Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I.

Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD(+) ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30µM EFV and submaximal effects at 50µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD(+) ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical.

Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury.

UNLABELLED: Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours. CONCLUSION: MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored.

Bypassing the compromised mitochondrial electron transport with methylene blue alleviates efavirenz/isoniazid-induced oxidant stress and mitochondria-mediated cell death in mouse hepatocytes.

Efavirenz (EFV) is an anti-retroviral drug frequently combined with isoniazid (INH) to treat HIV-1/tuberculosis co-infected patients. Both drugs have been associated with idiosyncratic liver injury (DILI), but combined anti-retroviral and anti-tubercular therapy can increase the risk for DILI as compared to either drug class alone. Because both EFV and INH have been implicated in targeting mitochondria, we aimed at exploring whether the two drugs might cause synergistic effects on the electron transport chain. We found that EFV inhibited complex I activity in isolated mouse liver mitochondria (IC50 Ëœ30 µM), whereas hydrazine, a major metabolite of INH generated by acylamidase-mediated hydrolytic cleavage, inhibited complex II activity (IC50 Ëœ30 µM). Neither INH alone (≤1000 µM) nor EFV alone (≤30 µM) was able to induce cell injury in cultured mouse hepatocytes. However, combined EFV/INH exposure resulted in increased superoxide formation and peroxynitrite stress, leading to the opening of the cyclosporine A-insensitive mode of the mitochondrial permeability transition (mPT), and necrotic cell death. The peroxynitrite scavengers, CBA or Fe-TMPyP, protected against mPT induction and alleviated cell injury. The acylamidase inhibitor bis-p-nitrophenyl phosphate prevented cell injury, suggesting that hydrazine greatly contributed to the toxicity. Methylene blue, a redox-active alternative electron acceptor/donor that bypasses complex I/II, effectively protected against EFV/INH-induced toxicity. These data demonstrate that, in murine hepatocytes, the mitochondrial electron transport chain is a critical target of combined EFV/INH exposure, and that this drug combination can lead to peroxynitrite stress-induced mPT and hepatocellular necrosis. These results are compatible with the concept that underlying silent mitochondrial dysfunction may be a key susceptibility factor contributing to idiosyncratic drug-induced liver injury.

Mechanisms of isoniazid-induced idiosyncratic liver injury: emerging role of mitochondrial stress.

Idiosyncratic drug-induced liver injury (DILI) is a significant adverse effect of antitubercular therapy with isoniazid (INH). Although the drug has been used for many decades, the underlying mode of action (both patient-specific and drug-specific mechanisms) leading to DILI are poorly understood. Among the patient-specific determinants of susceptibility to INH-associated DILI, the importance of HLA genetic variants has been increasingly recognized, whereas the role of polymorphisms of drug-metabolizing enzymes (NAT2 and CYP2E1) has become less important and remains controversial. However, these polymorphisms are merely correlative, and other molecular determinants of susceptibility have remained largely unknown. Regarding the drug-specific mechanisms underlying INH-induced liver injury, novel concepts have been emerging. Among these are covalent protein adduct formation via novel reactive intermediates, leading to hapten formation and a potential immune response, and interference with endogenous metabolism. Furthermore, INH and/or INH metabolites (e.g. hydrazine) can cause mitochondrial injury, which can lead to mitochondrial oxidant stress and impairment of energy homeostasis. Recent studies have revealed that underlying impairment of complex I function can trigger massive hepatocellular injury induced by otherwise nontoxic concentrations of INH superimposed on these mitochondrial deficiencies. This review discusses these emerging new paradigms of INH-induced DILI and highlights recent insights into the mechanisms, as well as points to the existing large gaps in our understanding of the pathogenesis.

Understanding and modulating mammalian-microbial communication for improved human health.

The fact that the bacteria in the human gastrointestinal (GI) tract play a symbiotic role was noted as early as 1885, well before we began to manage microbial infections using antibiotics. However, even with the first antimicrobial compounds used in humans, the sulfa drugs, microbes were recognized to be critically involved in the biotransformation of these therapeutics. Thus, the roles played by the microbiota in physiology and in the management of human health have long been appreciated. Detailed examinations of GI symbiotic bacteria that started in the early 2000s and the first phases of the Human Microbiome Project that were completed in 2012 have ushered in an exciting period of granularity with respect to the ecology, genetics, and chemistry of the mammalian-microbial axes of communication. Here we review aspects of the biochemical pathways at play between commensal GI bacteria and several mammalian systems, including both local-epithelia and nonlocal responses impacting inflammation, immunology, metabolism, and neurobiology. Finally, we discuss how the microbial biotransformation of therapeutic compounds, such as anticancer or nonsteroidal anti-inflammatory drugs, can be modulated to reduce toxicity and potentially improve therapeutic efficacy.

