All-trans-retinoic acid binds to and inhibits adenine nucleotide translocase and induces mitochondrial permeability transition

We investigated the effects of retinoic acids on mitochondrial permeability transition (MPT) measured as changes in rhodamine 123 fluorescence from both isolated heart mitochondria and HeLa cells. We report that all-trans-retinoic acid (atRA), 9-cis-retinoic acid, and 13-cis-retinoic acid induce a drop in mitochondrial membrane potential in isolated mitochondria. The atRA effect was done through the induction of MPT because it was dependent on Ca(2+), in a synergic mechanism, and inhibited by cyclosporin A (CsA). Furthermore, atRA also opened MPT in vivo, because treatment of HeLa cells with atRA results in a CsA-sensitive drop of mitochondrial membrane potential. We demonstrated for the first time that retinoic acids inhibit adenine nucleotide translocase (ANT) activity in heart and liver mitochondria. Kinetic studies revealed atRA as an uncompetitive inhibitor of ANT. Photoaffinity labeling of mitochondrial proteins with [3H]atRA demonstrated the binding of a 31-kDa protein to atRA. This protein was identified as ANT because the presence of carboxyatractyloside, a specific ANT inhibitor, prevented labeling. The specific photolabeling of ANT was also prevented in a concentration-dependent manner by nonlabeled atRA, whereas palmitic acid was ineffective. This study indicates that specific interaction between atRA and ANT takes place regulating MPT opening and adenylate transport. These observations establish a novel mechanism for atRA action, which could control both energetic and apoptotic mitochondrial processes in situations such as retinoic acid treatment.     

Roles of long chain fatty acids and carnitine in mitochondrial membrane permeability transition

Palmitoyl-CoA (Pal-CoA) lowered the respiratory control ratio (RCR), and induced mitochondrial membrane permeability transition (MPT) and cytochrome c (Cyt. c) release from isolated rat liver mitochondria. L-Carnitine suppressed the Pal-CoA-induced dysfunction, MPT, and Cyt. c release of isolated mitochondria. This suppression was inhibited by cephaloridine, an inhibitor of carnitine uptake into mitochondria. Cyclosporin A (CsA), an inhibitor of MPT, and BSA also suppressed the Pal-CoA-induced MPT. In the presence of inorganic phosphate (P(i)), Ca2+-induced MPT was suppressed by BSA, L-carnitine, and chlorpromazine, an inhibitor of phospholipase A2. In the presence of a low concentration of Ca2+, 3,3',5-triiodothyronine, long chain fatty acids, salicylic acid, and diclofenac induced MPT by a mechanism that was suppressed by BSA, L-carnitine, or chlorpromazine. During the incubation of mitochondria on ice, their respiratory competence decreased; L-carnitine and BSA also prevented this decrease. Mitochondrial depolarization in pheochromocytoma PC12 cells was induced by either serum deprivation or arachidonic acid by a mechanism that was suppressed by acetyl-L-carnitine. These results indicate that some MPTs may be regulated by fatty acid metabolism and that the Pal-CoA-induced MPT plays an important role in the induction of apoptosis.     

Dibenzofuran-induced mitochondrial dysfunction: Interaction with ANT carrier

Exposure to environmental pollutants such as dibenzofurans and furans is linked to the pathophysiology of several diseases. Dibenzofuran (DBF) is listed as a pollutant of concern due to its persistence in the environment, bioaccumulation and toxicity to humans, being associated with the development of lung diseases and cancers, due to its extremely toxic properties such as carcinogenic and teratogenic.Mitochondria play a key role in cellular homeostasis and keeping a proper energy supply for eukaryotic cells is essential in the fulfillment of the tissues energy-demand. Therefore, interference with mitochondrial function leads to cell death and organ failure. In this work, the effects of DBF on isolated rat liver mitochondria were analyzed.DBF exposure caused a markedly increase in the lag phase that follows depolarization induced by ADP, indicating an effect in the phosphorylative system. This was associated with a dose-dependent decrease in ATPase activity. Moreover, DBF also increased the threshold to the induction of the mitochondrial permeability transition (MPT) by calcium. Pretreatment of mitochondria with DBF also increased the concentration of carboxyatractyloside (CAT) necessary to abolish ADP phosphorylation and to induce the MPT, suggesting that DBF may interfere with mitochondria through an effect on the adenine nucleotide translocase (ANT). By co-immunoprecipitating ANT and Cyclophilin D (CypD) following MPT induction, we observed that in the presence of DBF, the ratio CypD/ANT was decreased. This demonstrates that DBF interferes with the ANT and so prevents CypD binding to the ANT, causing decreased phosphorylative capacity and inhibiting the MPT, which is also reflected by an increase in calcium retention capacity.Clarifying the role of pollutants in some mechanisms of toxicity, such as unbalance of bioenergetics status and mitochondrial function, may help to explain the progressive and chronic evolution of diseases derived from exposure to environmental pollutants.     

