Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

Berberine (BBR) shows promising effects in the treatment of nonalcoholic fatty liver disease (NAFLD) by influencing various metabolic aspects. Inhibition of mitochondrial ß-oxidation (ß-OX) participates in the pathogenesis of NAFLD. Silent mating-type information regulation 2 homolog 3 (SIRT3) has been reported to regulate mitochondrial ß-OX by deacetylating its substrate, long-chain acyl-coenzyme A dehydrogenase (LCAD). This study aimed to explore whether BBR can promote mitochondrial ß-OX and the role of SIRT3 as well as the mechanisms underlying the effects of BBR on hepatic lipid metabolism in mice fed a high-fat diet (HFD). BBR can significantly improve systematic and hepatic lipid metabolism in HFD-fed mice. Metabolomics analysis revealed that ß-OX was inhibited in HFD-induced mice, as indicated by the reduced production of short and medium carbon chain acyl-carnitines, the activated form of free fatty acids, via ß-OX, which was reversed by BBR intervention. Exploration of the mechanism found that BBR intervention reversed the down-regulation of SIRT3 and decreased the LCAD hyperacetylation level in HFD-fed mice. SIRT3 knockout (KO) mice were used to identify the role of SIRT3 in the BBR's influence of ß-OX. The beneficial effects of BBR on systemic and hepatic metabolism were profoundly attenuated in KO mice. Moreover, the promotive effect of BBR on ß-OX in HFD-induced mice was partially abolished in KO mice. These results suggested that BBR alleviates HFD-induced inhibition of fatty acid ß-OX partly through SIRT3-mediated LCAD deacetylation, which may provide a novel mechanism and support BBR as a promising therapeutic for NAFLD.-Xu, X., Zhu, X.-P., Bai, J.-Y., Xia, P., Li, Y., Lu, Y., Li, X.-Y., Gao, X. Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

Apoptosis in HepaRG and HL-7702 cells inducted by polyphyllin II through caspases activation and cell-cycle arrest.

Rhizoma Paridis, a traditional Chinese medicine, has shown promise in cancer prevention and therapy. Polyphyllin II is one of the most significant saponins in Rhizoma Paridis and it has toxic effects on kinds of cancer cells. However, our results in this study proved that the polyphyllin II has hepatotoxicity in vitro through caspases activation and cell-cycle arrest. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results indicated polyphyllin II inhibited proliferation, induced apoptosis in HepaRG cells and HL-7702 cells and showed a concentration and time-dependent. Then, we selected the innovative cell model-HepaRG cells to explore the mechanism of hepatotoxicity. Our data showed the reactive oxygen species (ROS) increased and the mitochondrial membrane potential decreased in HepaRG cells after administration of polyphyllin II. Besides, with the increase of concentration, the release of lactate dehydrogenase increased and the S phase of the cell cycle was arrested. Nevertheless, when pretreatment with antioxidant N-acetylcysteine, apoptotic cells decreased significantly, inhibited the production of ROS and improved the decrease of membrane potential in HepaRG cells. Moreover, polyphyllin II treatment increased levels of Fas, Bax, cytochrome c, activated caspase-3, -8, -9, cleaved poly(ADP-ribose) polymerase and decreased Bcl-2 expression levels. Finally, we identified two signal pathways of apoptosis induced by polyphyllin II including the death receptor pathway and the mitochondria pathway. This study confirmed the hepatotoxicity of the polyphyllin II in vitro, which has never been discovered and gave a wake-up call for the clinical application of Rhizoma Paridis.

Chemoproteomics reveals baicalin activates hepatic CPT1 to ameliorate diet-induced obesity and hepatic steatosis.

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.

Attenuation of liver cancer development by oral glycerol supplementation in the rat.

PURPOSE: Glycerol usage is increasing in food industry for human and animal nutrition. This study analyzed the impact of glycerol metabolism when orally supplemented during the early stage of rat liver carcinogenesis. METHODS: Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group). IP animals also received glycerol by gavage (200 mg/kg body weight, IPGly group). RESULTS: Glycerol treatment reduced the volume of preneoplastic lesions by decreasing the proliferative status of liver foci, increasing the expression of p53 and p21 proteins and reducing the expression of cyclin D1 and cyclin-dependent kinase 1. Besides, apoptosis was enhanced in IPGly animals, given by an increment of Bax/Bcl-2 ratio, Bad and PUMA mitochondrial expression, a concomitant increase in cytochrome c release and caspase-3 activation. Furthermore, hepatic levels of glycerol phosphate and markers of oxidative stress were increased in IPGly rats. Oxidative stress intermediates act as intracellular messengers, inducing p53 activation and changes in JNK and Erk signaling pathways, with JNK activation and Erk inhibition. CONCLUSION: The present work provides novel data concerning the preventive actions of glycerol during the development of liver cancer and represents an economically feasible intervention to treat high-risk individuals.

