The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: protection by albumin

Like other nonsteroidal anti-inflammatory drugs, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide) triggers hepatitis in a few recipients. Although nimesulide has been shown to uncouple mitochondrial respiration and cause hepatocyte necrosis in the absence of albumin, mechanisms for cell death are incompletely understood, and comparisons with human concentrations are difficult because 99% of nimesulide is albumin-bound. We studied the effects of nimesulide, with or without a physiological concentration of albumin, in isolated rat liver mitochondria or microsomes and in human hepatoma cells. Nimesulide did not undergo monoelectronic nitro reduction in microsomes. In mitochondria incubated without albumin, nimesulide (50 microM) decreased the mitochondrial membrane potential (DeltaPsim), increased basal respiration, and potentiated the mitochondrial permeability transition (MPT) triggered by calcium preloading. In HUH-7 cells incubated for 24 h without albumin, nimesulide (1 mM) decreased the DeltaPsim and cell NADPH and increased the glutathione disulfide/reduced glutathione ratio and cell peroxides; nimesulide triggered MPT, ATP depletion, high cell calcium, and caused mostly necrosis, with rare apoptotic cells. Coincubation with either cyclosporin A (an MPT inhibitor) or the combination of fructose-1,6-diphosphate (a glycolysis substrate) and oligomycin (an ATPase inhibitor) prevented the decrease in DeltaPsim, ATP depletion, and cell death. A physiological concentration of albumin abolished the effects of nimesulide on isolated mitochondria or HUH-7 cells. In conclusion, the weak acid, nimesulide, uncouples mitochondria and triggers MPT and ATP depletion in isolated mitochondria or hepatoma cells incubated without albumin. However, in the presence of albumin, only a fraction of the drug enters cells or organelles, and uncoupling and toxicity are not observed.     

Mechanisms of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes

Freshly isolated mouse hepatocytes were used to determine the role of mitochondrial permeability transition (MPT) in acetaminophen (APAP) toxicity. Incubation of APAP (1 mM) with hepatocytes resulted in cell death as indicated by increased alanine aminotransferase in the media and propidium iodide fluorescence. To separate metabolic events from later events in toxicity, hepatocytes were preincubated with APAP for 2 h followed by centrifugation of the cells and resuspension of the pellet to remove the drug and reincubating the cells in media alone. At 2 h, toxicity was not significantly different between control and APAP-incubated cells; however, preincubation with APAP followed by reincubation with media alone resulted in a marked increase in toxicity at 3 to 5 h that was not different from incubation with APAP for the entire time. Inclusion of cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine (NAC) in the reincubation phase prevented hepatocyte toxicity. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine methyl ester perchlorate fluorescence decreased during the reincubation phase indicating a loss of mitochondrial membrane potential. Inclusion of cyclosporine A, DTT, or NAC decreased oxidative stress and loss of mitochondrial membrane potential. Confocal microscopy studies with the dye calcein acetoxymethyl ester indicated that MPT had also occurred. These data are consistent with a hypothesis where APAP-induced cell death occurs by two phases, a metabolic phase and an oxidative phase. The metabolic phase occurs with GSH depletion and APAP-protein binding. The oxidative phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, and toxicity.     

NIM811 (N-methyl-4-isoleucine cyclosporine), a mitochondrial permeability transition inhibitor, attenuates cholestatic liver injury but not fibrosis in mice

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were approximately 1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor alpha, transforming growth factor-beta1, procollagen alpha1(I) mRNA, alpha-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.     

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.     

Increased susceptibility of fat-laden Zucker-rat hepatocytes to bile acid-induced oncotic necrosis: an in vitro model of steatocholestasis

Metabolic liver disorders cause chronic liver disease and liver failure in childhood. Many of these disorders share the histologic features of steatosis and cholestasis, or steatocholestasis. In this study we sought to (1) develop an in vitro model of steatocholestasis, (2) determine the mechanisms of cell death in this model, and (3) determine the role of mitochondrial disturbances in this model. Methods: Hepatocytes were isolated from 8-week-old obese (fa/fa) and lean Zucker rats. Cell suspensions were treated with glycochenodeoxycholic acid (GCDC), after which reactive oxygen species (ROS) generation, oncotic necrosis, apoptosis, and ATP content were assessed. Isolated liver mitochondria were exposed to GCDC and analyzed for ROS generation, mitochondrial membrane-permeability transition (MPT), and cytochrome c release. Oncotic necrosis was significantly increased and apoptosis reduced in fa/fa hepatocytes exposed to GCDC compared with that in lean hepatocytes. Necrosis occurred by way of an ROS- and MPT-dependent pathway. Basal and dynamic ATP content did not differ between fa/fa and lean hepatocytes. GCDC stimulated ROS generation, MPT, and cytochrome c release to a similar extent in purified mitochondria from both fa/fa and lean rats. These findings suggest that fat-laden hepatocytes favor a necrotic rather than an apoptotic cell death when exposed to low concentrations of bile acids. The protective effects of antioxidants and MPT blockers suggest novel therapeutic strategies for the treatment of steatocholestatic metabolic liver diseases.     

Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.     

Azathioprine acts upon rat hepatocyte mitochondria and stress-activated protein kinases leading to necrosis: protective role of N-acetyl-L-cysteine

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.     

Role of mitochondrial cytochrome c in cocaine-induced apoptosis in coronary artery endothelial cells

Cocaine induces apoptosis in coronary artery endothelial cells. Yet the cellular and molecular mechanisms are not clear. Given that cocaine has profound toxic effects on the mitochondria, the present study examined the role of mitochondrial cytochrome c in cocaine-mediated apoptosis. Using cultured bovine coronary artery endothelial cells, we found that cocaine-induced apoptosis was dose dependently inhibited by cyclosporin A with IC(50) of 0.2 microM. The maximum of 65% inhibition was obtained with 3 microM cyclosporin A. Cocaine induced a translocation of cytochrome c from the mitochondria to the cytosol with a 1.8-fold increase in cytosolic cytochrome c levels, and a corresponding decrease in mitochondrial cytochrome c. In accordance with its inhibition of cocaine-induced apoptosis, cyclosporin A blocked cocaine-induced cytochrome c translocation. Correspondingly, cocaine-induced activation of caspase-9 preceded that of caspase-3. Caspase-8 was not activated. Cocaine also produced a dose-dependent decrease in Bcl-2 protein levels, but had no effect on Bax protein levels. The cocaine-induced decrease in the Bcl-2 protein was not affected by cyclosporin A but was partially blocked by caspase-3 inhibitor Ac-DEVD-CHO. Collectively, these data indicate that the release of cytochrome c from the mitochondria and the subsequent activation of caspase-9 and caspase-3 play a key role in cocaine-induced apoptosis in these cells. Furthermore, the down-regulation of the Bcl-2 protein may play an important role in cocaine-induced release of cytochrome c.     

Antihypertrophic effect of Na+/H+ exchanger isoform 1 inhibition is mediated by reduced mitogen-activated protein kinase activation secondary to improved mitochondrial integrity and decreased generation of mitochondrial-derived reactive oxygen species

Although inhibition of Na+/H+ exchanger isoform 1 (NHE-1) reduces cardiomyocyte hypertrophy, the mechanisms underlying this effect are not known. Recent evidence suggests that this may be associated with improved mitochondrial function. To understand the mechanistic bases for mitochondrial involvement in the antihypertrophic effect of NHE-1 inhibition, we examined the effect of the NHE-1-specific inhibitor N-[2-methyl-4,5-bis(methylsulphonyl)-benzoyl]-guanidine, hydrochloride (EMD, EMD87580; 5 microM) on the hypertrophic phenotype, mitogen-activated protein kinase (MAPK) activity, mitochondrial membrane potential (Deltapsim), permeability transition (MPT) pore opening, and superoxide generation in phenylephrine (PE)-treated neonatal rat cardiomyocytes. EMD significantly suppressed markers of cell hypertrophy, including cell surface area and gene expression of atrial natriuretic peptide and alpha-skeletal actin. EMD inhibited the PE-induced MPT pore opening, prevented the loss in Deltapsim, and attenuated superoxide generation induced by PE. Moreover, the activation of p38 MAPK (p38) and extracellular signal-regulated kinase (ERK) 1/2 MAPKs induced by PE was significantly attenuated in the presence of EMD as well as the antioxidant catalase. To examine the role of MPT and mitochondrial Ca2+ uniport in parallel with EMD, the effects of cyclosporin A (0.2 microM) and ruthenium red (10 microM) were evaluated. Both agents significantly attenuated PE-induced hypertrophy and inhibited both mitochondrial dysfunction and p38 and ERK1/2 MAPK activation. Our results suggest a novel mechanism for attenuation of the hypertrophic phenotype by NHE-1 inhibition that is mediated by a reduction in PE-induced MAPK activation and superoxide production secondary to improved mitochondrial integrity.     

