Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells

The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 microM), decreased from Hg(2+) (most toxic) to Cu(2+) (least toxic). Hg(2+) and Cd(2+) produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu(2+) at concentrations up to 500 microM was weakly effective. Hg(2+) at concentration of 10 microM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu(2+) had inhibitory effect on cell respiration only at 500 microM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu(2+) induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg(2+) and Cd(2+) on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg(2+), Cd(2+) and Cu(2+) in AS-30D rat hepatoma cells.     

Adverse effect of tannery waste leachates in transgenic Drosophila melanogaster: role of ROS in modulation of Hsp70, oxidative stress and apoptosis

Leachate is a complex chemical mixture of chemicals produced as a result of leaching of solid wastes. The potential toxicity of leachates is a major environmental health concern. The present study evaluated the role of ROS in tannery leachates induced Hsp70 expression, antioxidant enzymes and apoptosis in Drosophila. Different concentrations (0.05-2.0%) of leachates prepared from tannery waste at different pH (7.00, 4.93 and 2.88) were mixed with Drosophila food and fed to the larvae for 2-48 h to examine the different stress and apoptotic markers. A concentration- and time-dependent significant increase in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content were observed in the exposed larvae. Activities of antioxidant enzymes were delayed compared with Hsp70 expression and MDA level in the exposed organisms. Apoptotic cell death was observed in the exposed larvae at higher concentrations concurrent with a significant regression in Hsp70 along with a higher level of ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression at these concentrations indicated the important role of ROS in the induction of cellular damage in the exposed organisms. There was a significant generation of ROS in the larvae exposed to 0.5% of leachates which did not interfere with the protection of their cells by Hsp70 and antioxidant enzymes. However, generation of significantly higher levels of ROS in the larvae exposed to 1.0% and 2.0% leachates may decrease Hsp70 expression thus leading to mitochondria-mediated caspase-dependent apoptotic cell death.     

Effects of single-wall carbon nanotubes in human cells of the oral cavity: Geno-cytotoxic risk

Single-wall carbon nanotubes (SWCNTs) are one of the most extensively produced carbon materials and the environmental, public and professional exposure is therefore dramatically increasing. Consequently the studies on bio-effects and safety of SWCNTs are highly needed. The goal of this study was investigate the effects in vitro of SWCNTs in cells of the oral cavity, never employed in this research field. We exposed human gingival fibroblasts to 50, 75, 100, 125, 150 μg/ml SWCNTs for 24 h and we investigated genotoxicity (Comet assay and micronucleus test), cytotoxicity, oxidative stress, as reactive oxygen species (ROS) generation, and stress response, as Heat shock protein 70 (Hsp70) expression. SWCNTs produced genotoxic effects at all doses, even if detected with different sensitiveness by the two tests, and at the two highest doses induced a strong decrease of the cell proliferation and cell survival, causing apoptosis too. Furthermore, we proved the ability of these nanomaterials to induce oxidative stress and Hsp70 expression. Finally, by inhibition of Hsp70 expression, we demonstrated that this heat shock protein conferred protection against SWCNT geno-cytotoxicity.     

Extracellular-superoxide dismutase expression in COS7 cells exposed to cadmium chloride

Cadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactive oxygen species (ROS) and induces cytotoxicity. Extracellular-superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the cells from damaging effects of ROS; however, the effect of Cd on the expression of EC-SOD in COS7 cells remains unclear. In this study, exposure to cadmium chloride (CdCl?) enhanced intracellular ROS generation and induced COS7 cell death. Moreover, exposure to Cd decreased the expression of EC-SOD at mRNA and protein levels, but not of other SOD isozymes, copper-and zinc-containing SOD and manganese-containing SOD. The reduction of EC-SOD and cell viability was partially attenuated by pretreatment with an antioxidant, N-acetylcysteine. Further, we determined the involvement of p38-mitogen-activated protein kinase (p38-MAPK) in the reduction of EC-SOD. From these observations, p38-MAPK signaling cascades activated by ROS play a pivotal role in the reduction of EC-SOD, and it is concluded that the reduction of EC-SOD leads to a decrease in the resistance to oxidative stress of Cd-exposed COS7 cells.     

Comparative mechanisms of zearalenone and ochratoxin A toxicities on cultured HepG2 cells: Is oxidative stress a common process?

