Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanism-based toxicity.

Steatosis is a liver lesion reported with numerous pharmaceuticals. Prior studies showed that severe impairment of mitochondrial fatty acid oxidation (mtFAO) constantly leads to lipid accretion in liver. However, much less is known about the mechanism(s) of drug-induced steatosis in the absence of severe mitochondrial dysfunction, although previous studies suggested the involvement of mild-to-moderate inhibition of mtFAO, increased de novo lipogenesis (DNL), and impairment of very low-density lipoprotein (VLDL) secretion. The objective of our study, mainly carried out in human hepatoma HepaRG cells, was to investigate these 3 mechanisms with 12 drugs able to induce steatosis in human: amiodarone (AMIO, used as positive control), allopurinol (ALLO), D-penicillamine (DPEN), 5-fluorouracil (5FU), indinavir (INDI), indomethacin (INDO), methimazole (METHI), methotrexate (METHO), nifedipine (NIF), rifampicin (RIF), sulindac (SUL), and troglitazone (TRO). Hepatic cells were exposed to drugs for 4 days with concentrations decreasing ATP level by less than 30% as compared to control and not exceeding 100 × Cmax. Among the 12 drugs, AMIO, ALLO, 5FU, INDI, INDO, METHO, RIF, SUL, and TRO induced steatosis in HepaRG cells. AMIO, INDO, and RIF decreased mtFAO. AMIO, INDO, and SUL enhanced DNL. ALLO, 5FU, INDI, INDO, SUL, RIF, and TRO impaired VLDL secretion. These seven drugs reduced the mRNA level of genes playing a major role in VLDL assembly and also induced endoplasmic reticulum (ER) stress. Thus, in the absence of severe mitochondrial dysfunction, drug-induced steatosis can be triggered by different mechanisms, although impairment of VLDL secretion seems more frequently involved, possibly as a consequence of ER stress.

Chronic and low exposure to a pharmaceutical cocktail induces mitochondrial dysfunction in liver and hyperglycemia: Differential responses between lean and obese mice.

Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.

Drug-induced impairment of mitochondrial fatty acid oxidation and steatosis: assessment of causal relationship with 45 pharmaceuticals.

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 non-steatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-L-carnitine, palmitoyl-CoA + L-carnitine, or octanoyl-L-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, while dexamethasone, olanzapine and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.

Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver.

Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis).

Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

A Fast, Simple, and Affordable Technique to Measure Oxygen Consumption in Living Zebrafish Embryos.

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Chemosensitization by knockdown of adenine nucleotide translocase-2.

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.

The Mitochondrion-lysosome Axis in Adaptive and Innate Immunity: Effect of Lupus Regulator Peptide P140 on Mitochondria Autophagy and NETosis.

Mitochondria deserve special attention as sensors of cellular energy homeostasis and metabolic state. Moreover, mitochondria integrate intra- and extra-cellular signals to determine appropriate cellular responses that range from proliferation to cell death. In autoimmunity, as in other inflammatory chronic disorders, the metabolism of immune cells may be extensively remodeled, perturbing sensitive tolerogenic mechanisms. Here, we examine the distribution and effects of the therapeutic 21-mer peptide called P140, which shows remarkable efficacy in modulating immune responses in inflammatory settings. We measured P140 and control peptide effects on isolated mitochondria, the distribution of peptides in live cells, and their influence on the levels of key autophagy regulators. Our data indicate that while P140 targets macro- and chaperone-mediated autophagy processes, it has little effect, if any, on mitochondrial autophagy. Remarkably, however, it suppresses NET release from neutrophils exposed to immobilized NET-anti-DNA IgG complexes. Together, our results suggest that in the mitochondrion-lysosome axis, a likely driver of NETosis and inflammation, the P140 peptide does not operate by affecting mitochondria directly.

Therapeutic peptides: Targeting the mitochondrion to modulate apoptosis.

For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation..., etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.

Aftins increase amyloid-ß42, lower amyloid-ß38, and do not alter amyloid-ß40 extracellular production in vitro: toward a chemical model of Alzheimer's disease?

Increased production of amyloid-ß (Aß)42 peptide, derived from the amyloid-ß protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-ß Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger ß-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aß42, and parallel down-regulation of Aß38, while Aß40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aß38/Aß40/Aß42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aß42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aß levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aß42 production and Aß38 down-regulation (potential 'pro-AD compounds').

Prediction of liver injury induced by chemicals in human with a multiparametric assay on isolated mouse liver mitochondria.

Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new compounds and marketed compounds.

Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-induced mitochondrial membrane permeabilization.

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells.

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

Pharmacological screening and enzymatic assays for apoptosis.

Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.