Effects of nimesulide and its reduced metabolite on mitochondria

1. We investigated the effects of nimesulide, a recently developed non-steroidal anti-inflammatory drug, and of a metabolite resulting from reduction of the nitro group to an amine derivative, on succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 20 microM Ca(2+), 1 microM cyclosporin A (CsA) or 5 microM ruthenium red. 2. Nimesulide uncoupled mitochondria through a protonophoretic mechanism and oxidized mitochondrial NAD(P)H, both effects presenting an EC(50) of approximately 5 microM. 3. Within the same concentration range nimesulide induced mitochondrial Ca(2+) efflux in a partly ruthenium red-sensitive manner, and induced mitochondrial permeability transition (MPT) when ruthenium red was added after Ca(2+) uptake by mitochondria. Nimesulide induced MPT even in de-energized mitochondria incubated with 0.5 mM Ca(2+). 4. Both Ca(2+) efflux and MPT were prevented to a similar extent by CsA, Mg(2+), ADP, ATP and butylhydroxytoluene, whereas dithiothreitol and N-ethylmaleimide, which markedly prevented MPT, had only a partial or no effect on Ca(2+) efflux, respectively. 5. The reduction of the nitro group of nimesulide to an amine derivative completely suppressed the above mitochondrial responses, indicating that the nitro group determines both the protonophoretic and NAD(P)H oxidant properties of the drug. 6. The nimesulide reduction product demonstrated a partial protective effect against accumulation of reactive oxygen species derived from mitochondria under conditions of oxidative stress like those resulting from the presence of t-butyl hydroperoxide. 7. The main conclusion is that nimesulide, on account of its nitro group, acts as a potent protonophoretic uncoupler and NAD(P)H oxidant on isolated rat liver mitochondria, inducing Ca(2+) efflux or MPT within a concentration range which can be reached in vivo, thus presenting the potential ability to interfere with the energy and Ca(2+) homeostasis in the liver cell.     

Critical role of organic anion transporters 1 and 3 in kidney accumulation and toxicity of aristolochic acid I

Ingestion of aristolochic acid (AA), especially its major constituent aristolochic acid I (AAI), results in severe kidney injury known as aristolochic acid nephropathy (AAN). Although hepatic cytochrome P450s metabolize AAI to reduce its kidney toxicity in mice, the mechanism by which AAI is uptaken by renal cells to induce renal toxicity is largely unknown. In this study, we found that organic anion transporters (OATs) 1 and 3, proteins known to transport drugs from the blood into the tubular epithelium, are responsible for the transportation of AAI into renal tubular cells and the subsequent nephrotoxicity. AAI uptake in HEK 293 cells stably transfected with human OAT1 or OAT3 was greatly increased compared to that in the control cells, and this uptake was dependent on the AAI concentration. Administration of probenecid, a well-known OAT inhibitor, to the mice reduced AAI renal accumulation and its urinary excretion and protected mice from AAI-induced acute tubular necrosis. Further, AAI renal accumulation and severe kidney lesions induced by AAl in Oat1 and Oat3 gene knockout mice all were markedly suppressed compared to those in the wild-type mice. Together, our results suggest that OAT1 and OAT3 have a critical role in AAl renal accumulation and toxicity. These transporters may serve as a potential therapeutic target against AAN.     

