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Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol induced malondialdehyde-deoxyguanosine (M1dG) adduct formation
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Abstracts / Alcohol 47 (2013) 567–576
25. Anti-inflammatory effects of ethanol and ethyl pyruvate B. Relja, M. Lehnert, I. Marzi, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany Objective: Exaggerated immune response in acute inflammatory conditions such as hypoxia results in end organ injury. We demonstrated the antiinflammatory and anti-oxidant effects of acute alcohol (ethanol, EtOH) gavage upon hemorrhagic shock with subsequent resuscitation (H/R) that were associated with improved outcome in vivo. Due to side-effects of EtOH its use as an anti-inflammatory drug in acute inflammation is limited. Here, we compare effects of EtOH to ethyl pyruvate (EtP) applied after the inflammatory “hit” using an in vitro experimental model of acute inflammation. Methods: 14 h before H/R, rats received EtOH (5 g/kg, 30%) or saline gavage. Two and 72 h after H/R hepatic and intestinal damage, local and systemic inflammation as well as oxidative stress and survival were evaluated. Human hepatocellular cell line Huh7 or pulmonary epithelial cell line A549 were stimulated with IL-1beta or TNF-alpha, respectively. Then, the cells were incubated for 1 h with various doses of EtP (2.5–10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85–170 mM). IL-8 and IL-6 release, neutrophil adhesion, ICAM-1 expression as well as the involvement of NF-kB were evaluated. Results: EtOH significantly reduced hepatic and intestinal injury and improved organ integrity compared to control animals resulting in higher survival rates after H/R. In vitro, both EtOH and EtP reduced the IL-8 (A549) and IL-6 release (Huh7) as well as the cytokine-induced adhesion of neutrophils to A549 or Huh7 monolayers. NaP exerted similar effects. EtOH and EtP reduced ICAM-1 expression in A549 without affecting ICAM-1 in Huh7 cells. Both in vivo and in vitro studies demonstrated enhanced expression of phosphorylated NF-kB p65 either in the liver tissue after H/R or in cells after the inflammatory stimuli. EtOH, EtP and NaP application was associated with reduced levels of phosphorylated NF-kB p65. Conclusions: EtOH-induced survival “benefit” after H/R compared to control animals was associated with its anti-inflammatory properties and probably the decreased NF-kB activation. In vitro results confirm further this antiinflammatory potential of EtOH but uncover the potential of EtP to mimic these effects suggesting EtP as a possible treatment for acute inflammatory conditions.
26. Alcohol and hepatitis C virus infection modulates monocytes to macrophage differentiation
27. Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol induced malondialdehyde-deoxyguanosine (M1dG) adduct formation M. Sapkota, T.K. Hottor, J.M. DeVasure, T.A. Wyatt, M.L. McCaskill, Department of Environmental, Agricultural and Occupational Health and Toxicology, College of Public Health, Department of Internal Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Nebraska Medical Center, Omaha, NE, USA; Global Environmental Health, School of Public Health and Tropical Medicine Sciences, Tulane University, New Orleans, LA, USA Alcohol use disorders are often associated with lung disease. High exposure to alcohol may lead to the production of reactive oxygen species, which can induce lipid peroxidation and formation of malondialdehyde (MDA) as well as induce the expression of cytochrome p-450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA is genotoxic and can lead to the formation of various DNA adducts such as malondialdehyde deoxyguanosine (M1dG) adducts which have been implicated in alcohol-related cancers and cardiovascular disease. Since the CYP2E1 pathway of alcohol metabolism is involved in the formation of MDA, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would regulate MDA production, M1dG adduct formation, and single strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue. C57BL/6 mice were administered 20% ethanol ad libitum in drinking water (Meadows-Cook model) for 8 weeks and exposed to whole body cigarette smoke for 5 weeks. Mice were also fed a CYP2E1 inhibitor, diallyl disulfide (DADS), at 1 mM/g of feed in their daily diet. Normal human bronchial epithelial cells (HBEC) were pre-treated with 10 mM DADS for 1 h, and treated with 80 mM ethanol 5% cigarette smoke extract (CSE) for 3 h for comet assay and 6 h for CYP2E1, MDA, and M1dG adduct assays. Ethanol exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBEC attenuated this ethanol effect. Ethanol also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE ethanol did not enhance these effects. In lung tissue homogenate of 8week alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison to control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5 weeks cigarette smoke-exposed mice. These findings suggest that cigarette smoke may not have a significant role in the formation of MDA and M1dG adduct, alcohol may be solely responsible for M1dG adduct formation. Thus CYP2E1 plays a pivotal role in alcohol induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation (Supported by NIH R01 AA017663 [TAW]).
