Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Maternal and neonatal outcomes and prognostic factors in acute fatty liver of pregnancy.

OBJECTIVE: To report complications of Acute Fatty Liver of pregnancy (AFLP), a rare liver disease of pregnancy, and identify prognostic factors for mothers and children. STUDY DESIGN: We conducted a retrospective descriptive study over 18 years in three French maternities. Demographic, clinical, biological data, and outcomes of patients and their infants were reviewed. RESULTS: 142,450 pregnancies from centers were studied. Eighteen patients with AFLP were identified The prevalence of AFLP was estimated as 1/7,914 pregnancies. Prolonged prothrombin time was identified as a risk factor of maternal complications (OR = 0.86, p = 0.0493). Gestational age at delivery was the only risk factor associated with fetal or neonate complications (OR = 0.37, p = 0.0417). One boy died of previously undiagnosed ß-oxidation deficiency at eight months. CONCLUSION: In AFLP, prothrombin time must be carefully monitored to anticipate major maternal complications. Infants born to mothers with ALFP should be screened as early as possible for mitochondrial fatty acid oxidation deficiency.

Indicators of iron status are correlated with adiponectin expression in adipose tissue of patients with morbid obesity.

AIM: The aim of this study was to assess interactions between glucose and iron homoeostasis in the adipose tissue (AT) of obese subjects. METHODS: A total of 46 obese patients eligible for bariatric surgery were recruited into the study. Anthropometric and biochemical characteristics were assessed, and biopsies of subcutaneous (SCAT) and visceral adipose tissue (VAT) performed. The mRNA levels of genes involved in iron and glucose homoeostasis were measured in their AT and compared with a pool of control samples. RESULTS: Gene expression of hepcidin (HAMP) was significantly increased in the SCAT and VAT of obese patients, while transferrin receptor (TFRC) expression was reduced, compared with non-obese controls, suggesting a higher iron load in obese patients. Also, mRNA levels of adiponectin (ADIPOQ) were decreased in both SCAT and VAT in obese patients, and correlated negatively with hepcidin expression, while adiponectin expression was positively correlated with TFRC expression in both SCAT and VAT. Interestingly, TFRC expression in VAT correlated negatively with several metabolic parameters, such as fasting blood glucose and LDL cholesterol. CONCLUSION: Iron content appears to be increased in the SCAT and VAT of obese patients, and negatively correlated with adiponectin expression, which could be contributing to insulin resistance and the metabolic complications of obesity.

Burn after feeding. An old uncoupler of oxidative phosphorylation is redesigned for the treatment of nonalcoholic fatty liver disease.

Uncoupling of oxidative phosphorylation (OXPHOS) in brown adipose tissue can be used by hibernating animals to produce heat at the expense of their fat mass. In a recent work, Dr Shulman et al. generated a liver-targeted derivative of the prototypical OXPHOS uncoupler 2,4-dinitrophenol that alleviated steatosis, hypertriglyceridemia and insulin resistance in several models of nonalcoholic fatty liver disease and type 2 diabetes.

Fat accretion in a subpopulation of hepatocytes as a strategy to protect the whole liver against oxidative stress and lipotoxicity.

Fatty liver can be induced by obesity, some drugs and alcohol intoxication. In this liver lesion, lipid accumulation can involve only some hepatocytes but the significance of this cell-to-cell heterogeneity is unknown. In a recent work, Dr Pol et al. propose that high-fat hepatocytes could protect the cell population against oxidative stress and lipotoxicity.

Bridging the gap between old and new concepts in drug-induced liver injury.

Recent studies have provided important information in the field of drug-induced liver injury (DILI), in particular regarding the pathogenesis of acetaminophen hepatotoxicity. However, these studies have sometimes left aside some old (but seminal) findings. Efforts should be made to bridge the gap between old and new concepts in DILI.

Pentoxifylline aggravates fatty liver in obese and diabetic ob/ob mice by increasing intestinal glucose absorption and activating hepatic lipogenesis.