Bacterial ß-glucuronidase inhibition protects mice against enteropathy induced by indomethacin, ketoprofen or diclofenac: mode of action and pharmacokinetics.

1. We have previously demonstrated that a small molecule inhibitor of bacterial ß-glucuronidase (Inh-1; [1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea]) protected mice against diclofenac (DCF)-induced enteropathy. Here we report that Inh-1 was equally protective against small intestinal injury induced by other carboxylic acid-containing non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin (10 mg/kg, ip) and ketoprofen (100 mg/kg, ip). 2. Inh-1 provided complete protection if given prior to DCF (60 mg/kg, ip), and partial protection if administered 3-h post-DCF, suggesting that the temporal window of mucosal protection can be extended for drugs undergoing extensive enterohepatic circulation. 3. Pharmacokinetic analysis of Inh-1 revealed an absolute bioavailability (F) of 21% and a short t1/2 of <1 h. This low F was shown to be due to hepatic first-pass metabolism, as confirmed with the pan-CYP inhibitor, 1-aminobenzotriazole. 4. Using the fluorescent probe 5 (and 6)-carboxy-2',7'-dichlorofluorescein, we demonstrated that Inh-1 did not interfere with hepatobiliary export of glucuronides in gall bladder-cannulated mice. 5. These data are compatible with the hypothesis that pharmacological inhibition of bacterial ß-glucuronidase-mediated cleavage of NSAID glucuronides in the small intestinal lumen can protect against NSAID-induced enteropathy caused by locally high concentrations of NSAID aglycones.

Stem Cell Strategies to Evaluate Idiosyncratic Drug-induced Liver Injury.

The host-dependent nature of idiosyncratic drug-induced liver injury (iDILI) suggests that rare genetic polymorphisms may contribute to the disease. Indeed, a few mutations in key genes have already been identified using conventional human genetics approaches. Over 50 commonly used drugs can precipitate iDILI, making this a substantial medical problem. Only recently have human induced pluripotent stem cells been used as a research tool to discover novel iDILI genes and to study the mechanisms of iDILI in vitro. Here we review the current state of stem cell use in the investigation of iDILI, with a special focus on genetics. In addition, the concerns and difficulties associated with genetics and animal model research are discussed. We then present the features of patient-specific pluripotent stem cells (which may be derived from iDILI patients themselves), and explain why these cells may be of great utility. A variety of recent approaches to produce hepatocyte-like cells from pluripotent cells and the associated advantages and limitations of such cells are discussed. Future directions for the use of stem cell science to investigate iDILI include novel ways to identify new iDILI genes, a consideration of epigenetic impacts on iDILI, and the development of new and improved strategies for the production of hepatocytes from human pluripotent cells.

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes.

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH (≤ 3000 µM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 µM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4(+/-) mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury.

Integrative toxicoproteomics implicates impaired mitochondrial glutathione import as an off-target effect of troglitazone.

Troglitazone, a first-generation thiazolidinedione of antihyperglycaemic properties, was withdrawn from the market due to unacceptable idiosyncratic hepatotoxicity. Despite intensive research, the underlying mechanism of troglitazone-induced liver toxicity remains unknown. Here we report the use of the Sod2(+/-) mouse model of silent mitochondrial oxidative-stress-based and quantitative mass spectrometry-based proteomics to track the mitochondrial proteome changes induced by physiologically relevant troglitazone doses. By quantitative untargeted proteomics, we first globally profiled the Sod2(+/-) hepatic mitochondria proteome and found perturbations including GSH metabolism that enhanced the toxicity of the normally nontoxic troglitazone. Short- and long-term troglitazone administration in Sod2(+/-) mouse led to a mitochondrial proteome shift from an early compensatory response to an eventual phase of intolerable oxidative stress, due to decreased mitochondrial glutathione (mGSH) import protein, decreased dicarboxylate ion carrier (DIC), and the specific activation of ASK1-JNK and FOXO3a with prolonged troglitazone exposure. Furthermore, mapping of the detected proteins onto mouse specific protein-centered networks revealed lipid-associated proteins as contributors to overt mitochondrial and liver injury when under prolonged exposure to the lipid-normalizing troglitazone. By integrative toxicoproteomics, we demonstrated a powerful systems approach in identifying the collapse of specific fragile nodes and activation of crucial proteome reconfiguration regulators when targeted by an exogenous toxicant.

Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious gastrointestinal (GI) injury including jejunal/ileal mucosal ulceration, bleeding, and even perforation in susceptible patients. The underlying mechanisms are largely unknown, but they are distinct from those related to gastric injury. Based on recent insights from experimental models, including genetics and pharmacology in rodents typically exposed to diclofenac, indomethacin, or naproxen, we propose a multiple-hit pathogenesis of NSAID enteropathy. The multiple hits start with an initial pharmacokinetic determinant caused by vectorial hepatobiliary excretion and delivery of glucuronidated NSAID or oxidative metabolite conjugates to the distal small intestinal lumen, where bacterial ß-glucuronidase produces critical aglycones. The released aglycones are then taken up by enterocytes and further metabolized by intestinal cytochrome P450s to potentially reactive intermediates. The "first hit" is caused by the NSAID and/or oxidative metabolites that induce severe endoplasmic reticulum stress or mitochondrial stress and lead to cell death. The "second hit" is created by the significant subsequent inflammatory response that would follow such a first-hit injury. Based on these putative mechanisms, strategies have been developed to protect the enterocytes from being exposed to the parent NSAID and/or oxidative metabolites. Among these, a novel strategy already demonstrated in a murine model is the selective disruption of bacteria-specific ß-glucuronidases with a novel small molecule inhibitor that does not harm the bacteria and that alleviates NSAID-induced enteropathy. Such mechanism-based strategies require further investigation but provide potential avenues for the alleviation of the GI toxicity caused by multiple NSAID hits.

Pharmacologic targeting of bacterial ß-glucuronidase alleviates nonsteroidal anti-inflammatory drug-induced enteropathy in mice.

Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial ß-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli ß-D-glucuronidase. Using a previously characterized novel bacteria-specific ß-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC50 ∼164 nM). We next hypothesized that pharmacologic inhibition of bacterial ß-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 µg per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the C(max), half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial ß-D-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure.

Acetaminophen overdose-induced liver injury in mice is mediated by peroxynitrite independently of the cyclophilin D-regulated permeability transition.

UNLABELLED: Acetaminophen (APAP) is safe at therapeutic dosage but can cause severe hepatotoxicity if used at overdose. The mechanisms of injury are not yet fully understood, but previous reports had suggested that the mitochondrial permeability transition (mPT) may be involved in triggering hepatocellular necrosis. We aimed at inhibiting mitochondrial cyclophilin D (CypD), a key regulator of the mPT, as a potential therapeutic target in APAP hepatotoxicity. Wildtype mice treated with a high dose of APAP (600 mg/kg, intraperitoneal) developed typical centrilobular necrosis, which could not, however, be prevented by cotreatment with the selective CypD inhibitor, Debio 025 (alisporivir, DEB025, a nonimmunosuppressive cyclosporin A analog). Similarly, genetic ablation of mitochondrial CypD in Ppif-null mice did not afford protection from APAP hepatotoxicity. To determine whether APAP-induced peroxynitrite stress might directly activate mitochondrial permeabilization, independently of the CypD-regulated mPT, we coadministered the peroxynitrite decomposition catalyst Fe-TMPyP (10 mg/kg, intraperitoneal, 90 minutes prior to APAP) to CypD-deficient mice. Liver injury was greatly attenuated by Fe-TMPyP pretreatment, and mitochondrial 3-nitrotyrosine adduct levels (peroxynitrite marker) were decreased. Acetaminophen treatment increased both the cytosolic and mitochondria-associated P-JNK levels, but the c-jun-N-terminal kinase (JNK) signaling inhibitor SP600125 was hepatoprotective in wildtype mice only, indicating that the JNK pathway may not be critically involved in the absence of CypD. CONCLUSION: These data support the concept that an overdose of APAP results in liver injury that is refractory to pharmacological inhibition or genetic depletion of CypD and that peroxynitrite-mediated cell injury predominates in the absence of CypD.

NSAID acyl glucuronides and enteropathy.