B. Saha, J. Bruneau, G. Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA Introduction: HCV infection and alcohol abuse are the leading cause of cirrhosis, requiring liver transplantation. Macrophage (MF) activation occurs in chronic hepatitis C virus (HCV) infection and alcohol exposure and dysfunctional MFs may contribute to liver fibrosis. Depending on signals from the tissue microenvironment, circulating monocytes differentiate into MFs with either M1 (classical) or M2 (alternative) polarization. However, the nature of MF polarization during HCV infection and alcohol exposure is not known. We hypothesized that HCV and alcohol provides signals for circulating monocytes to differentiate into “pathogenic macrophages”. Methods: Surface markers of circulating monocytes from HCV-infected patients, alcohol exposed individuals and healthy donors were studied by flow cytometry. In vitro co-culture experiments were performed with monocytes isolated from healthy donors (n ¼ 10–15) or 24 h alcohol exposed donors (4–7), and hepatoma cells (Huh7.5) infected with HCV (JFH-1/Huh7.5). Results: We found that circulating monocytes from chronic HCV-infected patients exhibit an M2 polarized phenotype with high expression of CD206 (mannose receptor) and CD163 (scavenger receptor) as compared to controls. However, the circulating monocytes from acute alcohol exposed donors predominantly expressed M1 markers (CD16 and CD40). We observed that JFH-1/Huh7.5 induced differentiation of normal monocytes to MF-like cells in vitro. These “HCV-educated” MFs displayed increased expression of M2 markers. However, co-culture of alcohol exposed monocytes with JFH-1/ Huh7.5, lowered the expression of MF markers. M1 markers on the alcohol exposed monocytes were not increased in the presence of HCV, but a synergistic effect on the expression of M2 marker (CD206) was observed. Healthy monocytes co-cultured with JFH-1/Huh7.5 cells secreted pro-inflammatory (IL-1b and TNF-a) and predominantly anti-inflammatory (IL-10 and TGF-b) cytokines. Alcohol exposed monocytes secreted higher IL-1b and TGF-b and lower IL-10 as compared to control monocytes co-cultured with JFH-1/ Huh7.5. The high level of TGF-b secreted by “HCV-educated” MF was profibrotic and led to activation of hepatic stellate (LX2) cells as this process could be blocked by anti-TGFb neutralizing antibody. Conclusion: Thus, alcohol exposure inhibits the process of HCV-mediated monocyte differentiation. This study demonstrates the inhibitory effect of alcohol on the innate immune response to HCV infection (Supported by NIH AA014372 [GS]).
28. The role of miR-122 in the pathogenesis of chronic alcoholic liver disease A. Satishchandran, S. Bala, G. Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA The pathological progression of alcoholic liver disease (ALD) is multifactorial involving both metabolic and immunological dysfunction. The development of steatosis from alcohol abuse sensitizes hepatocytes to injurious inflammatory stimuli such as IL-1B and TNF. Additionally, lipid-filled hepatocytes produce reactive oxygen species by way of normal ethanol metabolism and increased lipid peroxidation. miR-122 regulates essential functions in hepatic lipid metabolism, mitochondrial function, cell death pathways, fibrosis and carcinogenesis – major elements in alcoholic liver disease. Given the highly conserved role of this unique miRNA in hepatic homeostasis we hypothesize that miR-122 contributes to ALD progression and may be a therapeutic target. Utilizing an scAAV8 vector expressing antimiR-122 TuD, we inhibited miR-122 0.124-fold (p ¼ 0.0059) in mouse hepatocytes followed by a 4-week alcohol diet. The anti-miR-122 TuD + alcohol (TuD + et) treated groups demonstrated significant increases in ALT and weight loss (p ¼ 0.0091, p < 0.05 respectively) compared to scrambled controls (Scr). We also found significant increases in HIF-1a in 122TuD + et treated mice (p ¼ 0.011 vs. Scr + et). Further, we observed 20-, 5-, and 4–fold increases in MCP-1, TNF-a, and IL-1B, respectively, in PF and Et treated knockdown mice when compared to Scr controls (p < 0.05). Given that bioinformatic miRNA target predictions tools suggest miR-122 may also negatively regulate HIF1a we hypothesize that miR-122 inhibition in ALD increases HIF1a thereby potentiating inflammation. However, further studies into whether these increases are a direct result of miR-122 modulation in hepatocytes or a function of hepatocyte mediated immune cell activation are needed (Supported by NIH AA020744 [GS] and F30 AA022283-01 [AS]).
