The extraction, isolation and characterization of 29 natural products contained in Ginkgo biloba have been described, which we have now tested for their in-vitro capacity to inhibit the five major human cytochrome P450 (CYP) isoforms in human liver microsomes. Weak or negligible inhibitory activity was found for the terpene trilactones (ginkgolides A, B, C and J, and bilobalide), and the flavonol glycosides. However 50% inhibitory activity (IC50) was found at concentrations less than 10 microg L(-1) for the flavonol aglycones (kaempferol, quercetin, apigenin, myricetin, tamarixetin) with CYP1A2 and CYP3A. Quercetin, the biflavone amentoflavone, sesamin, as well as (Z,Z)-4,4'-(1,4-pentadiene-1,5-diyl)diphenol and 3-nonadec-8-enyl-benzene-1,2-diol, were also inhibitors of CYP2C9. The IC50 of amentoflavone for CYP2C9 was 0.019 microg mL(-1) (0.035 microM). Thus, the principal components of Ginkgo biloba preparations in clinical use (terpene trilactones and flavonol glycosides) do not significantly inhibit these human CYPs in-vitro. However, flavonol aglycones, the biflavonol amentoflavone and several other non-glycosidic constituents are significant in-vitro inhibitors of CYP. The clinical importance of these potential inhibitors will depend on their amounts in ginkgo preparations sold to the public, and the extent to which their bioavailability allows them to reach the CYP enzymes in-situ.

Hepatic ischemia-reperfusion (IRP) injury is a significant clinical problem during tumor resection surgery (Pringle maneuver), and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. In order to address this important issue in a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA-122, full-length cytokeratin-18 (FK18) and high mobility group box-1 (HMGB1) protein, and apoptosis including plasma caspase-3 activity and caspase-cleaved cytokeratin-18 (CK18), coupled with markers of inflammation (hyper-acetylated HMGB1) were compared with histological features in H&E- and TUNEL-stained liver sections. After 45 min of hepatic ischemia and 1–24h of reperfusion, all necrosis markers increased dramatically in plasma by 40-to->10,000-fold over baseline with a time course similar to ALT. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL-positive; initially (≤ 3h of RP) the staining was restricted to nuclei but later spread to the cytosol characteristic for karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except a minor increase at 3h RP) in plasma. The quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated the dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be on the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury.

HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs.

Overdose of acetaminophen (APAP) causes severe liver injury and even acute liver failure in both mice and human. A recent study by Kim et al. (2015, Metformin ameliorates acetaminophen hepatotoxicity via Gadd45β-dependent regulation of JNK signaling in mice. J. Hepatol. 63, 75-82) showed that metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP hepatotoxicity in mice. However, its exact protective mechanism has not been well clarified. To investigate this, C57BL/6J mice were treated with 400 mg/kg APAP and 350 mg/kg metformin was given 0.5 h pre- or 2 h post-APAP. Our data showed that pretreatment with metformin protected against APAP hepatotoxicity, as indicated by the over 80% reduction in plasma alanine aminotransferase (ALT) activities and significant decrease in centrilobular necrosis. Metabolic activation of APAP, as indicated by glutathione depletion and APAP-protein adducts formation, was also slightly inhibited. However, 2 h post-treatment with metformin still reduced liver injury by 50%, without inhibition of adduct formation. Interestingly, neither pre- nor post-treatment of metformin inhibited c-jun N-terminal kinase (JNK) activation or its mitochondrial translocation. In contrast, APAP-induced mitochondrial oxidant stress and dysfunction were greatly attenuated in these mice. In addition, mice with 2 h post-treatment with metformin also showed significant inhibition of complex I activity, which may contribute to the decreased mitochondrial oxidant stress. Furthermore, the protection was reproduced in JNK activation-absent HepaRG cells treated with 20 mM APAP followed by 0.5 or 1 mM metformin 6 h later, confirming JNK-independent protection mechanisms. Thus, metformin protects against APAP hepatotoxicity by attenuating the mitochondrial oxidant stress and subsequent mitochondrial dysfunction, and may be a potential therapeutic option for APAP overdose patients.

