Cytotoxic Effects of N-Hydroxyparacetamol in Suspensions of Isolated Rat Hepatocytes

Abstract: The cytotoxicity of N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of the hepatotoxic and nephrotoxic analgesic paracetamol, was studied in suspensions of hepatocytes isolated by collagen-perfusion of livers of male rats. Incubation of cells with 0.25–2.0 mM N-OH-pHAA led after 3–5 hours to increased cell permeability measured by increased trypan blue uptake, increased NADH penetration or leakage of prelabelled ⁵¹Cr. N-OH-pHAA rapidly depleted cellular glutathione, 16% of initial levels were seen after 30 min. incubation. ³H-N-OH-pHAA bound covalently to cellular proteins in a time- and concentration-dependent manner, considerably higher binding rates were seen with boiled cells compared to intact cells. Pretreatment of animals with the cytochrome P-450 inducer phenobarbital did not affect N-OH-pHAA cytotoxicity or covalent binding, whereas the cytochrome P-450 inhibitor metyrapone inhibited both cytotoxicity and binding. Lipid peroxidation in hepatocytes could be seen as a late event after a limited range of N-OH-pHAA concentrations. In contrast, lipid peroxidation was an early event in cells exposed to carbon tetrachloride. A minimal exposure time of 30 min. of the hepatocytes to N-OH-pHAA was sufficient to elicit cellular damage occurring after 3–5 hours.     

Cytotoxic effects of N-acetyl-p-benzoquinone imine,a common arylating intermediate of paracetamol and N-hydroxyparacetamol

The cytotoxic effects of N-acetyl-p-benzoquinone imine (NAPQI), a postulated ultimate reactive metabolite of paracetamol (pHAA), was studied in suspensions of isolated rat hepatocytes. Incubation of cells for 10–300 min with 0.1–0.5 mM NAPQI led to concentration dependent cell damage. as determined by increased trypan blue exclusion, lactate dehydrogenase release and glutathione (GSH) depletion. NAPQI and N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of pHAA, caused cytotoxic effects in the same concentration range. In contrast, no toxic effects of pHAA (? 20 mM) could be demonstrated. With the short half-life of NAPQI, less than 0.5% of the NAPQI added is expected to be left in the incubation medium after a 2 min incubated period. Nevertheless, 10–120 min (depending on the concentration of NAPQI) elapsed before the cells responded with increased membrane permeability. Clearly, the initial damage caused by NAPQI must be followed by subsequent cellular steps before toxicity becomes apparent. The addition of N-acetylcysteine, GSH or ascorbate during the NAPQI exposure period fully protected the hepatocytes from NAPQI damage. Lesser effects were demonstrated when these agents were added after the 5 min NAPQI exposure period. The results presented in this study further support the hypothesis that NAPQI is the ultimate reactive formed from pHAA.     

Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death

The toxic effect of thiram, a widely used dithiocarbamate fungicide, was investigated in cultured human skin fibroblasts. Cell survival assays demonstrated that thiram induced a dose-dependent decrease in the viable cell recovery. Thiram exposure resulted in a rapid depletion of intracellular reduced glutathione (GSH) content with a concomitant increase in oxidized glutathione (GSSG) concentration. Alteration of glutathione levels was accompanied by a dose-dependent decrease in the activity of glutathione reductase (GR), a key enzyme for the regeneration of GSH from GSSG. Thiram-exposed cells exhibited increased lipid peroxidation reflected by enhanced thiobarbituric acid reactive substances (TBARS) production, suggesting that GSH depletion and the lower GR activity gave rise to increased oxidative processes. To investigate the role of decreased GSH content in the toxicity of thiram, GSH levels were modulated prior to exposure. Pretreatment of fibroblasts with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented both lipid peroxidation and cell death induced by thiram exposure. In contrast, thiram cytotoxicity was exacerbated by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO). Taken together, these results strongly suggest that thiram induces GSH depletion, leading to oxidative stress and finally cell death.     