Abstracts / Alcohol 47 (2013) 567–576
25. Anti-inflammatory effects of ethanol and ethyl pyruvate B. Relja, M. Lehnert, I. Marzi, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany Objective: Exaggerated immune response in acute inflammatory conditions such as hypoxia results in end organ injury. We demonstrated the antiinflammatory and anti-oxidant effects of acute alcohol (ethanol, EtOH) gavage upon hemorrhagic shock with subsequent resuscitation (H/R) that were associated with improved outcome in vivo. Due to side-effects of EtOH its use as an anti-inflammatory drug in acute inflammation is limited. Here, we compare effects of EtOH to ethyl pyruvate (EtP) applied after the inflammatory “hit” using an in vitro experimental model of acute inflammation. Methods: 14 h before H/R, rats received EtOH (5 g/kg, 30%) or saline gavage. Two and 72 h after H/R hepatic and intestinal damage, local and systemic inflammation as well as oxidative stress and survival were evaluated. Human hepatocellular cell line Huh7 or pulmonary epithelial cell line A549 were stimulated with IL-1beta or TNF-alpha, respectively. Then, the cells were incubated for 1 h with various doses of EtP (2.5–10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85–170 mM). IL-8 and IL-6 release, neutrophil adhesion, ICAM-1 expression as well as the involvement of NF-kB were evaluated. Results: EtOH significantly reduced hepatic and intestinal injury and improved organ integrity compared to control animals resulting in higher survival rates after H/R. In vitro, both EtOH and EtP reduced the IL-8 (A549) and IL-6 release (Huh7) as well as the cytokine-induced adhesion of neutrophils to A549 or Huh7 monolayers. NaP exerted similar effects. EtOH and EtP reduced ICAM-1 expression in A549 without affecting ICAM-1 in Huh7 cells. Both in vivo and in vitro studies demonstrated enhanced expression of phosphorylated NF-kB p65 either in the liver tissue after H/R or in cells after the inflammatory stimuli. EtOH, EtP and NaP application was associated with reduced levels of phosphorylated NF-kB p65. Conclusions: EtOH-induced survival “benefit” after H/R compared to control animals was associated with its anti-inflammatory properties and probably the decreased NF-kB activation. In vitro results confirm further this antiinflammatory potential of EtOH but uncover the potential of EtP to mimic these effects suggesting EtP as a possible treatment for acute inflammatory conditions.
26. Alcohol and hepatitis C virus infection modulates monocytes to macrophage differentiation
27. Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol induced malondialdehyde-deoxyguanosine (M1dG) adduct formation M. Sapkota, T.K. Hottor, J.M. DeVasure, T.A. Wyatt, M.L. McCaskill, Department of Environmental, Agricultural and Occupational Health and Toxicology, College of Public Health, Department of Internal Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Nebraska Medical Center, Omaha, NE, USA; Global Environmental Health, School of Public Health and Tropical Medicine Sciences, Tulane University, New Orleans, LA, USA Alcohol use disorders are often associated with lung disease. High exposure to alcohol may lead to the production of reactive oxygen species, which can induce lipid peroxidation and formation of malondialdehyde (MDA) as well as induce the expression of cytochrome p-450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA is genotoxic and can lead to the formation of various DNA adducts such as malondialdehyde deoxyguanosine (M1dG) adducts which have been implicated in alcohol-related cancers and cardiovascular disease. Since the CYP2E1 pathway of alcohol metabolism is involved in the formation of MDA, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would regulate MDA production, M1dG adduct formation, and single strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue. C57BL/6 mice were administered 20% ethanol ad libitum in drinking water (Meadows-Cook model) for 8 weeks and exposed to whole body cigarette smoke for 5 weeks. Mice were also fed a CYP2E1 inhibitor, diallyl disulfide (DADS), at 1 mM/g of feed in their daily diet. Normal human bronchial epithelial cells (HBEC) were pre-treated with 10 mM DADS for 1 h, and treated with 80 mM ethanol 5% cigarette smoke extract (CSE) for 3 h for comet assay and 6 h for CYP2E1, MDA, and M1dG adduct assays. Ethanol exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBEC attenuated this ethanol effect. Ethanol also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE ethanol did not enhance these effects. In lung tissue homogenate of 8week alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison to control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5 weeks cigarette smoke-exposed mice. These findings suggest that cigarette smoke may not have a significant role in the formation of MDA and M1dG adduct, alcohol may be solely responsible for M1dG adduct formation. Thus CYP2E1 plays a pivotal role in alcohol induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation (Supported by NIH R01 AA017663 [TAW]).