Many nonsteroidal anti-inflammatory drugs (NSAIDs) are carboxylic acid-containing compounds that are conjugated in the liver to acyl glucuronides and excreted across the hepatocanalicular membrane into bile. Chronic and acute NSAID use has not only been associated with gastric injury but also increasingly recognized to cause small intestinal injury (enteropathy). The mechanisms of NSAID enteropathy are still unknown, but a combination of topical effects (including mitochondrial injury) combined with inhibition of COX1/2, followed by an inflammatory response triggered by LPS-mediated activation of LTR4 on macrophages, have been implicated in the pathogenesis. Some of the nucleophilic proteins that are targeted by the electrophilic NSAID acyl glucuronides or their iso-glucuronides have been identified both in bile canaliculi and on the apical membrane domain of enterocytes (e.g., aminopeptidase N); however, the mechanistic role of covalent adducts has remained enigmatic. In contrast, it has become increasingly clear that acyl glucuronide formation is a major toxicokinetic determinant, in that the drug conjugates are a transport form delivering the drug to the more distal parts of the jejunum/ileum, where the glucuronic acid moiety is cleaved off the aglycone due to higher local pH and the presence of bacterial ß-glucuronidase. Through this mechanism, high local concentrations of the parent NSAID can be attained, potentially leading to local tissue injury. Thus, even if one considers the formation of acyl glucuronides not as a potentially dangerous toxophore by virtue of their protein-reactivity, acyl glucuronides could still be a red flag in drug development if excreted at high rates into bile and delivered to more distal areas of the small intestine where high amounts of parent drug is released.

Trovafloxacin, a fluoroquinolone antibiotic with hepatotoxic potential, causes mitochondrial peroxynitrite stress in a mouse model of underlying mitochondrial dysfunction.

Trovafloxacin (TVX) is a fluoroquinolone antibiotic whose therapeutic use was severely restricted due to an unacceptable risk of idiosyncratic liver injury. Oxidative stress and mitochondrial injury have been implicated in fluoroquinolone toxicity, but the mechanisms underlying liver injury are poorly understood. Because TVX-induced hepatotoxicity cannot be modeled in normal healthy rodents, we asked whether an underlying genetic defect (heterozygous deficiency in mitochondrial superoxide dismutase, Sod2) might aggravate TVX-induced mitochondrial adverse effects. Wild-type and Sod2(+/-) mice were treated with vehicle or alatrofloxacin (the prodrug of TVX, 33mg/kg/day, ip) for 28 days. We found that hepatic protein carbonyls were increased by 2.5-fold and hepatic mitochondrial aconitase activity was decreased by 20% in mutant, but not wild-type mice. Because aconitase is a major target of peroxynitrite, we determined the extent of nitrotyrosine residues in hepatic mitochondrial proteins. Trovafloxacin significantly increased nitrotyrosine in Sod2(+/-) mice only. Using the NO-selective probe DAF-2, we found that TVX increased the production of mitochondrial NO in immortalized human hepatocytes. Similarly, mitochondrial Ca(2+) was increased by TVX, suggesting Ca(2+)-dependent activation of mitochondrial NOS activity. Furthermore, the transcript levels of the mtDNA-encoded gene Cox2/mtCo2 were decreased in Sod2(+/-) mice only, while the expression of nDNA-encoded mitochondrial genes was not significantly altered in both genotypes, suggesting selective effects on mtDNA expression. The amount of mtDNA (copy number) was, however, unchanged. These data indicate that TVX enhances hepatic mitochondrial peroxynitrite stress in mice with underlying increased basal levels of superoxide, leading to the disruption of critical mitochondrial enzymes and gene regulation.

Pharmacologic targeting or genetic deletion of mitochondrial cyclophilin D protects from NSAID-induced small intestinal ulceration in mice.