Epigallocatechin-3-gallate(-)protects Chang liver cells against ethanol-induced cytotoxicity and apoptosis

The objective of the study was to investigate the effect of epigallocatechin-3-gallate (EGCG) on ethanol (EtOH)-induced cytotoxicity in human Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in presence of (25 microM) EGCG for 24 hr. Assays were performed in treated cells to evaluate the ability of EGCG to prevent the toxic effects of EtOH. EtOH exposure suppressed the growth of Chang liver cells and induced lactate dehydrogenase leakage, oxygen radical formation, peroxidation of lipids, mitochondrial dysfunction and apoptosis. Reduced glutathione (GSH) concentration was significantly decreased (P < 0.05) while oxidized glutathione (GSSG) concentration was significantly elevated in EtOH-treated cells as compared to normal cells. Incubation of EGCG along with EtOH significantly prevented EtOH-dependent cell loss and lactate dehydrogenase leakage. This was associated with a reduction in oxidative damage as reflected by a reduction in the generation of reactive oxygen species, and in lipid peroxidation and maintenance of intracellular GSH/GSSG ratio. EGCG decreased the accumulation of sub-G(1) phase cells and reduced apoptosis. The findings suggest that EGCG exerts a protective action during EtOH-induced liver cell damage.     

Liv.52 attenuate copper induced toxicity by inhibiting glutathione depletion and increased antioxidant enzyme activity in HepG2 cells

Altered copper metabolism plays a pivotal role in the onset of several hepatic disorders and glutathione (GSH) plays an important role in its homeostasis. Hepatic diseases are often implicated with decreased content of intracellular GSH. GSH depleted cells are prone to increased oxidative damage eventually leading to its death. Liv.52 is used to treat hepatic ailments since long time. Hence, in the present study the potential cytoprotective effect of Liv.52 against toxicity induced by copper (Cu²⁺) was evaluated in HepG2 cells. Cu²⁺ at 750 μM induced cytotoxicity to HepG2 cells as determined by MTT assay. The toxicity was brought about by increased lipid peroxidation, DNA fragmentation and decreased GSH content. But, upon treatment with Liv.52 cell death induced by Cu²⁺ was significantly abrogated by inhibition of lipid peroxidation by 58% and DNA fragmentation by 37%. Liv.52 increased the GSH content by 74%. Activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase were increased by 46%, 22% and 81% respectively in Liv.52 treated cells. Thus, it is apparent from these results that Liv.52 abrogates Cu²⁺ induced cytotoxicity in HepG2 cells by inhibiting lipid peroxidation and increased GSH content and antioxidant enzyme activity.     

A 43-kDa protein from the leaves of the herb Cajanus indicus L. modulates chloroform induced hepatotoxicity in vitro

A 43-kDa protein isolated from the leaves of the herb Cajanus indicus L. has been shown to possess a protective role against drug- and toxin- induced hepatotoxicity both in vivo and in vitro. The current study was conducted to evaluate its protective action against chloroform (CHCl3)-induced cytotoxicity in hepatocytes. Cellular viability and biochemical parameters such as glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) release from the cells were measured. In addition, the antioxidant effect of the protein was investigated from the DPPH radical scavenging assay and by determining the levels of the antioxidant enzyme catalase (CAT), cellular reserves of reduced glutathione (GSH), and lipid peroxidation end products (measured as TBARS). Treatment of the cells with CHCl3 decreased cellular viability and increased GPT and LDH. Cells treated with the protein before and immediately after CHCl3 application showed a marked improvement in their viability and reduced leakage of GPT and LDH. The levels of CAT and GSH, which were diminished in cells treated with CHCl3, were restored by protein treatment. CHCl3 induced enhancement of lipid peroxidation in hepatocytes was significantly reduced by protein treatment. Results of the DPPH assay with the protein showed its radical scavenging activity. This data suggests that the protein possesses protective activity against CHCl3-induced cytotoxicity in hepatocytes and protects against CHCl3-induced hepatic damage.     

Glutathione level regulates HNE-induced genotoxicity in human erythroleukemia cells

4-hydroxy-trans-2-nonenal (HNE) is one of the most abundant and toxic lipid aldehydes formed during lipid peroxidation by reactive oxygen species. We have investigated the genotoxic effects of HNE and its regulation by cellular glutathione (GSH) levels in human erythroleukemia (K562) cells. Incubation of K562 cells with HNE (5-10 microM) significantly elicited a 3- to 5-fold increased DNA damage in a time- and dose-dependent manner as measured by comet assay. Depletion of GSH in cells by L-buthionine-[S,R]-sulfoximine (BSO) significantly increased HNE-induced DNA damage, whereas supplementation of GSH by incubating the cells with GSH-ethyl ester significantly decreased HNE-induced genotoxicity. Further, overexpression of mGSTA4-4, a HNE-detoxifying GST isozyme, significantly prevented HNE-induced DNA damage in cells, and ablation of GSTA4-4 and aldose reductase with respective siRNAs further augmented HNE-induced DNA damage. These results suggest that the genotoxicity of HNE is highly dependent on cellular GSH/GST/AR levels and favorable modulation of the aldehyde detoxification system may help in controlling the oxidative stress-induced complications.     

