A review on the mitochondrial toxicity of "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA).

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") is a drug of abuse used by millions worldwide. MDMA human abuse and dependence is well described, but addictive properties are not always consistent among studies. This amphetamine is a substrate type releaser, binding to monoamine transporters, leading to a pronounced release of serotonin and noradrenaline and to a minor extent dopamine. The toxicity of MDMA is well studied at the pre-clinical level, with neurotoxicity and hepatotoxicity being particularly described. In this review, we describe the most relevant MDMA effects at the mitochondrial level found in in vitro and in vivo models, these later conducted in mice and rats. Most of these reports focus on the mitochondria of brain or liver. In in vitro models, MDMA causes depletion of ATP levels and inhibition of mitochondrial complex I and III, loss in mitochondrial membrane potential (ΔΨm) and induction of mitochondrial permeability transition. The involvement of mitochondria in the apoptotic cell death evoked by MDMA has also been shown, such as the release of cytochrome c. Additionally, MDMA or its metabolites impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria. In animal studies, MDMA decreased mitochondrial complex I activity and decreased ATP levels. Moreover, MDMA-evoked oxidative stress has been shown to cause deletion on mitochondrial DNA and impairment in mitochondrial protein synthesis. Although the concentrations and doses used in some studies do not always correlate to the human scenario, the mitochondrial abnormalities evoked by MDMA are well described and are in part responsible for its mechanism of toxicity.

Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death.

Background & Aims: In cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging. Methods: To primary cocultures of astrocytes and neurons, low concentrations (1 and 5 µM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy. Results: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP. Conclusions: Our results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. Lay summary: The impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Iron Overload via Heme Degradation in the Endoplasmic Reticulum Triggers Ferroptosis in Myocardial Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is a promising therapeutic target to improve clinical outcomes after acute myocardial infarction. Ferroptosis, triggered by iron overload and excessive lipid peroxides, is reportedly involved in I/R injury. However, its significance and mechanistic basis remain unclear. Here, we show that glutathione peroxidase 4 (GPx4), a key endogenous suppressor of ferroptosis, determines the susceptibility to myocardial I/R injury. Importantly, ferroptosis is a major mode of cell death in I/R injury, distinct from mitochondrial permeability transition (MPT)-driven necrosis. This suggests that the use of therapeutics targeting both modes is an effective strategy to further reduce the infarct size and thereby ameliorate cardiac remodeling after I/R injury. Furthermore, we demonstrate that heme oxygenase 1 up-regulation in response to hypoxia and hypoxia/reoxygenation degrades heme and thereby induces iron overload and ferroptosis in the endoplasmic reticulum (ER) of cardiomyocytes. Collectively, ferroptosis triggered by GPx4 reduction and iron overload in the ER is distinct from MPT-driven necrosis in both in vivo phenotype and in vitro mechanism for I/R injury. The use of therapeutics targeting ferroptosis in conjunction with cyclosporine A can be a promising strategy for I/R injury.

Hexane fraction of Globimetula braunii induces mitochondria-mediated apoptosis in Plasmodium berghei-infected mice.

Background: Apoptosis is a common pathology in malaria and most antimalarial drugs induce apoptosis during chemotherapy. Globimetula braunii is an African mistletoe used for the treatment of malaria but its effect on mitochondria-mediated apoptosis is not known. Methods: Malarial infection was induced by the intraperitoneal injection of NK 65 strain Plasmodium berghei-infected erythrocytes into mice which were treated with graded doses (100-400 mg/kg) of methanol extract (ME), and fractions of n-hexane, dichloromethane, ethylacetate and methanol (HF, DF, EF and MF) for 9 days after the confirmation of parasitemia. Artequine (10 mg/kg) was used as control drug. The fraction with the highest antiplasmodial activity was used (same dose) to treat mice infected with chloroquine-resistant (ANKA) strain for 5 consecutive days after the confirmation of parasitemia. P-alaxin (10 mg/kg) was used as control drug. On the last day of the treatment, liver mitochondria were isolated and mitochondrial Permeability Transition (mPT) pore opening, mitochondrial F0F1 ATPase (mATPase) activity, lipid peroxidation (mLPO) and liver deoxyribonucleic acid (DNA) fragmentation were assessed spectrophotometrically. Caspases 3 and 9 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Cytochrome c, P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl2) were determined via immunohistochemistry. Phytochemical constituents of the crude methanol extract of Globimetula braunii were determined via the Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Results: There was large amplitude mPT induction by malaria parasites, extract and fractions of Globimetula braunii. At 400 mg/kg, HF significantly (p < 0.01) downregulated mATPase activity, and mLPO in both (susceptible and resistant) models, caused DNA fragmentation (P < 0.0001), induced caspases activation, P53, bax and cytochrome c release but downregulated Bcl2 in both models. The GC-MS analysis of methanol extract of Globimetula braunii showed that α-amyrin is the most abundant phytochemical. Conclusion: The n-hexane fraction of Globimetula braunii induced mitochondrial-mediated apoptosis through the opening of the mitochondrial pore, fragmentation of genomic DNA, increase in the levels of P53, bax, caspase 3 and 9 activation and cytochrome c release with concomitant decrease in the level of Bcl2. α-Amyrin is a triterpene with apoptotic effects.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Indian National Association for the Study of the Liver Consensus Statement on Acute Liver Failure (Part 1): Epidemiology, Pathogenesis, Presentation and Prognosis.