BACKGROUND AND PURPOSE: Pentoxifylline is in clinical trials for non-alcoholic fatty liver disease and diabetic nephropathy. Metabolic and hepatic effects of pentoxifylline were assessed in a murine model of obesity and type 2 diabetes. EXPERIMENTAL APPROACH: Pentoxifylline (100 mg·kg(-1) ·day(-1)) was administered for 4 days or 3 weeks in lean and obese/diabetic ob/ob mice. Plasma lipids, glucose, other metabolites and relevant enzymes were measured by standard assays. Hepatic lipids in vivo were assessed with magnetic resonance spectroscopy and by histology. Hepatic extracts were also analysed with RT-PCR and Western blotting. KEY RESULTS: Four days of pentoxifylline treatment slightly increased liver lipids in ob/ob mice. After 3 weeks, pentoxifylline exacerbated fatty liver and plasma transaminases in ob/ob mice but did not induce liver steatosis in lean mice. Plasma glucose was highest in fed, but not fasted, ob/ob mice treated with pentoxifylline. During the first 10 min of an oral glucose tolerance test, blood glucose increased more rapidly in pentoxifylline-treated mice. Jejunal expression of glucose transporter 2 isoform was increased in pentoxifylline-treated obese mice. Hepatic activity of carbohydrate response element binding protein (ChREBP) increased after pentoxifylline in ob/ob, but not lean, mice. Hepatic expression of lipogenic enzymes was highest in pentoxifylline-treated ob/ob mice. However, pentoxifylline reduced markers of oxidative stress and inflammation in ob/ob liver. CONCLUSION AND IMPLICATIONS: Pentoxifylline exacerbated fatty liver in ob/ob mice through enhanced intestinal glucose absorption, increased postprandial glycaemia and activation of hepatic lipogenesis. Long-term treatment with pentoxifylline could worsen fatty liver in some patients with pre-existing hyperglycaemia.

Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role.

Due to the worldwide surge in obesity and type 2 diabetes, the increased incidence of nonalcoholic fatty liver disease (NAFLD) is a major concern for the public health. Indeed, NAFLD encompasses a large spectrum of conditions ranging from fatty liver to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis in some patients. A better understanding of the mechanisms involved in fatty liver and its progression into NASH is important in order to develop efficient drugs able to alleviate these liver diseases. Although numerous investigations pointed to reactive oxygen species (ROS) as key players in the progression of fatty liver to NASH, their exact source is still uncertain. Besides the mitochondrial respiratory chain, cytochrome P450 2E1 (CYP2E1) has recently emerged as another potentially important cause of ROS overproduction. Indeed, higher hepatic CYP2E1 expression and activity have been frequently observed in the context of obesity and NAFLD. It is currently unknown why CYP2E1 is enhanced in these dysmetabolic diseases, although increased hepatic levels of fatty acids and insulin resistance might play a role. Nonetheless, higher hepatic CYP2E1 could play a significant role in the pathophysiology of NASH by inducing lipid peroxidation and oxidative damage of key cellular components. Moreover, CYP2E1-mediated overproduction of ROS could promote hepatic insulin resistance, which can further aggravate fatty liver. Since a significant amount of CYP2E1 can be located within liver mitochondria, higher levels of CYP2E1 in NAFLD could also have detrimental effects on mitochondrial function. Finally, increased CYP2E1 activity during NAFLD could enhance the susceptibility of some patients to the hepatotoxicity of different xenobiotics through the CYP2E1-mediated generation of harmful reactive metabolites.

The ins and outs of mitochondrial dysfunction in NASH.

Rich diet and lack of exercise are causing a surge in obesity, insulin resistance and steatosis, which can evolve into steatohepatitis. Steatosis and nonalcoholic steatohepatitis (NASH) can also be induced by drugs such as amiodarone, tamoxifen and some antiretroviral drugs. There is growing evidence that mitochondrial dysfunction, and more specifically respiratory chain deficiency, plays a role in the pathophysiology of NASH whatever its initial cause. In contrast, the B-oxidation of fatty acids can be either increased (as in insulin resistance-associated NASH) or decreased (as in drug-induced NASH). However, in both circumstances, the generation of reactive oxygen species (ROS) by the damaged respiratory chain is augmented, as components of this chain are over-reduced by electrons, which then abnormally react with oxygen to form increased amounts of ROS. Concomitantly, ROS oxidize fat deposits to release lipid peroxidation products that have detrimental effects on hepatocytes and other hepatic cells. In hepatocytes, ROS and lipid peroxidation products further impair the respiratory chain, either directly or indirectly through oxidative damage to the mitochondrial genome. This, in turn, leads to the generation of more ROS and a vicious cycle ensues. Mitochondrial dysfunction can also lead to apoptosis or necrosis depending on the energy status of the cell. ROS and lipid peroxidation products also activate stellate cells, thus resulting in fibrosis. Finally, ROS and lipid peroxidation increase the generation of several cytokines (TNF-alpha, TGF-B, Fas ligand) that play sundry roles in the pathogenesis of NASH. Recent investigations have shown that some genetic polymorphisms can significantly increase the risk of steatohepatitis and that several drugs can prevent or even reverse NASH. For the next decade, reducing the incidence of NASH will be a major challenge for hepatologists.

Acute ethanol administration oxidatively damages and depletes mitochondrial dna in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants.