Cerium oxide nanoparticles protects against acrylamide induced toxicity in HepG2 cells through modulation of oxidative stress

Acrylamide (AA) is a toxic chemical compound found in cooked foods. Considerable evidences suggest that oxidative stress and mitochondrial dysfunction are contributed to AA toxicity. Ceric oxide (CeO₂) nanoparticles (nano-ceria) have the potential to be developed as a therapeutic for oxidative stress insults due to their catalytic antioxidant properties. In this study we investigated, whether nano-ceria exerted a protective effect against AA-induced cytotoxicity and oxidative damage. HepG2 human cancer cell lines were exposed to nano-ceria (50, 100, and 200?µM) and after 30?min, AA in the half maximal inhibitory concentration (IC50) concentration (200?µM) was added to the cells. Twenty four hours later, cellular viability, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and cellular levels of glutathione (GSH) were assayed. AA decreased cell viability and pretreatment with nano-ceria significantly decreased AA-induced cytotoxicity. In addition, nano-ceria alleviated AA-induced ROS generation and LPO and depressed GSH level. Our results suggested that nano-ceria prevented cellular and oxidative damage induced by AA.     

The mechanism of elevated toxicity in HepG2 cells due to combined exposure to ethanol and ionizing radiation

Ethanol and ionizing radiation exposure are independently known to cause tissue damage through various mechanisms. The non-enzymatic and enzymatic metabolism of ethanol, the latter via the cytochrome P(450) 2E1-dependent pathway produces free radicals, which deplete cellular glutathione (GSH). Ionizing radiation exposure has been shown to induce lipid peroxidation, DNA damage, protein oxidation and GSH depletion. It was postulated that cells sensitized by ethanol will be susceptible to additional insult, such as by radiation through increased oxidative stress. In this investigation, cultured liver cells (HepG2, human hepatocellular liver carcinoma) were exposed to ethanol, followed by ionizing radiation. The antioxidant status of the cells was evaluated by an array of techniques. Levels of glutathione, cysteine (CYS), and malondialdehyde (MDA) were measured by HPLC. Activities of antioxidant enzymes, catalase and glutathione reductase (GR) were determined enzymatically. Apoptosis was evaluated by the caspases-3 assay and fluorescence microscopy. The data showed that combined treatment with ethanol and radiation resulted in the lowest levels of GSH, and highest MDA level compared with the control. The catalase activity was lower in the combined exposure groups, when compared with the single agent exposure groups, and the glutathione reductase activity was the highest in the combined exposure groups and lowest in the control. These findings suggest that a combination of ethanol and ionizing radiation results in greater toxicity in vitro through elevated oxidative stress.     

Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes

A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of acetaminophen and the toxicological significance of glutathione (GSH) depletion. The relative time of onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time- and dose-dependent depletion of cellular GSH. The acetaminophen-induced GSH depletion and ensuing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset, in contrast to that caused by iodoacetamide (IAA) and by diethylmaleate (DEM), 2 well-known depletors of GSH. There was comparable LDH leakage, irrespective of drug treatment, when GSH levels decreased to about 20% of normal. Reduction of GSH levels below the 20% threshold by IAA treatment resulted in marked LDH leakage and loss of viability. Maximal LDH leakage in response to IAA and acetaminophen preceded maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence of cell damage as well as GSH depletion. IAA and DEM produced a comparable, modest accumulation of MDA, yet IAA was much more cytotoxic. These findings indicate that lipid peroxidation does not play a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in vivo, the system can be a useful and valid model with which to study mechanisms of chemical toxicity.     

Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant

Oxidative stress has been implicated as an important factor in many neurological diseases. Oxidative toxicity in a number of these conditions is induced by excessive glutamate release and subsequent glutamatergic neuronal stimulation. This, in turn, causes increased generation of reactive oxygen species (ROS), oxidative stress, excitotoxicity, and neuronal damage. Recent studies indicate that the glutamatergic neurotransmitter system is involved in lead-induced neurotoxicity. Therefore, this study aimed to (1) investigate the potential effects of glutamate on lead-induced PC12 cell death and (2) elucidate whether the novel thiol antioxidant N-acetylcysteine amide (NACA) had any protective abilities against such cytotoxicity. Our results suggest that glutamate (1 mM) potentiates lead-induced cytotoxicity by increased generation of ROS, decreased proliferation (MTS), decreased glutathione (GSH) levels, and depletion of cellular adenosine-triphosphate (ATP). Consistent with its ability to decrease ATP levels and induce cell death, lead also increased caspase-3 activity, an effect potentiated by glutamate. Exposure to glutamate and lead elevated the cellular malondialdehyde (MDA) levels and phospholipase-A(2) (PLA(2)) activity and diminished the glutamine synthetase (GS) activity. NACA protected PC12 cells from the cytotoxic effects of glutamate plus lead, as evaluated by MTS assay. NACA reduced the decrease in the cellular ATP levels and restored the intracellular GSH levels. The increased levels of ROS and MDA in glutamate-lead treated cells were significantly decreased by NACA. In conclusion, our data showed that glutamate potentiated the effects of lead-induced PC12 cell death by a mechanism involving mitochondrial dysfunction (ATP depletion) and oxidative stress. NACA had a protective role against the combined toxic effects of glutamate and lead by inhibiting lipid peroxidation and scavenging ROS, thus preserving intracellular GSH.     

Potentiation of cadmium-induced cytotoxicity by sulfur amino acid deprivation through activation of extracellular signal-regulated kinase1/2 (ERK1/2) in conjunction with p38 kinase or c-jun N-terminal kinase (JNK). Complete inhibition of the potentiated toxicity by U0126 an ERK1/2 and p38 kinase inhibitor.

The mechanisms of cadmium-induced toxicity may include oxidative stress, altered redox homeostasis, and injuries to organelles. The current study was designed to study the effect of decreased cellular glutathione (GSH) content by sulfur amino acid deprivation on cadmium toxicity and to identify the signaling pathways responsible for the cytotoxicity. GSH content was increased by cadmium in H4IIE cells prior to cell death, which was prevented by excess GSH or cysteine. Cell viability, however, was not improved by GSH or cysteine complexation of cadmium. Cadmium-induced cytotoxicity was 40-fold potentiated in cells with decreased GSH by sulfur amino acid deprivation. Cadmium in combination with decreased GSH markedly increased apoptotic cell death. Mitogen-activated protein kinases including extracellular signal-regulated kinase 1/2, p38 kinase and c-Jun N-terminal kinase (JNK) were all activated 1-12 hr after sulfur amino acid deprivation. U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), which inhibited activation of extracellular signal-regulated kinase1/2 and p38 kinase in cells under sulfur amino acid deprivation, completely prevented potentiation in Cd-induced cytotoxicity and apoptosis. Potentiation of cadmium toxicity by sulfur amino acid deprivation was prevented in part by either PD98059 or SB203580, or in cells stably expressing dominant negative mutant of JNK1, and to greater extents by PD98059 in combination with either SB203580 or JNK1(-) transfection. These results demonstrated that decreased cellular GSH content potentiated cytotoxicity induced by cadmium at the level of human exposure, and that the potentiation of cytotoxicity resulted from activation of extracellular signal-regulated kinase1/2 in conjunction with p38 kinase or JNK.     

Effects of 1,2-dibromoethane on isolated hepatocytes: functional alterations and induction of lipid peroxidation

1. Isolated rat hepatocytes were exposed to the fumigant 1,2-dibromoethane (DBE) and cytotoxicity was evaluated by studying parameters of cellular function and lipid peroxidation. 2. DBE caused plasma membrane damage, as determined by leakage of lactate dehydrogenase, and was more severe in shaken suspensions than stationary suspensions, suggesting that cells were more fragile after DBE exposure. 3. DBE decreased hepatocyte glycogen content and stimulated albumin synthesis in hepatocyte suspensions. 4. Lipid peroxidation resulted from DBE exposure and was greater in cells isolated from phenobarbital-pretreated rats. Shaking the suspensions enhanced lipid peroxidation. Ethane production did not parallel formation of thiobarbituric acid reactants, suggesting that these parameters of lipid peroxidation reflect different mechanisms of molecular interaction of DBE.     