Acute liver failure (ALF) is an infrequent, unpredictable, potentially fatal complication of acute liver injury (ALI) consequent to varied etiologies. Etiologies of ALF as reported in the literature have regional differences, which affects the clinical presentation and natural course. In this part of the consensus article designed to reflect the clinical practices in India, disease burden, epidemiology, clinical presentation, monitoring, and prognostication have been discussed. In India, viral hepatitis is the most frequent cause of ALF, with drug-induced hepatitis due to antituberculosis drugs being the second most frequent cause. The clinical presentation of ALF is characterized by jaundice, coagulopathy, and encephalopathy. It is important to differentiate ALF from other causes of liver failure, including acute on chronic liver failure, subacute liver failure, as well as certain tropical infections which can mimic this presentation. The disease often has a fulminant clinical course with high short-term mortality. Death is usually attributable to cerebral complications, infections, and resultant multiorgan failure. Timely liver transplantation (LT) can change the outcome, and hence, it is vital to provide intensive care to patients until LT can be arranged. It is equally important to assess prognosis to select patients who are suitable for LT. Several prognostic scores have been proposed, and their comparisons show that indigenously developed dynamic scores have an edge over scores described from the Western world. Management of ALF will be described in part 2 of this document.

Neurological Recovery After Recovery From Acute Liver Failure: Is it Complete?

Neurologic dysfunction characterised by Hepatic Encephalopathy (HE) and cerebral oedema are the most dramatic presentations of Acute Liver Failure (ALF) and signify poor outcome. Improved critical care and wider availability of emergency Liver Transplantation (LT) has improved survivability in ALF. In most cases absence of clinically overt encephalopathy after spontaneous recovery from ALF or after LT is thought to indicate complete neurologic recovery. Recent data suggests that neurologic recovery may not always be complete. Instances of persistent neurologic dysfunction as well as neuropsychiatric abnormalities are now being recognised and warrant active follow up of these patients. Although evidences irreversible neurologic damage is uncommon after ALF, neuropsychiatric disturbances are not uncommon. Complex pathogenesis is involved in neurocognitive disorders seen after many other conditions including LT that require critical care. Structural damage and persistent neurological abnormalities seen after ALF are more likely to be related to cerebral edema, raised intracranial tension and cerebral hypoxemia, while neurocognitive dysfunctions may be a part of a wider spectrum of disorders commonly seen among those who recover from any critical illness.

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway.

Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

Hepatic Encephalopathy and Astrocyte Senescence.

Hepatic Encephalopathy (HE) is a severe complication of acute or chronic liver diseases with a broad spectrum of neurological symptoms including motor disturbances and cognitive impairment of different severity. Contrary to former beliefs, a growing number of studies suggest that cognitive impairment may not fully reverse after an acute episode of overt HE in patients with liver cirrhosis. The reasons for persistent cognitive impairment in HE are currently unknown but recent observations raise the possibility that astrocyte senescence may play a role here. Astrocyte senescence is closely related to oxidative stress and correlate with irreversible cognitive decline in aging and neurodegenerative diseases. In line with this, surrogate marker for oxidative stress and senescence were upregulated in ammonia-exposed cultured astrocytes and in post mortem brain tissue from patients with liver cirrhosis with but not without HE. Ammonia-induced senescence in astrocytes involves glutamine synthesis-dependent formation of reactive oxygen species (ROS), p53 activation and upregulation of cell cycle inhibitory factors p21 and GADD45α. More recent studies also suggest a role of ROS-induced downregulation of Heme Oxygenase (HO)1-targeting micro RNAs and upregulation of HO1 for ammonia-induced proliferation inhibition in cultured astrocytes. Further studies are required to identify the precise sequence of events that lead to astrocyte senescence and to elucidate functional implications of senescence for cognitive performance in patients with liver cirrhosis and HE.