Bavachin Induces Apoptosis through Mitochondrial Regulated ER Stress Pathway in HepG2 Cells.

As a traditional herbal medicine, the fruits of Psoralea corylifolia L. (Fructus Psoraleae (FP)) have been widely used for the treatment of various skin diseases for hundred years. Recently, the emerging FP-induced toxic effects, especially hepatotoxicity, in clinic are getting the public's attention. However, its exact toxic components and mechanisms underlying remain unclear. Bavachin, one of flavonoids in FP, has been documented as a hepatotoxic substance, and the present study aimed to determine the toxicity caused by bavachin and the possible toxic mechanisms involved using human hepatocellular carcinoma (HepG2) cells. Our results showed that bavachin could significantly inhibited cell proliferation and trigger the endoplasmic reticulum (ER) stress in a dose dependent manner. Downregulating ER stress using tauroursodeoxycholic acid (TUDCA) obvious attenuated bavachin-triggerd cell apoptosis. Then, small interfering RNA (siRNA) knock-down of Mitofusion2 (Mfn2) resulted in a remarkable aggravation of ER stress through the inhibition of the phosphorylation of protein kinase B (Akt). Additionally, suppression of reactive oxygen species (ROS) by ROS Scavenger (N-acetyl-l-cystein (NAC)) also reduced bavachin-induced ER stress. Taken together, our study demonstrated that bavachin-induced ER stress caused cell apoptosis by Mfn2-Akt pathway, and that ROS may participate upstream in this mechanism. Here, we not only provide a new understanding of ROS/Mfn2/Akt pathway in bavachin-induced cytotoxicity via the ER stress, but also identify a new specific intervention to prevent FP-induced hepatotoxicity in the future.

Ameliorative efficacy of quercetin against cisplatin­induced mitochondrial dysfunction: Study on isolated rat liver mitochondria.

The present study aimed to investigate the hepatoprotective effects of the bioflavonoid quercetin (QR) on cisplatin (CP)­induced mitochondrial oxidative stress in the livers of rats, to elucidate the role of mitochondria in CP­induced hepatotoxicity, and its underlying mechanism. Isolated liver mitochondria were incubated with 100 µg/ml CP and/or 50 µM QR in vitro. CP treatment triggered a significant increase in membrane lipid peroxidation (LPO) levels, protein carbonyl (PC) contents, and a decrease in reduced glutathione (GSH) and non­protein thiol (NP­SH) levels. In addition, CP caused a marked decline in the activities of enzymatic antioxidants and mitochondrial complexes (I, II, III and V) in liver mitochondria. QR pre­treatment significantly modulated the activities of enzymatic antioxidants and mitochondrial complex enzymes. Furthermore, QR reversed the alterations in LPO and PC levels, and GSH and NP­SH contents in liver mitochondria. The results of the present study suggested that QR supplementation may suppress CP­induced mitochondrial toxicity during chemotherapy, and provides a potential prophylactic and defensive candidate for anticancer agent­induced oxidative stress.

Dietary long-chain unsaturated fatty acids acutely and differently reduce the activities of lipogenic enzymes and of citrate carrier in rat liver.

The activities of lipogenic enzymes appear to fluctuate with changes in the level and type of dietary fats. Polyunsaturated fatty acids (PUFAs) are known to induce on hepatic de novo lipogenesis (DNL) the highest inhibitory effect, which occurs through a long-term adaptation. Data on the acute effects of dietary fatty acids on DNL are lacking. In this study with rats, the acute 1-day effect of high-fat (15 % w/w) diets (HFDs) enriched in saturated fatty acids (SFAs) or unsaturated fatty acids (UFAs), i.e., monounsaturated (MUFA) and PUFA, of the ω-6 and ω-3 series on DNL and plasma lipid level was investigated; a comparison with a longer time feeding (21 days) was routinely carried out. After 1-day HFD administration UFA, when compared to SFA, reduced plasma triacylglycerol (TAG) level and the activities of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), a decreased activity of the citrate carrier (CIC), a mitochondrial protein linked to lipogenesis, was also detected. In this respect, ω-3 PUFA was the most effective. On the other hand, PUFA maintained the effects at longer times, and the acute inhibition induced by MUFA feeding on DNL enzyme and CIC activities was almost nullified at 21 days. Mitochondrial fatty acid composition was slightly but significantly changed both at short- and long-term treatment, whereas the early changes in mitochondrial phospholipid composition vanished in long-term experiments. Our results suggest that in the early phase of administration, UFA coordinately reduced both the activities of de novo lipogenic enzymes and of CIC. ω-3 PUFA showed the greatest effect.

Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

Hypolipidemic effect of dietary water-soluble protein extract from chicken: impact on genes regulating hepatic lipid and bile acid metabolism.

BACKGROUND: Amount and type of dietary protein have been shown to influence blood lipids. The present study aimed to evaluate the effects of a water-soluble fraction of chicken protein (CP) on plasma and hepatic lipid metabolism in normolipidemic rats. METHODS: Male Wistar rats were fed either a control diet with 20 % w/w casein as the protein source, or an experimental diet where casein was replaced with CP at 6, 14, or 20 % w/w for 4 weeks. RESULTS: Rats fed CP had markedly reduced levels of triacylglycerols (TAG) and cholesterol in both plasma and liver, accompanied by stimulated hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase 2 activity in the 20 % CP group compared to the control group. In addition, reduced activities and gene expression of hepatic enzymes involved in lipogenesis were observed. The gene expression of sterol regulatory element-binding transcription factor 1 was reduced in the 20 % CP-fed rats, whereas gene expression of peroxisome proliferator-activated receptor alpha was increased. Moreover, 6, 14, and 20 % CP-fed rats had significantly increased free carnitine and acylcarnitine plasma levels compared to control rats. The plasma methionine/glycine and lysine/arginine ratios were reduced in 20 % CP-treated rats. The mRNA level of ATP-binding cassette 4 was increased in the 20 % CP group, accompanied by the increased level of plasma bile acids. CONCLUSIONS: The present data suggest that the hypotriglyceridemic property of a water-soluble fraction of CP is primarily due to effects on TAG synthesis and mitochondrial fatty acid oxidation. The cholesterol-lowering effect by CP may be linked to increased bile acid formation.

Desmodeleganine, a new alkaloid from the leaves of Desmodium elegans as a potential monoamine oxidase inhibitor.

Desmodeleganine (1), a new potential monoamine oxidase inhibitor, along with three known alkaloids, bufotenin (2), hydroxy-N, N-dimethyltryptamine N(12)-oxide (3), 2-(5-methoxy-1H-indol-3-yl)-N, and N-dimethylethylamine (4) were isolated from the leaves of Desmodium elegans. Their structures were elucidated by IR, MS, 1D and 2D NMR spectra. 1 showed strong monoamine oxidase inhibitory activity with IC50 value of 13.92 ± 1.5 µM, when the IC50 value of iproniazid as a standard was 6.5 ± 0.5 µM. The molecular modeling was also performed to explore the binding mode of compounds 1, 2 at the active site of MAO-A and MAO-B.

Curcumin attenuates Cr(VI)-induced ascites and changes in the activity of aconitase and F(1)F(0) ATPase and the ATP content in rat liver mitochondria.

Occupational and environmental exposure to potassium dichromate (K2Cr2O7), a hexavalent chromium compound, can result in liver damage associated with oxidative stress and mitochondrial dysfunction. The purpose of this study was to evaluate the effect of the antioxidant curcumin (400 mg/kg b.w.) on the K2Cr2O7-induced injury, with special emphasis on ascitic fluid accumulation and oxidative phosphorylation mitochondrial enzymes and the adenosine triphosphate (ATP) levels in isolated mitochondria from livers of rats treated with K2Cr2O7 (15 mg/kg b.w.). Thus, curcumin attenuated the ascites generation, prevented the decrease in the activities of aconitase and F1F0 ATPase, and maintained the ATP levels. The activity of complex II was not completely reestablished by curcumin, whereas complexes III and IV activities were unchanged.

Phosphate-dependent glutaminase response to liver injury and hyperbaric oxygenation.

Activity of phosphate-dependent glutaminase was determined in hepatocytes of white female rats, both in healthy animals and in rats with chronic CCl4-hepatitis on day 3 after liver resection and hyperbaric oxygenation. In healthy animals, activity of phosphate-dependent glutaminase was not altered after hepatic resection, but it was elevated in animals with chronic CCl4-hepatitis. Hyperbaric oxygenation inhibited activity of hepatocytic phosphate-dependent glutaminase in non-operated healthy rats but stimulated it after hepatic resection. In animals with chronic CCl4-hepatitis; hyperbaric oxygenation restricted the stimulating effect of hepatic resection on phosphate-dependent glutaminase activity.