The protective role of intracellular GSH status in the arsenite-induced vascular endothelial dysfunction

In this study, we used porcine aortic endothelial cells (PAECs) as an in vitro system to investigate the role of intracellular GSH status in arsenite-induced vascular endothelial damage. Exposure of PAECs to l-buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), markedly enhanced the arsenite-induced cytotoxicity. The data implied that intracellular GSH might play an important role in protection of PAECs from arsenite-induced cytotoxicity. Low concentrations of arsenite exposure increased intracellular GSH concentrations, whereas high concentrations of arsenite exposure decreased intracellular GSH concentrations. We further modulated intracellular GSH concentration by using GSH modulators. N-Acetyl cysteine (NAC) and l-cystine (oxidized l-cysteine), by up-regulating intracellular GSH concentrations, were shown to protect PAECs from arsenite-induced cytotoxicity. On the other hand, BSO and monosodium glutamate (MSG), which down-regulated the intracellular GSH concentrations, further potentiated arsenite-induced cytotoxicity. Moreover, exposure of PAECs to NAC alleviated the arsenite-induced JNK/AP-1 activation and apoptosis, whereas exposure of PAECs to BSO enhanced the arsenite-induced JNK/AP-1 activation and apoptosis. These results indicated that an increase in GSH content represented one of the detoxification mechanisms responding to arsenite exposure and probably played critical roles in the regulation of stress-response signaling molecules as well as in protection of PAECs from arsenite attack.     

Cytotoxicity,oxidative stress,and inflammation in human Hep G2 liver epithelial cells following exposure to gold nanorods

The gold nanorods (GNRs) are great potentials in imaging, therapy, biosensing, and many other commercial applications. However, GNRs interactions with human cells and potential health risks remain not well known. The present investigation aimed to evaluate the in vitro toxicity of 10 and 25?nm GNRs (10–50?μg/mL) following exposure for 48?h in human Hep G2 liver epithelial cells using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) leakage, glutathione (GSH) estimation, lipid peroxidation (TBARS), caspase-3 levels, and interleukin-8 (IL-8) release assays. Exposure of GNRs to cells results in decrease in cell viability and causes cell membrane damage through LDH leakage results in cytotoxicity. The IC₅₀ (concentration required to inhibit 50% of cells) values of 10?nm GNRs, 25?nm GNRs, and quartz (toxic control)-treated cells were found to be 19.9, 26.8, and 36.35?μg/mL, suggesting the higher cytotoxicity of GNRs. The GNRs exposure to liver cells found in depleted GSH levels, increased lipid peroxidation, and increased caspase-3 levels leads to induction of oxidative stress. In addition, enhanced levels of IL-8 were found, a sign of inflammation. The 10?nm GNRs have shown significant toxicity against all biochemical assays when compare to 25?nm GNRs and quartz-treated cells. Finally, the data indicate that the concentration size-dependent in vitro toxicity of GNRs toward liver Hep G2 cells. The toxicity of GNRs may be due to cell membrane damage, induction of oxidative stress, and inflammatory mediator release. Further investigations are necessitated to elucidate the in vivo toxicity of GNRs.     

Contribution of glutathione and metallothioneins to protection against copper toxicity and redox cycling: quantitative analysis using MT+/+ and MT-/- mouse lung fibroblast cells