Mitochondria in innate immune signaling.

Mitochondria are functionally versatile organelles. In addition to their conventional role of meeting the cell's energy requirements, mitochondria also actively regulate innate immune responses against infectious and sterile insults. Components of mitochondria, when released or exposed in response to dysfunction or damage, can be directly recognized by receptors of the innate immune system and trigger an immune response. In addition, despite initiation that may be independent from mitochondria, numerous innate immune responses are still subject to mitochondrial regulation as discrete steps of their signaling cascades occur on mitochondria or require mitochondrial components. Finally, mitochondrial metabolites and the metabolic state of the mitochondria within an innate immune cell modulate the precise immune response and shape the direction and character of that cell's response to stimuli. Together, these pathways result in a nuanced and very specific regulation of innate immune responses by mitochondria.

Lactated Ringer's solution for preventing myocardial reperfusion injury.

Reperfusion of ischemic myocardium is crucial for salvaging myocardial cells from ischemic cell death. However, reperfusion itself induces various deleterious effects on the ischemic myocardium. These effects, known collectively as reperfusion injury, comprise stunned myocardium, reperfusion-induced arrhythmia, microvascular reperfusion injury, and lethal reperfusion injury. No approach has proven successful in preventing any of these injuries in the clinical setting. My colleagues and I recently proposed a new postconditioning protocol, postconditioning with lactate-enriched blood (PCLeB), for the prevention of reperfusion injury. This new approach consists of intermittent reperfusion and timely coronary injections of lactated Ringer's solution, aiming to achieve controlled reperfusion with cellular oxygenation and minimal lactate washout from the cells. This approach appeared to be effective in preventing all types of reperfusion injury in patients with ST-segment elevation myocardial infarction (STEMI), and we have already reported excellent in-hospital outcomes of patients with STEMI treated using PCLeB. In this review article, I discuss a possible mechanism of reperfusion injury, which we believe to be valid and which we targeted using this new approach, and I report how the approach worked in preventing each type of reperfusion injury.

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death.

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 µM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.

Pathogenesis of hepatic encephalopathy: role of ammonia and systemic inflammation.

The syndrome we refer to as Hepatic Encephalopathy (HE) was first characterized by a team of Nobel Prize winning physiologists led by Pavlov and Nencki at the Imperial Institute of Experimental Medicine in Russia in the 1890's. This focused upon the key observation that performing a portocaval shunt, which bypassed nitrogen-rich blood away from the liver, induced elevated blood and brain ammonia concentrations in association with profound neurobehavioral changes. There exists however a spectrum of metabolic encephalopathies attributable to a variety (or even absence) of liver hepatocellular dysfunctions and it is this spectrum rather than a single disease entity that has come to be defined as HE. Differences in the underlying pathophysiology, treatment responses and outcomes can therefore be highly variable between acute and chronic HE. The term also fails to articulate quite how systemic the syndrome of HE can be and how it can be influenced by the gastrointestinal, renal, nervous, or immune systems without any change in background liver function. The pathogenesis of HE therefore encapsulates a complex network of interdependent organ systems which as yet remain poorly characterized. There is nonetheless a growing recognition that there is a complex but influential synergistic relationship between ammonia, inflammation (sterile and non-sterile) and oxidative stress in the pathogenesis HE which develops in an environment of functional immunoparesis in patients with liver dysfunction. Therapeutic strategies are thus moving further away from the traditional specialty of hepatology and more towards novel immune and inflammatory targets which will be discussed in this review.

Activation of autophagy via Ca(2+)-dependent AMPK/mTOR pathway in rat notochordal cells is a cellular adaptation under hyperosmotic stress.

