Maria D. Ybanez

Signal Transduction Pathways Involved in Drug-Induced Liver Injury

Hepatocyte death following drug intake is the critical event in the clinical manifestation of drug-induced liver injury (DILI). Traditionally, hepatocyte death caused by drugs had been attributed to overwhelming oxidative stress and mitochondria dysfunction caused by reactive metabolites formed during drug metabolism. However, recent studies have also shown that signal transduction pathways activated/inhibited during oxidative stress play a key role in DILI. In acetaminophen (APAP)-induced liver injury, hepatocyte death requires the sustained activation of c-Jun kinase (JNK), a kinase important in mediating apoptotic and necrotic death. Inhibition of JNK using chemical inhibitors or knocking down JNK can prevent hepatocyte death even in the presence of extensive glutathione (GSH) depletion, covalent binding, and oxidative stress. Once activated, JNK translocates to mitochondria, to induce mitochondria permeability transition and trigger hepatocyte death. Mitochondria are central targets where prodeath kinases such as JNK, prosurvival death proteins such as bcl-xl, and oxidative damage converge to determine hepatocyte survival. The importance of mitochondria in DILI is also observed in the Mn-SOD heterozygous (+/-) model, where mice with less mitochondrial Mn-SOD are sensitized to liver injury caused by certain drugs. An extensive body of research is accumulating suggesting a central role of mitochondria in DILI. Drugs can also cause redox changes that inhibit important prosurvival pathways such as NF-kappaB. The inhibition of NF-kappaB by subtoxic doses of APAP sensitizes hepatocyte to the cytotoxic actions of tumor necrosis factor (TNF). Many drugs will induce liver injury if simultaneously treated with LPS, which promotes inflammation and cytokine release. Drugs may be sensitizing hepatocytes to the cytotoxic effects of cytokines such as TNF, or vice versa. Overall many signaling pathways are important in regulating DILI, and represent potential therapeutic targets to reduce liver injury caused by drugs.

Silencing glycogen synthase kinase-3β inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1

Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver.

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice BIOGENESIS, REMODELING, AND FUNCTIONAL ALTERATIONS

Background: Mitochondrial respiration plays an important role in alcohol metabolism by regenerating NAD+ needed for alcohol/acetaldehyde metabolism. Results: Chronic alcohol feeding caused many mitochondrial alterations, such as increased mitochondrial respiration, that enhanced acetaldehyde metabolism. Conclusion: Mitochondria in the liver adapt to the metabolic stress of alcohol. Significance: Mitochondrial alterations may play a role in many vital functions of the liver. Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD+ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD+, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)

Determination of GSH, GSSG, and GSNO Using HPLC with Electrochemical Detection

GSNO is an important intermediate in nitric oxide metabolism and mediates many ()NO-mediated signaling pathways through the post-translational modification of redox-sensitive proteins. The detection of GSNO in biological samples has been hampered by a lack of sensitive and simple assays. In this work, we describe the utilization of HPLC with electrochemical detection for the identification and quantification of GSNO in biological samples. GSNO requires a high potential (>700 mV) for its electrochemical detection, similar to that of GSSG. A simple isocratic HPLC system can be used to separate and simultaneously detect GSH, GSSG, and GSNO electrochemically. This HPLC system can be utilized to measure the redox profile of biological samples and applied for the measurement of GSNO reductase activity in cells. Proper sample preparation is essential in GSNO measurements, because artifactual formation of GSNO occurs in acidic conditions due to the reaction between GSH and nitrite. Treatment of samples with ammonium sulfamate or N-ethylmaleimide (NEM) can prevent the artifactual formation of GSNO and accurately detect GSNO in biological samples. Overall, the HPLC with electrochemical detection is a powerful tool to measure redox status in cells and tissues.

Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)−/− mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase−/− hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase−/− hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase−/− mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent signaling pathways

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways: Saberi, Ybanez, et al.

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)