Yongke Lu

Role of oxidative stress in alcohol-induced liver injury

Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in small amounts during the body’s metabolic reactions and can react with and damage complex cellular molecules such as lipids, proteins, or DNA. Acute and chronic ethanol treatments increase the production of ROS, lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury. Special emphasis is placed on CYP2E1, which is induced by alcohol and is reactive in metabolizing and activating many hepatotoxins, including ethanol, to reactive products, and in generating ROS.

Cytochrome P450 2E1 contributes to ethanol‐induced fatty liver in mice

Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol‐feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild‐type mice but not in CYP2E1‐knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1‐knockout mice but not in wild‐type mice. CYP2E1 was induced by ethanol in wild‐type mice, and oxidative stress induced by ethanol was higher in wild‐type mice than in CYP2E1‐knockout mice. Peroxisome proliferator‐activated receptor α (PPARα), a regulator of fatty acid oxidation, was up‐regulated in CYP2E1‐knockout mice fed ethanol but not in wild‐type mice. A PPARα target gene, acyl CoA oxidase, was decreased by ethanol in wild‐type but not in CYP2E1‐knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up‐regulated PPARα protein level in wild‐type mice fed ethanol. The introduction of CYP2E1 to CYP2E1‐knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1‐derived oxidative stress may inhibit oxidation of fatty acids by preventing up‐regulation of PPARα by ethanol, resulting in fatty liver. (HEPATOLOGY 2008.)

Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice.

A major pathway for chronic ethanol-induced liver injury is ethanol-induced oxidant stress. Several pathways contribute to mechanisms by which ethanol induces oxidant stress. Although some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. Most previous studies were conducted in the intragastric infusion model of ethanol administration. There is a need to develop oral models of significant liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. We evaluated chronic ethanol-induced liver injury, steatosis, and oxidant stress in wild-type (WT) mice, CYP2E1 knock out (KO) mice, and humanized CYP2E1 knock-in (KI) mice, in which the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and the CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, National Cancer Institute) were fed a high-fat Lieber-DeCarli ethanol liquid diet for 3weeks; pair-fed controls received dextrose. Ethanol produced fatty liver and oxidant stress in WT mice but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice, with elevated transaminases, necrosis, and increased levels of collagen type 1 and smooth muscle actin. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK and decreased levels of Bcl-2 and Bcl-XL were observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in the WT and the KI mice was associated with lower levels of PPARα and acyl-CoA oxidase. No such changes were found in the ethanol-fed KO mice. These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. It is the absence of CYP2E1 in the KO mice that is responsible for the blunting of steatosis and oxidant stress because restoring the CYP2E1 restores the fatty liver and oxidant stress. Moreover, it is the human CYP2E1 that restores these effects of ethanol, which suggests that results for fatty liver and oxidant stress from rodent models of ethanol intake and mouse CYP2E1 can be extrapolated to human models of ethanol intake and to human CYP2E1.

OSTEOPONTIN, AN OXIDANT STRESS-SENSITIVE CYTOKINE, UP-REGULATES COLLAGEN-I VIA INTEGRIN αVβ3 ENGAGEMENT AND PI3K-pAkt-NFκB SIGNALING

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen‐I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro‐fibrogenic phenotype and Collagen‐I expression. Recombinant OPN (rOPN) up‐regulated Collagen‐I protein in primary HSCs in a transforming growth factor beta (TGFβ)–independent fashion, whereas it down‐regulated matrix metalloprotease‐13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α‐smooth muscle actin (αSMA) expression and enhanced their invasive and wound‐healing potential. HSCs isolated from wild‐type (WT) mice were more profibrogenic than those from OPN knockout (Opn−/−) mice and infection of primary HSCs with an Ad‐OPN increased Collagen‐I, indicating correlation between both proteins. OPN induction of Collagen‐I occurred via integrin αvβ3 engagement and activation of the phosphoinositide 3‐kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)–signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70‐kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin αvβ3 prevented the OPN‐mediated activation of the PI3K/pAkt/NFκB–signaling cascade and Collagen‐I up‐regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN‐mediated Collagen‐I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen‐I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl4 injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up‐regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (OpnHEP Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl4 injection and TAA treatment caused more liver fibrosis to WT than to Opn−/− mice and the reverse occurred in OpnHEP Tg mice. Conclusion: OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen‐I protein contributing to scarring and liver fibrosis. (HEPATOLOGY 2012)