Nucleus pulposus (NP) cells experience hyperosmotic stress in spinal discs; however, how these cells can survive in the hostile microenvironment remains unclear. Autophagy has been suggested to maintain cellular homeostasis under different stresses by degrading the cytoplasmic proteins and organelles. Here, we explored whether autophagy is a cellular adaptation in rat notochordal cells under hyperosmotic stress. Hyperosmotic stress was found to activate autophagy in a dose- and time-dependent manner. SQSTM1/P62 expression was decreased as the autophagy level increased. Transient Ca(2+) influx from intracellular stores and extracellular space was stimulated by hyperosmotic stress. Activation of AMPK and inhibition of p70S6K were observed under hyperosmotic conditions. However, intercellular Ca(2+) chelation inhibited the increase of LC3-II and partly reversed the decrease of p70S6K. Hyperosmotic stress decreased cell viability and promoted apoptosis. Inhibition of autophagy led to SQSTM1/P62 accumulation, reduced cell viability, and accelerated apoptosis in notochordal cells under this condition. These evidences suggest that autophagy induction via the Ca(2+)-dependent AMPK/mTOR pathway might occur as an adaptation mechanism for notochordal cells under hyperosmotic stress. Thus, activating autophagy might be a promising approach to improve viability of notochordal cells in intervertebral discs.

Novel insights into the mitochondrial permeability transition.

Alavian and colleagues recently provided further evidence in support of the notion that the c subunit of the mitochondrial F1FO ATP synthase constitutes the long-sought pore-forming unit of the supramolecular complex responsible for the so-called 'mitochondrial permeability transition' (MPT). Besides shedding new light on the molecular mechanisms that underlie the MPT, these findings corroborate the notion that several components of the cell death machinery, including cytochrome c and the F1FO ATP synthase, mediate critical metabolic activities.

Naringenin accords hepatoprotection from streptozotocin induced diabetes in vivo by modulating mitochondrial dysfunction and apoptotic signaling cascade.

Diabetic complications cause noticeable liver damage, which finally progresses to diabetic hepatopathy. Nutritive antioxidants not only reduce the liver damage, but also prevent it by modulating the release of various proteins involved in apoptotic signaling cascades. This study explores the molecular mechanisms underlying diabetes-induced liver damage and its modulation by naringenin. Antioxidant status, liver & kidney biomarker enzymes, reactive oxygen species (ROS) generation, mitochondrial membrane potential, expression of apoptotic proteins like Bax (bcl-2 associated X), Bcl-2 (b-cell Lymhoma-2), Caspase-3, Caspase-9, AIF (Apoptosis inducing factor) and Endo-G (Endonuclease-G) were studied in streptozotocin induced diabetic rats. Significant hyperglycemia, disturbed antioxidant status, altered carbohydrate metabolizing enzymes, increased ROS and lipid peroxidation; decreased mitochondrial membrane potential and enhanced release of AIF and Endo-G were observed. Hyperglycemia also affected apoptosis and its related genes at both transcriptional and translational level (Caspase-3 & 9, Bax and Bcl-2) in the liver of diabetic rats. Naringenin, a flavonone, exerted anti-hyperglycemic effect and was able to prevent oxidative stress and resultant apoptotic events caused due to diabetes-induced hepatotoxicity. Thus, our study shows, a protective effect of naringenin against diabetes induced liver damage and redox imbalance, which could further be exploited for the management of diabetic hepatopathy.

Hepatotoxicity Related to Anti-tuberculosis Drugs: Mechanisms and Management.

Development of idiosyncratic hepatotoxicity is an intricate process involving both concurrent as well as sequential events determining the direction of the pathways, degree of liver injury and its outcome. Decades of clinical observation have identified a number of drug and host related factors that are associated with an increased risk of antituberculous drug-induced hepatotoxicity, although majority of the studies are retrospective with varied case definitions and sample sizes. Investigations on genetic susceptibility to hepatotoxicity have so far focused on formation and accumulation reactive metabolite as well as factors that contribute to cellular antioxidant defense mechanisms and the environment which can modulate the threshold for hepatocyte death secondary to oxidative stress. Recent advances in pharmacogenetics have promised the development of refined algorithms including drug, host and environmental risk factors that allow better tailoring of medications based on accurate estimates of risk-benefit ratio. Future investigations exploring the pathogenesis of hepatotoxicity should be performed using human tissue and samples whenever possible, so that the novel findings can be translated readily into clinical applications.