Ergosta-4,6,8(14),22-tetraen-3-one isolated from Polyporus umbellatus prevents early renal injury in aristolochic acid-induced nephropathy rats

Objectives Aristolochic acid (AA) nephropathy, first reported as Chinese herbs nephropathy, is a rapidly progressive tubulointerstitial nephropathy that results in severe anemia, interstitial fibrosis and end‐stage renal disease. Tubulointerstitial injury was studied in a rat model of AA nephropathy to determine whether ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) treatment prevents early renal injury in rats with aristolochic acid I‐induced nephropathy. Methods Early renal injury via renal interstitial fibrosis was induced in rats by administration of aristolochic acid I (AAI) solution intragastrically for 8 weeks. Ninety‐six rats were randomly divided into four groups (n = 24/group): (1) control (2) AAI (3) AAI + ergone (10 mg/kg) and (4) AAI + ergone (20 mg/kg). Blood and urine samples were collected and rat were sacrificed for histological assessment of the kidneys on at the end of weeks 2, 4, 6 and 8. Key findings AAI caused progressive elevation of blood urea nitrogen, creatinine, potassium, sodium, chlorine, proteinuria and urinary N‐acetyl‐β‐D‐glucosaminidase (NAG). Ergone suppressed elevation of blood urea, nitrogen, creatinine, proteinuria and urinary NAG to some degree, but the AAI–ergone‐treated group did not differ from AAI‐treated group for body weight, serum potassium, sodium and chlorine. The progress of the lesions in the kidney after AAI administration was also observed by histopathological examinations, but kidneys from rats of AAI–ergone‐treated group displayed fewer lesions. Conclusions Ergone treatment conferred protection against early renal injury in a rat model of AA nephropathy. Early administration of ergone may prevent the progression of renal injury and the subsequent renal fibrosis in AA nephropathy.     

Mitochondrial dysfunction induced by sertraline, an antidepressant agent

Sertraline, a selective serotonin reuptake inhibitor, has been used for the treatment of depression. Although it is generally considered safe, cases of sertraline-associated liver injury have been documented; however, the possible mechanism of sertraline-associated hepatotoxicity is entirely unknown. Here, we report that mitochondrial impairment may play an important role in liver injury induced by sertraline. In mitochondria isolated from rat liver, sertraline uncoupled mitochondrial oxidative phosphorylation and inhibited the activities of oxidative phosphorylation complexes I and V. Additionally, sertraline induced Ca(2+)-mediated mitochondrial permeability transition (MPT), and the induction was prevented by bongkrekic acid (BA), a specific MPT inhibitor targeting adenine nucleotide translocator (ANT), implying that the MPT induction is mediated by ANT. In freshly isolated rat primary hepatocytes, sertraline rapidly depleted cellular adenosine triphosphate (ATP) and subsequently induced lactate dehydrogenase leakage; both were attenuated by BA. Our results, including ATP depletion, induction of MPT, inhibition of mitochondrial respiration complexes, and uncoupling oxidative phosphorylation, indicate that sertraline-associated liver toxicity is possibly via mitochondrial dysfunction.     

The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury

Objective

Many nonsteroidal anti-inflammatory drugs (NSAIDs) with diphenylamine structure induce severe hepatotoxicities. We evaluated the role of diphenylamine structure in liver injuries induced by these NSAIDs.

Methods

Effects of diphenylamine, diclofenac and tolfenamic acid on mitochondrial permeability transition (MPT) and efflux of calcium in isolated liver mitochondria as well as on cellular ATP content and mitochondrial membrane depolarization in rat primary hepatocyte cultures were examined.

Results

Diclofenac and tolfenamic acid induced cyclosporine A (CsA)-sensitive mitochondrial swelling and membrane depolarization in isolated liver mitochondria. Only diclofenac caused the release of calcium in isolated liver mitochondria. Diphenylamine had no effects on isolated liver mitochondria. All three compounds decreased ATP content and induced mitochondrial membrane depolarization. CsA attenuated these effects, suggesting MPT might be involved in the hepatotoxicities caused by diphenylamine, diclofenac and tolfenamic acid. SKF-525A, a general inhibitor of CYP450, markedly inhibited the injury induced by diphenylamine, but not diclofenac or tolfenamic acid.

Conclusion

The hepatotoxicities caused by diclofenac and tolfenamic acid may be attributed to the mitochondrial dysfunction induced by these drugs instead of the diphenylamine structure per se.     