Background and Aims Hypoxic hepatitis is a clinical condition precipitated by prolonged periods of oxygen deprivation to the liver. It can have several underlying causes. Despite its prevalence in critically ill patients, which can reach upwards of 10%, very little is known about the mechanisms of injury. Thus, we set out to measure previously identified circulating biomarkers in an attempt to describe mechanisms of injury following hypoxic hepatitis. Methods Plasma from patients diagnosed with hypoxic hepatitis was collected for this study. Biomarkers of hepatocellular injury, mitochondrial damage, and cell death were measured. These results were compared against results obtained from well characterized acetaminophen overdose patients. Results At peak injury, ALT measured 4082±606 U/L and gradually decreased over 5 days, corresponding to the clinically observed pattern of hypoxic hepatitis. Levels of GDH showed a similar pattern, but neither ALT nor GDH were significantly higher in these patients than in acetaminophen patients. Plasma levels of DNA fragments mimicked hepatocellular injury as measured by ALT and miRNA-122. Interestingly, we found a significant increase in caspase-cleaved cytokeratin-18; however, the full-length form greatly exceeded the cleaved form at the time of maximum injury (45837±12085 vs. 2528±1074 U/L). We also found an increase in acHMGB1 at later time points indicating a possible role of inflammation, but cytokine levels at these times were actually decreased relative to early time points. Conclusions The mechanism of injury following hypoxic hepatitis involves mitochondrial damage and DNA fragmentation. Importantly, necrosis, rather than apoptosis, is the main mode of cell death.

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondrial targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300mg/kg APAP followed by 20mg/kg MT 1.5h or 3h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10mg/kg) 2h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine (NAC), the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

Background and aims Pannexins constitute a relatively new family of transmembrane proteins that form channels linking the cytoplasmic compartment with the extracellular environment. Presence of pannexin1 in the liver has been documented previously, where it underlies inflammatory responses, such as those occurring upon ischemia-reperfusion injury. In the present study, we investigated whether pannexin1 plays a role in acute drug-induced liver toxicity. Methods Hepatic expression of pannexin1 was characterized in a mouse model of acetaminophen-induced hepatotoxicity. Subsequently, mice were overdosed with acetaminophen followed by treatment with the pannexin1 channel inhibitor 10Panx1. Sampling was performed 1, 3, 6, 24 and 48 hours after acetaminophen administration. Evaluation of the effects of pannexin1 channel inhibition was based on a number of clinically relevant read-outs, including protein adduct formation, measurement of aminotransferase activity and histopathological examination of liver tissue as well as on a series of markers of inflammation, oxidative stress and regeneration. Results Although no significant differences were found in histopathological analysis, pannexin1 channel inhibition reduced serum levels of alanine and aspartate aminotransferase. This was parallelled by a reduced amount of neutrophils recruited to the liver. Furthermore, alterations in the oxidized status were noticed with upregulation of glutathione levels upon suppression of pannexin1 channel opening. Concomitant promotion of regenerative activity was detected as judged on increased proliferating cell nuclear antigen protein quantities in 10Panx1-treated mice. Conclusions Pannexin1 channels are important actors in liver injury triggered by acetaminophen. Inhibition of pannexin1 channel opening could represent a novel approach for the treatment of drug-induced hepatotoxicity.

Mouse models of acetaminophen (APAP) hepatotoxicity are considered relevant for the human pathophysiology. The C57BL/6 strain is most popular because it is the background strain of gene knock-out mice. However, conflicting results in the literature may have been caused by sub-strain mismatches, e.g. C57BL/6J and C57BL/6N. This study was initiated to determine the mechanism behind the sub-strain susceptibility to APAP toxicity. C57BL/6N and C57BL/6J mice were dosed with 200 mg/kg APAP and sacrificed at different time points. C57BL/6N mice developed significantly more liver injury as measured by plasma ALT activities and histology. Although there was no difference in glutathione depletion or cytochrome P450 activity between groups, C57BL/6N had a higher glutathione disulfide-to-glutathione ratio and more APAP protein adducts. C57BL/6N showed more mitochondrial translocation of phospho-JNK and BAX, and more release of mitochondrial intermembrane proteins (apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), which caused more DNA fragmentation. The increased mitochondrial dysfunction was confirmed in vitro as C57BL/6N hepatocytes had a more precipitous drop in JC-1 fluorescence after APAP exposure. Conclusion: C57BL/6N mice are more susceptible to APAP-induced hepatotoxicity, likely due to increased formation of APAP-protein adducts and a subsequent enhancement of mitochondrial dysfunction associated with aggravated nuclear DNA fragmentation.