Glutathione (GSH) and metallothioneins (MT) are believed to play important roles in protecting cells against high copper (Cu) concentrations. Little is known, however, about their specific intracellular interactions and the coordination of protective functions. We investigated contributions of GSH and MT to protection against Cu toxicity in fibroblasts derived from wild-type (MT+/+) and knockout (MT-/-) mice that were challenged with cupric nitrilotriacetate (Cu-NTA). Endogenous levels of GSH and MT were manipulated using an inhibitor of gamma-glutamylcysteine synthetase, buthionine sulfoximine (BSO, 5 microM), as GSH depletor and ZnCl(2) (100 microM) as inducer of MT expression. BSO pretreatment markedly decreased cellular GSH levels in MT+/+ and MT-/- cells, by 65% and 70%, respectively, which resulted in Cu cytotoxicity accompanied by its elevated redox-cycling activity and enhanced Cu-induced membrane phospholipid peroxidation. BSO-pretreated MT-/- cells were markedly more sensitive to Cu despite the fact that the residual levels of GSH were similar in both BSO-pretreated MT+/+ and MT-/- cells. Zn pretreatment resulted in more than 10-fold induction of MT in MT+/+ cells but not in MT-/- cells. Accordingly, Zn pretreatment afforded significant protection of MT+/+ cells against Cu cytotoxicity, likely associated with MT-dependent suppression of Cu redox-cycling activity and phospholipid peroxidation, but it exerted no protection in MT-/- cells (as compared to naive cells). To determine whether MT functions specifically in Cu regulation or rather acts as a nonspecific Cu-binding cysteine-rich nucleophile, experiments were performed using MT+/+ and MT-/- cells pretreated with both BSO and Zn. BSO pretreatment did not affect Zn-induced MT expression in MT+/+ cells. As compared with BSO pretreatment alone, exposure to Cu of MT+/+ cells after Zn/BSO pretreatment resulted in the following: (i) a significantly higher viability; (ii) attenuated Cu-dependent redox-cycling activity; and (iii) a lower level of phospholipid peroxidation. In BSO/Zn-pretreated MT-/- cells, the redox-cycling activity of Cu and the level of phospholipid peroxidation remained remarkably higher than in naive cells and were not significantly different from those in cells pretreated with BSO alone. Cu-induced toxicity was remarkably higher in BSO/Zn-pretreated MT-/- cells than in naive or Zn-pretreated cells, although slightly lower than in the MT-/- cells pretreated with BSO alone.     

Oxidative stress mediated cytotoxicity of cyanide in LLC-MK2 cells and its attenuation by alpha-ketoglutarate and N-acetyl cysteine

Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.     

Cytotoxicity in ciprofloxacin-treated human fibroblast cells and protection by vitamin E

Quinolones (Qs) were shown to have cytotoxic effects in various cell lines including human carcinoma cells; however, mechanism of these effects was not fully understood. To investigate the possibility of the involvement of an oxidative stress induction in this mechanism of action, we examined viability of human fibroblast cells exposed to a Q antibiotic, ciprofloxacin (CPFX), and measured lipid peroxidation and total glutathione (GSH) levels, and activities of catalase (Cat), superoxide dismutases (SODs), glutathione peroxidase (GPx). The effects of vitamin E pretreatment on those parameters were also examined. Our results showed that the effect of CPFX on the viability of the cells, as determined by neutral red uptake assay, was time dependent. Cytotoxicity was not observed in the concentration range of 0.0129-0.387 mM CPFX when the cells were incubated for 24 hours. However, significant level of cytotoxicity was observed at concentrations 0.129 and 0.194 mM, and >0.129 mM, following 48 and 72 hours of exposure, respectively. When the cells were exposed to 0.194 mM CPFX for 48 hours, the level of lipid peroxidation increased and the content of total GSH decreased significantly; activities of total SOD, Mn SOD and CuZn SOD did not change; the decrease observed in the activity of Cat was not significant; and the activity of GPx was highly variable. Vitamin E pretreatment of the cells provided significant protection against CPFX-induced cytotoxicity; lowered the level of lipid peroxidation significantly, but increased the total GSH content only moderately; no change was observed in the activities of Cat and total SOD, but a significant increase in Mn SOD and a significant decrease in CuZn SOD were noticed. These results suggested that CPFX-induced cytotoxicity on human fibroblast cell cultures is related to oxidative stress, and vitamin E pretreatment can afford a protection.     

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40-50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50-100 microM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO).     

Effects of 1,2-dibromoethane on isolated hepatocytes: Functional alterations and induction of lipid peroxidation

1. Isolated rat hepatocytes were exposed to the fumigant 1,2-dibromoethane (DBE) and cytotoxicity was evaluated by studying parameters of cellular function and lipid peroxidation.

2. DBE caused plasma membrane damage, as determined by leakage of lactate dehydrogenase, and was more severe in shaken suspensions than stationary suspensions, suggesting that cells were more fragile after DBE exposure.

3. DBE decreased hepatocyte glycogen content and stimulated albumin synthesis in hepatocyte suspensions.

4. Lipid peroxidation resulted from DBE exposure and was greater in cells isolated from phenobarbital-pretreated rats. Shaking the suspensions enhanced lipid peroxidation. Ethane production did not parallel formation of thiobarbituric acid reactants, suggesting that these parameters of lipid peroxidation reflect different mechanisms of molecular interaction of DBE.