Zearalenone (ZEN) and Ochratoxin A (OTA) are structurally diverse fungal metabolites that can contaminate feed and foodstuff and can cause serious health problems for animals as well as for humans. In this study, we get further insight of the molecular aspects of ZEN and OTA toxicities in cultured human HepG2 hepatocytes. In this context, we have monitored the effects of ZEN and OTA on (i) cell viability, (ii) heat shock protein (Hsp) 70 and Hsp 27 gene expressions as a parameter of protective and adaptive response, (iii) oxidative damage, and (iv) cell death pathways. Our results clearly showed that both ZEN and OTA inhibit cell proliferation. For ZEN, a significant induction of Hsp 70 and Hsp 27 was observed. In the same conditions, ZEN generated an important amount of reactive oxygen species (ROS). Antioxidant supplements restored the major part of cell mortality induced by ZEN. However, OTA treatment downregulated Hsp 70 and Hsp 27 protein and mRNA levels and did not induce ROS generation. Antioxidant supplements did not have a significant effect on OTA‐induced cell mortality. Using another cell system (Vero monkey kidney cells), we demonstrated that OTA downregulates three members of HSP 70 family: Hsp 70, Hsp 75, and Hsp 78. Our findings showed that oxidative damage seemed to be the predominant toxic effect for ZEN, while OTA toxicity seemed to be rather because of the absence of Hsps protective response. Furthermore, the two mycotoxins induced an apoptotic cell death. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Induction of metallothionein synthesis in cultured human trophoblasts by cadmium and zinc

A system of primary cultures of human chorionic trophoblasts has been used for studying the effects of heavy metals on human reproductive tissue. Using this system, changes in cellular concentration of metallothionein (MT) in response to exposure to Cd or Zn were determined. Trophoblasts were isolated from term chorion leave, grown in RPMI-1640 medium, and exposed to Cd or Zn. Cellular content of MT was measured using the Cd/heme radioassay. MT increased in a concentration- and time-dependent manner after exposure to either metal. Cd increased the content of MT in trophoblasts at concentrations as low as 0.5 microM during a 24-h exposure. Moreover, extending exposure to Cd (2 microM) to 72 h resulted in a 3-4-fold increase in the concentration of MT. On a molar basis, Zn was not as potent a stimulus for MT synthesis as Cd, and required a concentration of 2.5 microM to increase the concentration of MT over a 24-h period. However, a 48- or 72-h exposure to Zn (10 microM) increased concentrations of MT nearly 8-fold over control values. Simultaneous exposure to Cd (2 microM) and inhibitors of protein synthesis, cycloheximide and actinomycin D, prevented the typical increase in MT concentration, suggesting that the metals act to increase the synthesis of MT. In another series of experiments, trophoblasts were exposed to Cd (2 microM) for 24 h, after which the cells were challenged with cytotoxic concentrations of Cd. Cells pretreated with Cd and then challenged with toxic concentrations of Cd had higher levels of MT and showed less toxicity, as indicated by leakage of lactic dehydrogenase. These results suggest that MT serves to sequester the metals in trophoblasts and reduce the toxicity of heavy metals. Thus, this system should be useful for studying the effects of heavy metals and characterizing the induction of MT in human reproductive tissue in vitro.     

Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 microM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 microM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages.     

The cytotoxic effects of cadmium chloride and mercuric chloride mixtures in rat primary hepatocyte cultures

The toxic effects of Cd and Hg mixtures were studied using primary monolayer cultures of rat hepatocytes. Cytotoxicity was assessed by measuring the release of lactic dehydrogenase from the cells. Cytotoxic and non-cytotoxic metal levels were used. At the higher exposure concentrations (0.2 micrograms Cd.ml-1 and 2.0 micrograms Hg.ml-1), Cd was very toxic to hepatocytes whereas Hg was only marginally toxic. The combination of Cd and Hg was more toxic than predicted by summation of the individual metal toxicities. The incorporation of [35S]cysteine into protein of the cytosol and insoluble cell fraction was increased in response to Cd or Hg exposure and was directly related to cell 35S accumulation. Combinations of Cd and Hg significantly increased the proportion of total 35S which was incorporated in cell protein, an effect that was attributed to the accumulation of protein in the insoluble cell fraction. Cd uptake by hepatocytes was related to exposure concentration but was lower when Hg was also present in the incubation medium. Gel chromatography of the cytosol from Cd-exposed cells showed 3 Cd containing fractions which corresponded to the elution positions of high Mr proteins, metallothionein (MT) and low Mr molecules. When hepatocytes were exposed to Hg in combination with Cd, the MT-like fraction was no longer evident and Cd in the low Mr fraction was greatly reduced. Regardless of the presence or absence of Cd in the exposure medium, 98% of cytosol Hg in Hg-exposed cells was found to elute after the low Mr fraction, at a position equivalent to inorganic salts. This indicates that the enhanced cytotoxicity of Cd and Hg may be related to a decrease in the MT-like protein in the cytosol and not due to a direct competitive binding interaction in relation to the protein.     

Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes

Severe chronic liver disease results from the hepatic accumulation of copper nickel, cobalt or iron in humans and on the other hand cadmium, dichromate and arsenic may induce lung or kidney cancer. Acute or chronic CdCl₂, HgCl₂ or dichromate administration induces hepatic and nephrotoxicity in rodents. Oxidative stress is often cited as a possible cause but has not yet been measured. For the first time we have measured the reactive oxygen species (ROS) formation induced when cells are incubated with metals and determined its source. Hepatocytes incubated with 2′,7′-dichlorofluorescin diacetate resulted in its rapid uptake and deacetylation by intracellular esterases to form 2′,7′-dichlorofluorescin. A marked increase in ROS formation occurred with LD₅₀ concentrations of cadmium [Cd(II)], Hg(II) or arsenite [As(III)] which was released by proton ionophores that uncouple oxidative phosphorylation. Uncouplers or oxidative phosphorylation also inhibited ROS formation induced by these metals, which suggests that mitochondria are major contributors to endogenous ROS formation. Glycolytic substrates also inhibited Cd(II)/Hg(II)/As(III)-induced ROS formation and confirms that mitochondria are the site of ROS formation. By contrast ROS formation by LD₅₀ concentrations of Cu(II), Ni(II), Co(II) or dichromate [Cr(VI)] were not affected by uncouplers or glycolytic substrates. However they were inhibited by lysosomotropic agents or endogenous inhibitors [in contrast to Hg(II), Cd(II) or As(III)]. Furthermore Cu(II), Ni(II), Co(II) or Cr(VI) accumulated in the lysosomes and the ROS formed caused a loss of lysosomal membrane integrity. The release of lysosomal proteases and phospholipases also contributed to hepatocyte cytotoxicity. ROS formation and cytotoxicity induced by added H₂O₂ or generated by the intracellular redox cycling of nitrofurantoin was also inhibited by lysosomotropic agents and ferric chelators suggesting that lysosomal Fe(II) contributes to H₂O₂-induced cytotoxicity. In conclusion, lysosomes are sites of cytotoxic ROS formation with redox transition metals (CuII, CrVI, NiII, CoII) whereas mitochondria are the ROS sites for non-redox or poor redox cycling transition metals (CdII, HgII, AsIII).     

Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture

Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.     

Modulation of aryl hydrocarbon receptor-regulated gene expression by arsenite, cadmium, and chromium

Aryl hydrocarbon receptor (AhR) ligands and heavy metals are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of AhR-dependent phase I and II drug metabolizing enzymes. To determine the effect of heavy metals on the AhR-regulated genes: cytochrome P4501A1 (Cyp1a1), NAD(P)H: quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were treated with increasing concentrations of As3+ (1-10 microM), Cd2+ (1-25 microM) and Cr6+ (1-25 microM) with or without the AhR ligands: 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 uM), or benzo[a]pyrene (1 microM). Our results show that AhR ligands alone and As3+ or Cd2+ alone increased the catalytic activities and mRNA levels of all AhR-regulated genes. When metals were co-administered with an AhR ligand, all three metals inhibited the induction of Cyp1a1 activity by the AhR ligands but potentiated its mRNA and protein expression. In addition, all metals enhanced QOR and GST Ya at the activity and mRNA levels but modulated their induction by AhR ligands in a concentration, metal, and AhR ligand-dependent manner. Generally, Cr6+ inhibited while As3+ and Cd2+ potentiated the induction of QOR and GST Ya activities and mRNA levels. The three metals enhanced the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results show that the ability of metals to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II enzymes will influence the carcinogenicity and mutagenicity of the AhR ligands.     