外周苯二氮受体参与调控大鼠心肌线粒体通透性转换

目的探讨外周苯二氮受体(peripheral benzodiaz-epine receptor,PBR)在调控心肌线粒体通透性转换(mito-chondrial permeability transition,MPT)中的作用。方法分离SD大鼠心肌线粒体,电镜观察其形态证实线粒体结构完整性。将线粒体分别和不同浓度(50,100,200μmol·L-1)的PBR拮抗剂PK11195孵育,部分线粒体和100mmol·L-1PK11195孵育之前5min加入5μmol·L-1MPT抑制剂环孢菌素A(CsA组)。不给任何处理的线粒体为阴性对照(Con组),单纯给150μmol·L-1Ca2+作阳性对照(Ca2+组)。分光光度法测520nm处吸光度的改变反映MPT变化,电镜观察线粒体超微结构改变,Western blot检测线粒体细胞色素C(CytoC)释放。结果PK11195剂量依赖性诱发MPT,不同浓度组间比较差异有显著性(P<0.05,P<0.01);PK11195导致线粒体出现空泡变性、线粒体肿胀、线粒体脊膜破裂,CytoC释放明显增多(vsCon组,P<0.01);CsA能阻断PK11195的上述作用,CsA组线粒体超微结构基本完好,线粒体CytoC释放较少(vs100μmol·L-1组,P<0.05)。结论PBR参与调控大鼠心肌MPT,其拮抗剂PK11195可剂量依赖性诱导心肌MPT,导致线粒体超微结构损伤和线粒体CytoC释放。     

Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca²⁺-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.     

Mitochondrial permeability transition induced by the anticancer drug etoposide.

Etoposide (VP-16) is widely used for the treatment of several forms of cancer. The cytotoxicity of VP-16 has been assigned to the induction of apoptotic cell death but the signaling pathway for VP-16-induced apoptosis is essentially unknown. There is some evidence that this process depends on events associated with the loss of mitochondrial membrane potential (Delta Psi) and/or release of apoptogenic factors, putatively as a consequence of mitochondrial permeability transition (MPT) induction. This work evaluates the interference of VP-16 with MPT in vitro, which is characterized by the Ca(2+)-dependent depolarization of Delta Psi, the release of matrix Ca(2+) and by extensive swelling of mitochondria. Delta Psi depolarization and Ca(2+) release were measured with ion-selective electrodes, and mitochondrial swelling was monitored spectrophotometrically. Incubation of rat liver mitochondria with VP-16 results in a concentration-dependent induction of MPT, evidenced by an increased sensitivity to Ca(2+)-induced swelling, depolarization of Delta Psi, Ca(2+) release by mitochondria and stimulation of state 4 oxygen consumption. All of these effects are prevented by preincubating the mitochondria with cyclosporine A, a potent and specific inhibitor of the MPT. Therefore, VP-16 increases the sensitivity of isolated mitochondria to the Ca(2+)-dependent induction of the MPT. Together, these data provide a possible mechanistic explanation for the previously reported effects of VP-16 on apoptosis induction.     

Cysteinyl leukotrienes synthesis is involved in aristolochic acid I-induced apoptosis in renal proximal tubular epithelial cells

Aristolochic acid I (AAI) is a primary nephrotoxin and carcinogen that is found in some Chinese herbal medicines, and AAI is responsible for the progression of aristolochic acid nephropathy. The membrane associated proteins in the eicosanoid and glutathione metabolism (MAPEG) superfamily are associated with cysteinyl leukotrienes (cysLTs) synthesis. The present study investigated whether cysLTs synthesis was involved in AAI-induced renal proximal tubular epithelial cell injury in LLC-PK1 cells. Based on MAPEG and related molecular events, the potential mechanisms of AAI-induced LLC-PK1 cell injury were explored. AAI triggered the mitochondrial/caspase apoptotic pathway in LLC-PK1 cells, which was indicated by an enhanced Bax/Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome C release, and caspase 3 activation. In addition, AAI-induced cysLTs release was accompanied by selective upregulation of 5-lipoxygenase activating protein (FLAP) and microsomal glutathione S-transferase 3 (mGST3) in a concentration-dependent manner. The FLAP inhibitor MK866 significantly protected cells from AAI-induced apoptosis. Furthermore, activation of extracellular signal-regulated kinase (ERK) 1/2 and inhibition of phosphorylated p38-MAPK were demonstrated at the early phase of AAI treatment. Notably, the MEK/ERK inhibitor U0126 reversed AAI-induced apoptosis and reduced both FLAP, mGST3 and mitochondrial/caspase protein expression. Taken together, these findings suggest that cysLTs synthesis is involved in AAI-induced apoptosis via an ERK activation way.     