High Mobility Group Box-1 (HMGB1) Participates in the Pathogenesis of Alcoholic Liver Disease (ALD)

Background: HMGB1 is a proinflammatory cytokine produced in response to tissue injury, but its role in ALD is unknown. Results: HMGB1 increases; translocates; and undergoes acetylation, phosphorylation, and oxidation in ALD. HMGB1 ablation in hepatocytes protects against steatosis and injury in ALD. Conclusion: HMGB1 plays a key role in ALD. Significance: Dissecting how the increase in HMGB1 causes hepatotoxicity is key for understanding the pathogenesis of ALD. Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5′AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.

Induction of cytochrome P450 2E1 increases hepatotoxicity caused by Fas agonistic Jo2 antibody in mice

Cytochrome P450 2E1 (CYP2E1) may be a central pathway in generating oxidative stress, reactive oxygen species, and causing hepatotoxic injury by alcohol and various hepatotoxins. This study evaluated the ability of CYP2E1 to potentiate or synergize the hepatotoxicity of Fas in vivo. C57BL/6 mice were injected intraperitoneally with pyrazole (Pyr) to induce CYP2E1. Then, 16‐hour fasted mice were administered agonistic Jo2 anti‐Fas antibody ip. Other mice were treated with Pyr or Jo2 alone. Levels of serum aminotransferase were 8.3‐ and 6.3‐fold higher in the Pyr/Jo2 group compared with Jo2 alone, respectively. Histological evaluation of liver showed more extensive acidophilic necrosis and severe pathological changes in the Pyr/Jo2‐treated mice. DNA fragmentation and caspase‐8 and ‐3 activities were more elevated in the Pyr/Jo2 group compared with Jo2 alone. CYP2E1 activity and protein levels were higher in the Pyr/Jo2 group than in Jo2 alone. Levels of inducible nitric oxide synthase, 3‐nitrotyrosine protein adducts, malondialdehyde, and protein carbonyls were also higher in the Pyr/Jo2 group compared with Jo2 alone. Glutathione and activities of catalase and Cu‐Zn superoxide dismutase were decreased in the Pyr/Jo2 group. Administration of chlormethiazole, an inhibitor of CYP2E1, to the Pyr/Jo2‐treated mice caused a significant decrease of alanine aminotransferase and liver pathological changes in association with a decrease in CYP2E1 protein and activity. In conclusion, enhanced hepatotoxicity of Fas was found in mice with elevated levels of CYP2E1. We speculate that overexpression of CYP2E1 might synergize and increase the susceptibility to Fas induced‐liver injury. (HEPATOLOGY 2005;42:400–410.)

Osteopontin induces ductular reaction contributing to liver fibrosis

Objective In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring. Results In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-β+ BEC, which were lacking in TAA-treated Opn−/− mice and in healthy human explants, this suggested that OPN could regulate TGF-β, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-β expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-β (rTGFβ) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-β reduced collagen-I expression in co-cultured HSC. Conclusions OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-β.