Different induction of metallothioneins and Hsp70 and presence of the membrane transporter ZnT-1 in HepG2 cells exposed to copper and zinc

Eukaryotic cells respond to stressful environmental stimuli, such as toxic concentrations of heavy metals, by rapidly synthesising defence proteins: the metallothioneins (MT) and the heat shock protein 70 (Hsp70). In this study we have analysed how the human hepatoblastoma cell line HepG2 responds to exposure to excess copper (30 μg/ml) and zinc (50 μg/ml) for long exposure times (48 and 72 h). Accumulation of the two metals, as measured by ICP-AES, was time-dependent reaching a plateau after 72 h. HepG2 cells responded by dramatically increasing levels of MT during stress, mostly during zinc exposure. A time lag in Hsp70 induction was observed as the levels of this protein increased only after removal of the stress from culture medium (recovery) for 24 h, thus suggesting that the two defence mechanisms are not coordinated in a metal-induced stress response. Moreover in HepG2 cells, immunochemical and fluorescence techniques showed the presence and the localisation of the zinc membrane exporter ZnT-1 as a further mechanism of defence/homeostasis against zinc toxicity.     

Co-exposure to nickel and cobalt chloride enhances cytotoxicity and oxidative stress in human lung epithelial cells

Nickel and cobalt are heavy metals found in land, water, and air that can enter the body primarily through the respiratory tract and accumulate to toxic levels. Nickel compounds are known to be carcinogenic to humans and animals, while cobalt compounds produce tumors in animals and are probably carcinogenic to humans. People working in industrial and manufacturing settings have an increased risk of exposure to these metals. The cytotoxicity of nickel and cobalt has individually been demonstrated; however, the underlying mechanisms of co-exposure to these heavy metals have not been explored. In this study, we investigated the effect of exposure of H460 human lung epithelial cells to nickel and cobalt, both alone and in combination, on cell survival, apoptotic mechanisms, and the generation of reactive oxygen species and double strand breaks. For simultaneous exposure, cells were exposed to a constant dose of 150 μM cobalt or nickel, which was found to be relatively nontoxic in single exposure experiments. We demonstrated that cells exposed simultaneously to cobalt and nickel exhibit a dose-dependent decrease in survival compared to the cells exposed to a single metal. The decrease in survival was the result of enhanced caspase 3 and 7 activation and cleavage of poly (ADP-ribose) polymerase. Co-exposure increased the production of ROS and the formation of double strand breaks. Pretreatment with N-acetyl cysteine alleviated the toxic responses. Collectively, this study demonstrates that co-exposure to cobalt and nickel is significantly more toxic than single exposure and that toxicity is related to the formation of ROS and DSB.     

Cadmium stimulates MAPKs and Hsp27 phosphorylation in bovine adrenal chromaffin cells

Cadmium (Cd(2+)) is a common environmental pollutant, which is widely used in industry and is a constituent of tobacco smoke. Exposure to this heavy metal has been linked to a wide range of detrimental effects on mammalian cells. In this study, the action of Cd(2+) on protein phosphorylation in bovine adrenal chromaffin cells (BACCs) was examined. Cells were incubated with (32)Pi in the presence of Cd(2+) (1-50 microM) and proteins were separated by one- or two-dimensional electrophoresis. An increase in the phosphorylation of BACCs proteins, without changing cell viability, was observed in response to Cd(2+) (5-50 microM). Particularly at three spots, with molecular weight of 25kDa and isoeletric point range 4.0-4.5, which were identified as phosphorylated isoforms of the heat shock protein of 27kDa (Hsp27). Phosphorylation of the p38(MAPK), a member of mitogen-activated protein kinase (MAPK) family, was stimulated by Cd(2+) over the same concentration range and it was the major upstream protein kinase involved in the phosphorylation of all three spots of Hsp27. Cd(2+) also stimulated the phosphorylation of other MAPK family member, the extracellular signal-regulated kinase (ERK)-1/2. Therefore, primary adrenal chromaffin cells are a target for Cd(2+) and both the ERK1/2 and the p38(MAPK) are activated. Additionally, Hsp27 is highly phosphorylated in response to the metal exposure, due to p38(MAPK) activation. These biochemical effects of Cd(2+) might disrupt the normal secretory function of these cells.