Bitter gourd inhibits the development of obesity-associated fatty liver in C57BL/6 mice fed a high-fat diet.

Bitter gourd (BG) is a popular fruit in Asia with numerous well-known medicinal uses, including as an antidiabetic. In the current study, we aimed to explore the effects of BG on mitochondrial function during the development of obesity-associated fatty liver. C57BL/6 mice were divided into 4 experimental groups: mice fed a normal diet (control; included for reference only), mice fed a high-fat diet (HFD), and mice fed an HFD supplemented with freeze-dried BG powder through daily gavage at doses of 0.5 (HFD+0.5BG) and 5 (HFD+5BG) g/kg, respectively. After 16 wk, mice in the HFD+5BG group showed less body and tissue weight gain and less hyperglycemia and hyperlipidemia compared with those in the HFD group (P < 0.05). In both HFD+0.5BG and HFD+5BG groups, serum interleukin-6 concentration was lower than that in the HFD group (P < 0.02). The serum C-reactive protein concentration was lower in the HFD+5BG group compared with the HFD group (P < 0.04). An analysis of liver tissue revealed lower liver triglyceride and cholesterol concentrations in both HFD+0.5BG and HFD+5BG groups than in the HFD group (P < 0.01). The HFD+5BG group had less activation of the sterol regulatory element binding protein/fatty acid synthase (SREBP-1/FAS) pathway, greater superoxide dismutase activity, and less total protein and mitochondrial protein oxidation than did the HFD group (P < 0.05). Mitochondrial complex I, II, III, and V activity was greater in the HFD+0.5BG group than in the HFD group (P < 0.03). The HFD+5BG group only had greater complex V activity compared with the HFD group (P < 0.05). Mitochondrial dynamics regulators, including dynamin related protein 1 (DRP1) and mitofusin 1 (MFN1), as well as proapoptotic protein expression levels were restored by BG treatment (P < 0.02). Taken together, our results suggest that BG prevents inflammation and oxidative stress, modulates mitochondrial activity, suppresses apoptosis activation, and inhibits lipid accumulation during the development of fatty liver.

Arsenic-induced hepatic mitochondrial toxicity in rats and its amelioration by diallyl trisulfide.

The present investigation was aimed to investigate the possible protective role of diallyl trisulfide (DATS) against arsenic (As)-induced hepatic mitochondrial toxicity in rats. Mitochondria were isolated from the liver tissue of rats from all the groups. Lipid profile, lipid peroxidation, antioxidant enzyme activities, hepatic function enzymes, mitochondrial swelling, cytochrome c oxidase activity, mitochondrial Ca(+)-ATPase and Na(+)/K(+)-ATPase activity, mitochondrial calcium content and mitochondrial enzyme activities were measured. Short-term As exposure (5 mg/kg bw/d for 28 d) caused liver damage as evidenced by changes in activities of liver enzymes. The effects of As were coupled with enhanced reactive oxygen species generation, mitochondrial swelling, inhibition of cytochrome c oxidase, complex I-mediated electron transfer, decreased Ca(2+)-ATPase and Na(+)/K(+)-ATPase activity, a reduction in mitochondrial calcium content, changes in indices of hepatic mitochondrial oxidative stress, significant increase in mitochondrial lipid peroxidation products and alterations in mitochondrial lipid profile. Significant decreases in mitochondrial antioxidants and tricarboxylic acid cycle enzymes were also found in the liver mitochondria of As-induced hepatic mitochondrial toxicity in rats. As also increased hepatic caspase-3 activity and DNA fragmentation. All these apoptosis-related molecular changes caused by As could be alleviated by supplementation with DATS, which likely suggests a protective role against As-induced hepatotoxic changes and hepatic mitochondrial toxicity. The protective effect of DATS on the liver mitochondria was evidenced by altering all the changes induced by As. Free radical scavenging and metal chelating activities of DATS may be the mechanism, responsible for the protective action against As-induced mitochondrial damage in liver.

Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study.

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology.

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice.

BACKGROUND: Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice. RESULTS: Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice. CONCLUSIONS: These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

BACKGROUND: Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice. RESULTS: Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice. CONCLUSIONS: These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.

Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes.

To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify "toxic" and "non-toxic" drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.

The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases.

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.

Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner.

NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1 mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9 mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.