Endoplasmic reticulum stress mediates aristolochic acid I-induced apoptosis in human renal proximal tubular epithelial cells

Aristolochic acid (AA), derived from the Aristolochia species, has been associated with aristolochic acid nephropathy (AAN), which has emerged as a worldwide disease. Aristolochic acid I (AAI) is the main ingredient of AA, and the underlying mechanisms for AAI-induced nephrotoxicity are still unclear. In this study, we investigated whether endoplasmic reticulum (ER) stress was involved in AAI-induced nephrotoxicity. The results showed that treatment of HK-2 cells (a human proximal tubular epithelial cell line) with AAI caused an increase in eukaryotic initiation factor-2α (eIF2α) phosphorylation, X-box binding protein 1 (XBP1) mRNA splicing and the expression of glucose-regulated protein (GRP) 78 and CAAT/enhancer-binding protein-homologous protein (CHOP). These events represent typical markers of the ER stress-related signaling pathway. Pretreatment with 4-phenylbutyrate (4-PBA) or salubrinal (Sal) significantly inhibited AAI-induced apoptosis, indicating the role of ER stress in AAI-induced apoptosis. In addition, AAI-induced cell death followed an increase of reactive oxygen species (ROS) formation in HK-2 cells. Pretreatment with N-acetyl cysteine (NAC) or glutathione (GSH) significantly inhibited AAI-induced ER stress proteins and cell death, suggesting that ROS mediate AAI-induced ER stress. Taken together, these results suggest that the ER stress response is involved in apoptosis induced by AAI in HK-2 cells, thus offering a new insight into the nephrotoxicity of AAI.     

Mitochondrial permeability transition as the critical target of N-acetyl perfluorooctane sulfonamide toxicity in vitro.

Perfluorooctanyl compounds with active functional groups have been shown to disrupt mitochondrial bioenergetics by three distinct mechanisms: protonophoric uncoupling of mitochondrial respiration, induction of the mitochondrial permeability transition (MPT), or a nonselective increase in membrane permeability. The purpose of this investigation was to identify the initial target and specific sequence of events associated with the N-acetyl substituted perfluorooctanesulfonamides induced MPT. N-acetyl-perfluorooctanesulfonamide (FOSAA), N-ethyl-N-acetyl-perfluorooctanesulfonamide (N-Et FOSAA), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and N-ethyl-N-(2-ethoxy)-perfluorooctanesulfonamide (N-Et FOSE) were added individually to liver mitochondria freshly isolated from Sprague-Dawley rats. Mitochondrial swelling and cytochrome c release were recorded spectrophotometrically, oxygen uptake was monitored with a Clark-type oxygen electrode, and reactive oxygen species (ROS) were monitored by dichlorodihydrofluorescein diacetate (H(2)DCFDA) fluorescence. FOSAA (45 microM) and N-Et FOSAA (7.5 microM) induced calcium-dependent mitochondrial swelling, the release of cytochrome c, inhibition of uncoupled mitochondrial respiration, and ROS generation, all of which were inhibited by cyclosporin-A (CsA). PFOA (200 microM) displayed slight CsA sensitive activity, but neither PFOS (10 microM) nor N-Et FOSE (70 microM) induced the MPT. Results of this investigation demonstrate two important findings: (1) MPT induction is specific to the N-acetyl substituted perfluorooctanesulfonamides and, (2) the sequence of events is initiated by induction of the MPT, which causes the release of cytochrome c as well as other cofactors leading to inhibition of respiration and ROS generation. The toxicity of N-acetyl perfluorooctanyl compounds may therefore reflect the mitochondrial dysfunction, which is compounded by the ensuing oxidative injury.     