Ethanol metabolism causes oxidative stress and lipid peroxidation not only in liver but also in extra-hepatic tissues. Ethanol administration has been shown to cause oxidative degradation and depletion of hepatic mitochondrial DNA (mtDNA) in rodents, but its in vivo effects on the mtDNA of extra-hepatic tissues have not been assessed. We studied the effects of an acute intragastric ethanol administration (5 g/kg) on brain, heart, skeletal muscle, and liver mtDNA in mice. Ethanol administration caused mtDNA depletion and replacement of its supercoiled form by linearized forms in all tissues examined. Maximal mtDNA depletion was about similar (ca. 50%) in all organs studied. It occurred 2 h after ethanol administration in heart, skeletal muscle, and liver but after 10 h in brain. This mtDNA depletion was followed by increased mtDNA synthesis. A secondary, transient increase in mtDNA levels occurred 24 h after ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by vitamin E, melatonin, or coenzyme Q, three antioxidants. In conclusion, our study shows for the first time that ethanol metabolism also causes oxidative degradation of the mitochondrial genome in brain, heart, and skeletal muscles. These effects could contribute to the development of (cardio)myopathy and brain injury in some alcoholic patients. Antioxidants prevent these effects in mice and could be useful in persevering drinkers.

Homozygosity for alanine in the mitochondrial targeting sequence of superoxide dismutase and risk for severe alcoholic liver disease.

BACKGROUND & AIMS: For similar ethanol consumption, some subjects only develop macrovacuolar steatosis whereas others develop severe liver lesions. A genetic dimorphism encodes for either alanine or valine in the mitochondrial targeting sequence of manganese superoxide dismutase and could modulate its mitochondrial import. METHODS: The DNA of 71 white patients with alcoholic liver disease and 79 white blood donors was amplified and genotyped. RESULTS: The frequency of the alanine-encoding allele and the percentage of alanine homozygotes were higher in all patients than in controls and increased with the severity of liver lesions. The percentage of alanine homozygotes was 19% in controls, 17% in alcoholic patients with macrovacuolar steatosis, 43% in patients with microvesicular steatosis, 58% in patients with alcoholic hepatitis, and 69% in patients with cirrhosis. Alcohol consumption in alcoholics was similar whatever the genotype. Alanine homozygosity did not change the risk of developing macrovacuolar steatosis in alcoholics, but increased by 3-fold that of microvesicular steatosis, and 6- and 10-fold that of alcoholic hepatitis and cirrhosis. CONCLUSIONS: Homozygosity for alanine in the mitochondrial targeting sequence of manganese superoxide does not modify alcohol consumption and the risk of macrovacuolar steatosis in alcoholics but is a major risk factor for severe alcoholic liver disease.

Mitochondria in steatohepatitis.

For the first time in history, populations in affluent countries may concomitantly indulge in rich food and physical idleness. Various combinations of obesity, diabetes, and hypertriglyceridemia, with insulin resistance as the common feature, cause hepatic steatosis, which can trigger necroinflammation and fibrosis. Patients with "primary" steatohepatitis exhibit ultrastructural mitochondrial lesions, decreased activity of respiratory chain complexes, and have impaired ability to resynthesize ATP after a fructose challenge. Mitochondria play a major role in fat oxidation and energy production but also leak reactive oxygen species (ROS) and are the main cellular source of ROS. In patients with steatosis, mitochondrial ROS may oxidize hepatic fat deposits, as suggested in animal models. Lipid peroxidation products impair the flow of electrons along the respiratory chain, which may cause overreduction of respiratory chain components, further increasing mitochondrial ROS formation and lipid peroxidation. Another vicious circle could involve ROS-induced depletion of antioxidants, impairing ROS inactivation. Blood vitamin E is decreased in some obese children with steatohepatitis, and serum transaminases improve after vitamin E supplementation. Steatohepatitis is also caused by alcohol abuse, drugs, and other causes. In "secondary" steatohepatitis, mitochondrial ROS formation is further increased as the causative disease itself directly increases ROS or first impairs respiration, which secondarily increases mitochondrial ROS formation. This "second hit" could cause more lipid peroxidation, cytokine induction, Fas ligand induction, and fibrogenesis than in primary steatohepatitis.

Effect of stavudine on mitochondrial genome and fatty acid oxidation in lean and obese mice.

