS formation in CAA- induced hepatotoxicity

Uranyl acetate induces oxidative stress and mitochondrial membrane potential collapse in the human dermal fibroblast primary cells

Cytotoxicity of depleted uranium, as a byproduct of military has been came to spotlight in recent decades. DU is known as a chemical rather than radioactive hazard and efforts to illustrating its mechanism is undergo, but the precise complete molecular mechanisms are still unclear. Recent studies showed that uranium induces biological changes in many different target tissues, such as the kidney, brain and skin. The aim of this study was to assess the impact of depleted uranium exposure at the cellular level in the human dermal fibroblast primary cells. The human dermal fibroblast primary cells incubated with different concentration [250-750 microM] of depleted uranium. Cytotoxicity and mitochondrial function in this cell lines were determined with the LDH leakage assay and the MTT test respectively. MDA levels were measured for determination of Lipid peroxidation in DU treated cells. Besides glutathione depletion and apoptosis phenotype detection were also assessed to complete the mechanistic screening. Results showed that the cell viability ameliorates in concentration and time dependent manners following in 24, 48 and 72 h incubation with DU. Moreover the significant increase in lipid peroxidation and significant decrease in cellular GSH recorded in DU treated human dermal fibroblast primary cells suggesting the preoxidant effect of uranyl ions. Cytoprotective effects of N-acetylcysteine [NAC] and dramatic decrease of cell viability in buthionin sulfoxamid [BSO] pretreated cells indicated the possibility of a critical role for glutathione system in DU detoxification. Death pattern, in fibroblast cells following DU treatment was varied from apoptosis to necrosis while the time and concentration increased. Since ROS formation is the initiation step for cell apoptosis, the present studies suggest Uranyl-induced toxicity in the human dermal fibroblast primary cells originated from oxidative stress and lead to occurrence of programmed cell death

Application of cell-based assay systems for the early screening of human drug hepatotoxicity in the discovery phase of drug development

While drug toxicity [especially hepatotoxicity] is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in-vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index [TI] may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in-vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase

Lysosomal oxidative stress cytotoxicity induced by para-phenylenediamine redox cycling in hepatocytes

It has already been reported that muscle necrosis induced by various phenylenediamine derivatives are correlated with their autoxidation rate. Now in a more detailed investigation of the cytotoxic mechanism using a model system of isolated hepatocytes and ring-methylated structural isomer durenediamine [DD] we have shown that under aerobic conditions, phenylenediamine induced cytotoxicity and ROS formation were markedly increased by inactivating DT-diaphorase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that the H[2]O[2] generation could be attributed to a futile two electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT- diaphorase. The subcellular organelle oxidative stress effects leading to cytotoxicity has not yet been identified. Hepatocyte mitochondrial membrane potential was only slightly decreased by phenylenediamine before cytotoxicity ensued. However phenylenediamine induced lysosomal damage and hepatocyte protease activation. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented phenylenediamine induced cytotoxicity. Furthermore desferoxamine [a ferric chelator], antioxidants or ROS scavengers [catalase, mannitol, tempol or dimethylsulfoxide] prevented phenylenediamine cytotoxicity. It is concluded that H[2]O[2] reacts with lysosomal Fe[2+] to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death