Protective effect of BMP-7 against aristolochic acid-induced renal tubular epithelial cell injury

Aristolochic acid nephropathy (AAN) is regarded as a kind of rapidly progressive renal fibrosis caused by the ingestion of herbal remedies containing aristolochic acid (AA). Recent studies showed that bone morphogenetic protein-7 (BMP-7) exerts beneficial effects on acute and chronic kidney injuries induced by different pathological conditions. We examined whether BMP-7 protects human renal tubular epithelial cells (HK-2) against AA-induced injury in vitro. HK-2 cells were cultured with different concentrations of AA and BMP-7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) release. The apoptosis rate and the activity of caspase 3 protease were also examined. Epithelial-to-mesenchymal transition (EMT) was determined by cell morphology, E-cadherin and α-smooth muscle actin (α-SMA) protein expression, and TGF-β₁ and collagen III secretion. Additionally, the effect of anti-TGF-β1 antibody on AA-induced EMT was assessed. Our results indicated that BMP-7 significantly increased cell proliferation, decreased apoptosis rate and attenuated activation of caspase-3, resulting in the protection of HK-2 cells from AA-induced cytotoxicity. In addition, studies on EMT revealed that BMP-7 could inhibit AA-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through reducing the activation of a myofibroblast phenotype and production TGF-β1. Treatment with neutralizing anti-TGF-β1 antibody also blocked AA-induced EMT and collagen III secretion. Together, these observations strongly suggest that BMP-7 is a potent inhibitor of AA-induced renal tubular epithelial cell injury and might be a promising agent for aristolochic acid-induced kidney damage.     

In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney.

In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl2), phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in Bel-7402 cells and HK-2 cells (R2 = 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using specific cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity effect after long-term exposure to a chemical.     

Acrylic Acid Induces the Glutathione-lndependent Mitochondrial Permeability Transition in Vitro

Acrylic acid (AA) is used widely in the synthesis of estersessential in the production of paints, adhesives, plastics,and coatings. The minimal systemic toxicity of AA is attributedto its rapid oxidation to acetyl-CoA and CO₂ via the vitaminB₁₂-independent beta-oxidation pathway. This oxidation is localizedto the mitochondria and preliminary evidence suggests a possibleinhibition of mitochondrial metabolism by acrylic acid. Thepurpose of this investigation was to evaluate whether AA interfereswith mitochondrial bioenergetics in vitro. Incubation of isolatedrat liver mitochondria with AA resulted in a dose-dependentinduction of the mitochondrial permeability transition (MPT).This was evidenced by an increased sensitivity to calcium-inducedstimulation of state 4 oxygen consumption, depolarization ofmembrane potential, and swelling, all of which were preventedby preincubating the mitochondria with cyclosporine A, a potentand specific inhibitor of the mitochondrial permeability transitionpore. Both N-ethylmaleimide (NEM) and dithiothreitol (DTT) showedonly partial protection against induction of the MPT by AA.Associated with the induction of the MPT by AA was the lossof mitochondrial glutathione (GSH), which was due to effluxfrom the matrix rather than oxidation to GSSG. CyclosporineA, by inhibiting the permeability transition, prevented theAA-induced loss of mitochondrial GSH. In conclusion, AA increasesthe sensitivity of isolated mitochondria in vitro to the calcium-dependentinduction of the MPT. Although the molecular mechanism has yetto be defined, it does not appear to be related to the oxidationof critical thiols.     

Calcineurin-independent inhibition of mitochondrial Ca2+ uptake by cyclosporin A

1. Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1-10 microm) has an additional effect on Ca(2+) homeostasis in mitochondria that cannot be attributed to inhibition of PTP. 2. By measuring specifically mitochondrial [Ca(2+)] with targeted aequorin, we show that CsA inhibited Ca(2+) entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca(2+) entry was triggered by low cytosolic [Ca(2+)], below 5 microm. 3. Inhibition of mitochondrial Ca(2+) uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid. 4. CsA blocked the increase in mitochondrial Ca(2+) uptake rate induced by the mitochondrial Ca(2+) uniporter activator SB202190. 5. Our results suggest that CsA inhibits Ca(2+) entry through the Ca(2+) uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca(2+) overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.