Like other antihuman immunodeficiency virus dideoxynucleosides, stavudine may occasionally induce lactic acidosis and perhaps lipodystrophy in metabolically or genetically susceptible patients. We studied the effects of stavudine on mitochondrial DNA (mtDNA), fatty acid oxidation, and blood metabolites in lean and genetically obese (ob/ob) mice. In lean mice, mtDNA was depleted in liver and skeletal muscle, but not heart and brain, after 6 weeks of stavudine treatment (500 mg/kg/day). With 100 mg/kg/day, mtDNA transiently decreased in liver, but was unchanged at 6 weeks in all organs, including white adipose tissue (WAT). Despite unchanged mtDNA levels, lack of significant oxidative mtDNA lesions (as assessed by long polymerase chain reaction experiments), and normal blood lactate/pyruvate ratios, lean mice treated with stavudine for 6 weeks had increased fasting blood ketone bodies, due to both increased hepatic fatty acid beta-oxidation and decreased peripheral ketolysis. In obese mice, basal WAT mtDNA was low and was further decreased by stavudine. In conclusion, stavudine can decrease hepatic and muscle mtDNA in lean mice and can also cause ketoacidosis during fasting without altering mtDNA. Stavudine depletes WAT mtDNA only in obese mice. Fasting and ketoacidosis could trigger decompensation in patients with incipient lactic acidosis, whereas WAT mtDNA depletion could cause lipodystrophy in genetically susceptible patients.

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates.

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice.

BACKGROUND & AIMS: Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks. METHODS: Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively. RESULTS: Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant. CONCLUSIONS: After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Hepatotoxicity due to mitochondrial dysfunction.

Mitochondria are involved in fatty acid beta-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation, which provide most of the cell energy. Mitochondria are also the main source of reactive oxygen species in the cell and are involved in cell demise through opening of the mitochondrial permeability transition pore. It was therefore to be expected that mitochondrial dysfunction could be a major mechanism of drug-induced liver disease. Microvesicular steatosis (which may cause liver failure, coma, and death) is the consequence of severe impairment of mitochondrial beta-oxidation. Endogenous compounds (such as cytokines or female sex hormones) or xenobiotics (including toxins such as ethanol and drugs such as aspirin, valproic acid, ibuprofen, or zidovudine) can inhibit beta-oxidation directly or through a primary effect on the mitochondrial genome or the respiratory chain itself. In some patients, infections and cytokines, or inborn errors of beta-oxidation enzymes or the mitochondrial genome, may favor the appearance of drug-induced microvesicular steatosis. Nonalcoholic steatohepatitis may develop under conditions causing prolonged, microvesicular, and/or macrovacuolar steatosis. In this condition, chronic impairment of mitochondrial beta-oxidation (causing steatosis) and the respiratory chain (increasing the production of ROS) lead to lipid peroxidation, which, in turn, may cause the diverse lesions of steatohepatitis, namely, necrosis, inflammation, Mallory's bodies, and fibrosis. Finally, mitochondria are involved in several forms of drug-induced cytolytic hepatitis, through inhibition or uncoupling of respiration or through a drug-induced or reactive metabolite-induced mitochondrial permeability transition. The latter effect commits hepatocytes to either apoptosis or necrosis, depending on the number of organelles that have undergone the permeability transition.

Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.

BACKGROUND & AIMS: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms. METHODS: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined. RESULTS: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats. CONCLUSIONS: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

Multiple mtDNA deletions features in autosomal dominant and recessive diseases suggest distinct pathogeneses.

Multiple mitochondrial DNA (mtDNA) deletions have been described in patients with autosomal dominant progressive external ophthalmoplegia (AD-PEO) and in autosomal recessive disorders including mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and autosomal recessive cardiomyopathy ophthalmoplegia (ARCO). The pathogenic bases of these disorders are unknown. We studied three patients with AD-PEO and three patients with autosomal recessive (AR)-PEO (two patients with MNGIE and one patient with ARCO). Histochemistry and Southern blot analyses of DNA were performed in skeletal muscle from the patients. Muscle mtDNA was used to characterize the pattern and amounts of the multiple mtDNA rearrangements; PCR analysis was performed to obtain finer maps of the deleted regions in both conditions. The patients with AD-PEO had myopathic features; the patients with AR-PEO had multisystem disorders. The percentage of ragged-red and cytochrome c oxidase-negative fibers tended to be higher in muscle from the patients with AD-PEO (19% +/- 13.9, 29.7 +/- 26.3) than in muscle from the patients with AR-PEO (1.4% +/- 1.4, 3.3% +/- 3.2; p < 0.10). The sizes of the multiple mtDNA deletions ranged from approximately 4.0 to 10.0 kilobases in muscle from both groups of patients, and in both groups, we identified only deleted and no duplicated mtDNA molecules. Patients with AD-PEO harbored a greater proportion of deleted mtDNA species in muscle (31% +/- 5.3) than did patients with AR-PEO (9.7% +/- 9.1; p < 0.05). In the patients with AD-PEO, we identified a deletion that included the mtDNA heavy strand promoter (HSP) region, which had been previously described as the HSP deletion. The HSP deletion was not present in the patients with AR-PEO. Our findings show the clinical, histologic, and molecular genetic heterogeneity of these complex disorders. In particular, the proportions of multiple mtDNA deletions were higher in muscle samples from patients with AD-PEO than in those from patients with AR-PEO.