Fe(III) shifts the mitochondria permeability transition-eliciting capacity of mangiferin to protection of organelle

Mangiferin acts as a strong antioxidant on mitochondria. However, when in the presence of Ca(2+), mangiferin elicits mitochondrial permeability transition (MPT), as evidenced by cyclosporin A-sensitive mitochondrial swelling. We now provide evidence, by means of electrochemical and UV-visible spectroscopical analysis, that Fe(III) coordinates with mangiferin. The resulting mangiferin-Fe(III) complex does not elicit MPT and prevents MPT by scavenging reactive oxygen species. Indeed, the complex protects mitochondrial membrane protein thiols and glutathione from oxidation. Fe(III) also significantly increases the ability of mangiferin to scavenge the 2,2-diphenyl-1-picrylhydrazyl radical, as well as to display antioxidant activity toward antimycin A-induced H(2)O(2) production and t-butyl hydroperoxide-promoted membrane lipid peroxidation in mitochondria. We postulate that coordination with Fe(III) constitutes a potential protective mechanism toward the prooxidant action of mangiferin and other catechol-containing antioxidants regarding MPT induction. Potential therapeutic relevance of this finding for conditions of pathological iron overload is discussed.     

Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity

Amiodarone (AM), a potent antidysrhythmic agent, can cause potentially life-threatening pulmonary fibrosis. In the present investigation of mechanisms of initiation of AM lung toxicity, we found that 100 microM AM decreased mitochondrial membrane potential in intact hamster lung alveolar macrophages and preparations enriched in isolated alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells, following 2 h of incubation. This was followed by a drop in cellular ATP content (by 32--77%) at 4 to 6 h, and 30 to 55% loss of viability at 24 h. Supplementation of incubation media with 5.0 mM glucose or 2.0 mM niacin did not reduce AM-induced ATP depletion or cell death in macrophages, and the mitochondrial permeability transition inhibitor cyclosporin A (1.0 microM) did not affect AM cytotoxicity. At 50 microM, the AM metabolite N-desethylamiodarone (DEA) produced effects similar to those of AM, but more rapidly and extensively, with the Clara cell-enriched preparation being particularly susceptible. In isolated whole lung mitochondria, DEA was accumulated to a greater extent than AM. Both AM and DEA inhibited complex I- and complex II-supported respiration, but DEA inhibited complex II to a greater degree than AM. These results demonstrate that AM and DEA disrupt mitochondrial membrane potential prior to ATP depletion and subsequent lung cell death, that DEA is more potent than AM, and that the mitochondrial permeability transition is not involved in mitochondrial perturbation by AM. This suggests that AM- and DEA-induced perturbations of mitochondrial function may initiate AM-induced pulmonary toxicity.     

The critical role of mitochondrial energetic impairment in the toxicity of nimesulide to hepatocytes

We described the effects of nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) and its reduced metabolite in isolated rat hepatocytes. Nimesulide stimulated the succinate-supported state 4 respiration of mitochondria, indicating an uncoupling effect of the drug. Incubation of hepatocytes with nimesulide (0.1-1 mM) elicited a concentration- and time-dependent decrease in cell viability as assessed by lactate dehydrogenase leakage, a decrease of mitochondrial membrane potential as assessed by rhodamine 123 retention, and cell ATP depression. Nimesulide also decreased the levels of NAD(P)H and glutathione in hepatocytes, but the extent of the effects was less pronounced in relation to the energetic parameters; in addition, these effects did not imply the peroxidation of membrane lipids. The decrease in the viability of hepatocytes was prevented by fructose and, to a larger extent, by fructose plus oligomycin; it was stimulated by proadifen, a cytochrome P450 inhibitor. In contrast, the reduced metabolite of nimesulide did not present any of the effects observed for the parent drug. These results indicate that: 1) nimesulide causes injury to the isolated rat liver cells, 2) this effect is mainly mediated by impairment of ATP production by mitochondria due to uncoupling, and 3) on account of the activity of its nitro group, the parent drug by itself is the main factor responsible for its toxicity to the hepatocytes.     

Protective effect of glycyrrhizin on 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death in differentiated PC12 cells

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against the toxicity of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and role of oxidative stress. MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species, and the depletion of glutathione (GSH) in differentiated PC12 cells. Glycyrrhizin up to 100 microM significantly attenuated cell death and depletion of GSH due to MPP+ concentration-dependently. Meanwhile, 18beta-glycyrrhetinic acid showed a maximal inhibitory effect at 10 microM; beyond this concentration, the inhibitory effect declined. The protective effect of licorice compounds was also detected in the rotenone-treated PC12 cells. Glycyrrhizin and 18beta-glycyrrhetinic acid prevented the MPP+-induced formation of the mitochondrial permeability transition. The results show that both glycyrrhizin and a metabolite, 18beta-glycyrrhetinic acid, exhibit a depressant effect against the MPP+ toxicity. Glycyrrhizin and 18beta-glycyrrhetinic acid may prevent the cytotoxicity of MPP+ by suppressing the mitochondrial permeability transition formation. The preventive effect seems to be ascribed to the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.     

Diglycolic acid,the toxic metabolite of diethylene glycol,chelates calcium and produces renal mitochondrial dysfunction in vitro

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. Materials and methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5?mmol/L) than succinate respiration (100?mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.     