B. Saha, J. Bruneau, G. Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA Introduction: HCV infection and alcohol abuse are the leading cause of cirrhosis, requiring liver transplantation. Macrophage (MF) activation occurs in chronic hepatitis C virus (HCV) infection and alcohol exposure and dysfunctional MFs may contribute to liver fibrosis. Depending on signals from the tissue microenvironment, circulating monocytes differentiate into MFs with either M1 (classical) or M2 (alternative) polarization. However, the nature of MF polarization during HCV infection and alcohol exposure is not known. We hypothesized that HCV and alcohol provides signals for circulating monocytes to differentiate into “pathogenic macrophages”. Methods: Surface markers of circulating monocytes from HCV-infected patients, alcohol exposed individuals and healthy donors were studied by flow cytometry. In vitro co-culture experiments were performed with monocytes isolated from healthy donors (n ¼ 10–15) or 24 h alcohol exposed donors (4–7), and hepatoma cells (Huh7.5) infected with HCV (JFH-1/Huh7.5). Results: We found that circulating monocytes from chronic HCV-infected patients exhibit an M2 polarized phenotype with high expression of CD206 (mannose receptor) and CD163 (scavenger receptor) as compared to controls. However, the circulating monocytes from acute alcohol exposed donors predominantly expressed M1 markers (CD16 and CD40). We observed that JFH-1/Huh7.5 induced differentiation of normal monocytes to MF-like cells in vitro. These “HCV-educated” MFs displayed increased expression of M2 markers. However, co-culture of alcohol exposed monocytes with JFH-1/ Huh7.5, lowered the expression of MF markers. M1 markers on the alcohol exposed monocytes were not increased in the presence of HCV, but a synergistic effect on the expression of M2 marker (CD206) was observed. Healthy monocytes co-cultured with JFH-1/Huh7.5 cells secreted pro-inflammatory (IL-1b and TNF-a) and predominantly anti-inflammatory (IL-10 and TGF-b) cytokines. Alcohol exposed monocytes secreted higher IL-1b and TGF-b and lower IL-10 as compared to control monocytes co-cultured with JFH-1/ Huh7.5. The high level of TGF-b secreted by “HCV-educated” MF was profibrotic and led to activation of hepatic stellate (LX2) cells as this process could be blocked by anti-TGFb neutralizing antibody. Conclusion: Thus, alcohol exposure inhibits the process of HCV-mediated monocyte differentiation. This study demonstrates the inhibitory effect of alcohol on the innate immune response to HCV infection (Supported by NIH AA014372 [GS]).
28. The role of miR-122 in the pathogenesis of chronic alcoholic liver disease A. Satishchandran, S. Bala, G. Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA The pathological progression of alcoholic liver disease (ALD) is multifactorial involving both metabolic and immunological dysfunction. The development of steatosis from alcohol abuse sensitizes hepatocytes to injurious inflammatory stimuli such as IL-1B and TNF. Additionally, lipid-filled hepatocytes produce reactive oxygen species by way of normal ethanol metabolism and increased lipid peroxidation. miR-122 regulates essential functions in hepatic lipid metabolism, mitochondrial function, cell death pathways, fibrosis and carcinogenesis – major elements in alcoholic liver disease. Given the highly conserved role of this unique miRNA in hepatic homeostasis we hypothesize that miR-122 contributes to ALD progression and may be a therapeutic target. Utilizing an scAAV8 vector expressing antimiR-122 TuD, we inhibited miR-122 0.124-fold (p ¼ 0.0059) in mouse hepatocytes followed by a 4-week alcohol diet. The anti-miR-122 TuD + alcohol (TuD + et) treated groups demonstrated significant increases in ALT and weight loss (p ¼ 0.0091, p < 0.05 respectively) compared to scrambled controls (Scr). We also found significant increases in HIF-1a in 122TuD + et treated mice (p ¼ 0.011 vs. Scr + et). Further, we observed 20-, 5-, and 4–fold increases in MCP-1, TNF-a, and IL-1B, respectively, in PF and Et treated knockdown mice when compared to Scr controls (p < 0.05). Given that bioinformatic miRNA target predictions tools suggest miR-122 may also negatively regulate HIF1a we hypothesize that miR-122 inhibition in ALD increases HIF1a thereby potentiating inflammation. However, further studies into whether these increases are a direct result of miR-122 modulation in hepatocytes or a function of hepatocyte mediated immune cell activation are needed (Supported by NIH AA020744 [GS] and F30 AA022283-01 [AS]).