Small intestinal ulceration is a frequent and potentially serious condition associated with nonselective cyclooxygenase 1/2 inhibitors (nonsteroidal anti-inflammatory drugs, NSAIDs) including diclofenac (DCF). An initial topical effect involving mitochondria has been implicated in the pathogenesis, but the exact mechanisms of NSAID-induced enteropathy are unknown. We aimed at investigating whether DCF caused enterocyte demise via the mitochondrial permeability transition (mPT) and whether inhibition of critical mPT regulators might protect the mucosa from DCF injury. Cultured enterocytes (IEC-6) exposed to DCF readily underwent mPT-mediated cell death. We then targeted mitochondrial cyclophilin D (CypD), a key regulator of the mPT, in a mouse model of NSAID enteropathy. C57BL/6J mice were treated with an ulcerogenic dose of DCF (60 mg/kg, ip), followed (+ 1 h) by a non-cholestatic dose (10 mg/kg, ip) of the CypD inhibitor, cyclosporin A (CsA). CsA greatly reduced the extent of small intestinal ulceration. To avoid potential calcineurin-mediated effects, we used the non-immunosuppressive cyclosporin analog, D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025). Debio 025 similarly protected the mucosa from DCF injury. To exclude drug-drug interactions, we exposed mice genetically deficient in mitochondrial CypD (peptidyl-prolyl cis-trans isomerase F [Ppif(-/-)]) to DCF. Ppif-null mice were largely protected from the ulcerogenic effects of DCF, whereas their wild-type littermates developed typical enteropathy. Enterocyte injury was preceded by upregulation of the proapoptotic transcription factor C/EBP homologous protein (Chop). Chop-null mice were refractory to DCF enteropathy, suggesting a critical role of endoplasmic reticulum stress induced by DCF. In conclusion, mitochondrial CypD plays a key role in NSAID-induced enteropathy, lending itself as a potentially new therapeutic target for cytoprotective intervention.

Nimesulide-induced electrophile stress activates Nrf2 in human hepatocytes and mice but is not sufficient to induce hepatotoxicity in Nrf2-deficient mice.

Nimesulide is a widely prescribed nitroaromatic sulfoanilide-type nonsteroidal anti-inflammatory drug that, despite its favorable safety profile, has been associated with rare cases of idiosyncratic drug-induced liver injury (DILI). Because reactive metabolites have been implicated in DILI, we aimed at investigating whether hepatic bioactivation of nimesulide produces a protein-reactive intermediate in hepatocytes. Also, we explored whether nimesulide can activate the transcription factor Nrf2 that would protect from drug-induced hepatocyte injury. We found that [(14)C]-nimesulide covalently bound to human liver microsomes (<50 pmol/mg under standard conditions) or immortalized human hepatocytes in a sulfaphenazole-sensitive, rifampicin-inducible manner; yet the overall extent of binding was modest. Although exposure of hepatocytes to nimesulide was not associated with increased net levels of superoxide anion, nimesulide (100 microM, 24 h) caused nuclear translocation of Nrf2 in a sulfaphenazole-sensitive manner, indicating a role of electrophilic metabolites. However, knockdown of Nrf2 with siRNA did not make the cells more sensitive to nimesulide-induced cell injury. Similarly, exposure of wild-type C57BL/6x129 Sv mice to nimesulide (100 mg/kg/day, po, for 5 days) was associated with nuclear translocation of immunoreactive Nrf2 in a small number of hepatocytes and induced >2-fold the expression levels of the Nrf2-target gene Nqo1 in wild-type but not Nrf2-null mice. Nimesulide administered to Nrf2(-/-) knockout mice did not cause increases in serum ALT activity or any apparent histopathological signs of liver injury. In conclusion, these data indicate that nimesulide is bioactivated by CYP2C to a protein-reactive electrophilic intermediate that activates the Nrf2 pathway even at nontoxic exposure levels.

Mechanisms of pathogenesis in drug hepatotoxicity putting the stress on mitochondria.

Mitochondria play key roles in aerobic life and in cell death. Thus, interference of normal mitochondrial function impairs cellular energy and lipid metabolism and leads to the unleashing of mediators of cell death. The role of mitochondria in cell death due to drug hepatotoxicity has been receiving renewed attention and it is therefore timely to assess the current status of this area.

The Sod2 mutant mouse as a model for oxidative stress: a functional proteomics perspective.

Oxidative stress has been implicated in the pathogenesis of numerous human diseases and disorders, but the mechanistic basis often remains enigmatic. The Sod2 mutant mouse, which is sensitized to mitochondrial stress, is an ideal mutant model for studying the role of oxidative stress in a diverse range of complications arising from mitochondrial dysfunction and diminished antioxidant defense. To fully appreciate the widespread molecular consequences under increased oxidative stress, a systems approach utilizing proteomics is able to provide a global overview of the complex biological changes, which a targeted single biomolecular approach cannot address fully. This review focuses on the applications of mass spectrometry and functional proteomics in the Sod2 mouse. The combinatorial approach provides novel insights into the interplay of chemistry and biology, free radicals and proteins, thereby augmenting our understanding of how redox perturbations influence protein dynamics. Ultimately, this knowledge can lead to the development of free radical-targeted therapies.