Mechanism of inhibition of rat liver mitochondrial respiration by oxmetidine, an H2-receptor antagonist

Suspensions of rat liver hepatocytes exposed to oxmetidine rapidly lose viability, an event preceded by a marked and rapid inhibition of cell respiration and depletion of ATP. In isolated rat liver mitochondria (RLM), oxmetidine inhibits pyruvate/malate- but not succinate-supported, ADP-stimulated oxygen consumption (state 3). The purpose of this investigation was to determine the exact molecular site of oxmetidine-induced inhibition of RLM electron transport. Oxmetidine did not significantly inhibit succinate-supported, ADP-stimulated state 3 oxygen consumption in isolated RLM at concentrations up to 0.5 mM. In contrast, oxmetidine significantly inhibited beta-hydroxybutyrate- or isocitrate-supported mitochondrial state 3 oxygen consumption at concentrations above 10 microM and 25 microM, respectively. In RLM electron transport particles (ETP), oxmetidine inhibited NADH-oxidase and NADH-CoQ reductase activity (IC50 of 3.4 microM and 2.6 microM, respectively). However, oxmetidine did not significantly affect NADH-Fe3(CN)6 reductase activity (at concentrations up to 200 microM). SK&F 92058, a thiourea analog of oxmetidine approximately 24-fold less toxic to hepatocytes, produced a similar pattern of inhibition of respiration, although far less potent (IC50 of 0.8 mM and 0.6 mM for NADH-oxidase and NADH-CoQ reductase, respectively). SK&F 92058 did not significantly inhibit NADH-Fe3(CN)6 reductase activity at concentrations up to 3.0 mM. Studies with [14C]oxmetidine failed to show any specific, saturable binding to rat liver ETP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for the hepatotoxicity of the antiandrogen, nilutamide. Evidence suggesting that redox cycling of this nitroaromatic drug leads to oxidative stress in isolated hepatocytes.

The nitroaromatic drug nilutamide has been shown previously to undergo redox cycling in aerobic rat liver microsomes, being reduced by NADPH-cytochrome P-450 reductase to a nitro anion-free radical which reacts with oxygen, to regenerate the parent drug, and form a superoxide anion dismuted to hydrogen peroxide. In the present study, the effects of nilutamide on isolated rat hepatocytes have been determined. After 6 and 8 hr of incubation with 0.5 mM nilutamide, lactate dehydrogenase was released in the incubation medium, and cell viability was decreased markedly. Consistent with a redox cycle producing reactive oxygen species, nilutamide increased nonmitochondrial (cyanide-resistant) oxygen consumption; the toxicity of nilutamide occurred sooner and was more extensive in the presence of sodium azide (an inhibitor of catalase). Consistent with an oxidative stress, the toxicity of nilutamide was associated with depletion of reduced glutathione, increased levels of glutathione disulfide, increased Ca(++)-dependent phosphorylase a activity, oxidation and accumulation of cytoskeleton-associated proteins and formation of blebs; toxicity was prevented by glutathione precursors, thiol reductants and/or antioxidants, such as L-cystine, L-cysteine, N-acetyl-L-cysteine, dithiothreitol, N,N'-diphenyl-p-phenylene-diamine and alpha-tocopherol. Feeding the animals with a diet supplemented with 2% L-cystine increased the initial glutathione stores of hepatocytes and prevented nilutamide toxicity. It is concluded that nilutamide is toxic to isolated rat hepatocytes, as a probable consequence of an oxidative stress due to the redox cycling of this nitroaromatic compound.     

Lysophosphatidic acid induces rapid and sustained decreases in epidermal growth factor receptor binding via different signaling pathways in BEAS-2B airway epithelial cells

L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.     

Structure, function and regulation of the mitochondrial peripheral-type benzodiazepine receptor.

Steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Through in vitro and in vivo studies using various steroidogenic cell models and with the help of pharmacological, biochemical, morphological and molecular approaches we demonstrated that the peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial protein that interacts with other proteins in the outer mitochondrial membrane to form a multimeric complex. PBR is required for the binding, uptake and release, upon ligand activation, of the substrate cholesterol. Thus, cholesterol becomes available to the inner mitochondrial membrane P450scc where steroid biosynthesis begins. The presence of mitochondrial PBR is also critical in maintaining outer mitochondrial membrane stability and in preventing apoptosis. Considering these functions of PBR and the fact that PBR is a ubiquitous protein, it is suggested that this drug receptor may serve as a target to control various mitochondrial and cell functions and to protect against experimentally or pathologically induced mitochondrial and cell toxicity.     

The myxoma poxvirus protein,M11L,prevents apoptosis by direct interaction with the mitochondrial permeability transition pore

M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.