Enhancement by pyrazole of lipopolysaccharide‐induced liver injury in mice: Role of cytochrome P450 2E1 and 2A5

The mechanisms by which alcohol causes liver injury are still not certain. Either LPS or CYP2E1 are considered independent risk factors involved in alcoholic liver disease, but mutual relationships or interactions between them are unknown. In the present study, the possible synergistic action of CYP2E1 and LPS in liver injury was investigated by evaluating the effects of pyrazole (inducer of CYP2E1), Chlormethiazole (CMZ), an inhibitor of CYP2E1, and CYP2E1‐knockout mice. Mice were injected with pyrazole (150 mg/kg, ip) daily for 2 days, followed by LPS injection (4 mg/kg, ip). CMZ (50mg/kg, ip) was administered 15 h before and 30 min after LPS treatment, respectively. LPS‐induced liver injury was enhanced by pyrazole, as indicated by pathological changes and increases in ALT and AST, and positive TUNEL staining. LPS‐induced oxidative stress was also enhanced by pyrazole as indicated by increases in 4‐hydroxy‐2‐nonenal and 3‐nitrotyrosine adduct formation. CMZ protected against the pyrazole enhanced LPS liver injury and oxidative stress. CYP2E1 but also CYP2A5 were increased by the pyrazole/LPS treatment. CMZ decreased the elevated CYP2E1 activity by 90%, but CYP2A5 activity was also lowered (30%‐50%). CYP2E1‐knockout mice exhibited only minor liver injury after treatment with pyrazole/LPS, but wild‐type mice exhibited severe liver injury. While no CYP2E1 was present in the CYP2E1 knockout mice, CYP2A5 activity was also lower. In conclusion, induction of CYP2E1 plays an important role in the enhancement of LPS liver injury by pyrazole, but some contribution by CYP2A5 cannot be excluded. (HEPATOLOGY 2006;44:263–274.)

Fibromodulin, an oxidative stress-sensitive proteoglycan, regulates the fibrogenic response to liver injury in mice.

BACKGROUND & AIMS Collagen I deposition contributes to liver fibrosis, yet little is known about other factors that mediate this process. Fibromodulin is a liver proteoglycan that regulates extracellular matrix organization and is induced by fibrogenic stimuli. We propose that fibromodulin contributes to the pathogenesis of fibrosis by regulating the fibrogenic phenotype of hepatic stellate cells (HSCs). METHODS We analyzed liver samples from patients with hepatitis C-associated cirrhosis and healthy individuals (controls). We used a coculture model to study interactions among rat HSCs, hepatocytes, and sinusoidal endothelial cells. We induced fibrosis in livers of wild-type and Fmod(-/-) mice by bile duct ligation, injection of CCl(4), or administration of thioacetamide. RESULTS Liver samples from patients with cirrhosis had higher levels of fibromodulin messenger RNA and protein than controls. Bile duct ligation, CCl(4), and thioacetamide each increased levels of fibromodulin protein in wild-type mice. HSCs, hepatocytes, and sinusoidal endothelial cells produced and secreted fibromodulin. Infection of HSCs with an adenovirus that expressed fibromodulin increased expression of collagen I and α-smooth muscle actin, indicating increased activation of HSCs and fibrogenic potential. Recombinant fibromodulin promoted proliferation, migration, and invasion of HSCs, contributing to their fibrogenic activity. Fibromodulin was sensitive to reactive oxygen species. HepG2 cells that express cytochrome P450 2E1 produced fibromodulin, and HSCs increased fibromodulin production in response to pro-oxidants. In mice, administration of an antioxidant prevented the increase in fibromodulin in response to CCl(4). Coculture of hepatocytes or sinusoidal endothelial cells with HSCs increased the levels of reactive oxygen species in the culture medium, along with collagen I and fibromodulin proteins; this increase was prevented by catalase. Fibromodulin bound to collagen I, but the binding did not prevent collagen I degradation by matrix metalloproteinase 13. Bile duct ligation caused liver fibrosis in wild-type but not Fmod(-/-) mice. CONCLUSIONS Fibromodulin levels are increased in livers of patients with cirrhosis. Hepatic fibromodulin activates HSCs and promotes collagen I deposition, which leads to liver fibrosis in mice.

Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 (-/-)) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 (-/-) mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 (-/-) KI) mice but not in CYP2E1 knockout (Cyp2e1 (-/-)) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 (-/-) KI mice but not in Cyp2e1 (-/-) mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 (-/-)) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 (-/-) mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 (-/-) KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis.