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Macke AJ, Divita TE, Pachikov AN, Mahalingam S, Bellamkonda R, Rasineni K, Casey CA, Petrosyan A. Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D. Autophagy 2024; 20:1537-1558. [PMID: 38591519 PMCID: PMC11210917 DOI: 10.1080/15548627.2024.2329476] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Revised: 02/21/2024] [Accepted: 03/07/2024] [Indexed: 04/10/2024] Open
Abstract
The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: trans-Golgi network; SIM: structured illumination super-resolution microscopy.
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Affiliation(s)
- Amanda J. Macke
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA
| | - Taylor E. Divita
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA
| | - Artem N. Pachikov
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA
| | - Sundararajan Mahalingam
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- Omaha Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Ramesh Bellamkonda
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- Omaha Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Karuna Rasineni
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- Omaha Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Carol A. Casey
- Omaha Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA
| | - Armen Petrosyan
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
- The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA
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Huang J, Chan EOT, Liu X, Lok V, Ngai CH, Zhang L, Xu W, Zheng ZJ, Chiu PKF, Vasdev N, Enikeev D, Shariat SF, Ng CF, Teoh JYC, Wong MCS. Global Trends of Prostate Cancer by Age, and Their Associations With Gross Domestic Product (GDP), Human Development Index (HDI), Smoking, and Alcohol Drinking. Clin Genitourin Cancer 2023; 21:e261-e270.e50. [PMID: 36878752 DOI: 10.1016/j.clgc.2023.02.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Accepted: 02/05/2023] [Indexed: 02/17/2023]
Abstract
BACKGROUND We aimed to examine the global disease burden and trends of prostate cancer incidence and mortality by age, and their associations with gross domestic product (GDP), human development index (HDI), smoking, and alcohol drinking. MATERIALS AND METHODS We retrieved the Global Cancer Observatory (GLOBOCAN) database for the incidence and mortality of prostate cancer in 2020; the World Bank for GDP per capita; the United Nations for HDI; the WHO Global Health Observatory for prevalence of smoking and alcohol drinking; the Cancer Incidence in 5 Continents (CI5), WHO mortality database, for trend analysis. We presented the prostate cancer incidence and mortality using age-standardized rates. We examined their associations with GDP, HDI, smoking, and alcohol drinking by Spearman's correlations and multivariable regression. We estimated the 10-year trend of incidence and mortality by joinpoint regression analysis with average annual percent change with 95% confidence intervals in different age groups. RESULTS A wide variation in the burden of prostate cancer with the highest mortality found in low-income countries while the highest incidence was observed in high-income countries. We found moderate to high positive correlations for GDP, HDI, and alcohol drinking with prostate cancer incidence, whilst a low negative correlation was observed for smoking. Globally, there was an increasing incidence but decreasing mortality of prostate cancer, and such trends were particularly prominent in Europe. Notably, the incidence increase was also found in the younger population aged <50 years. CONCLUSIONS There was a global variation in the burden of prostate cancer associated with GDP, HDI, smoking, and alcohol drinking.
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Affiliation(s)
- Junjie Huang
- Jockey Club School of Public Health and Primary Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; Centre for Health Education and Health Promotion, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Erica On-Ting Chan
- S.H. Ho Urology Centre, Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Xianjing Liu
- Department of Radiology and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Veeleah Lok
- Department of Global Public Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden
| | - Chun Ho Ngai
- Jockey Club School of Public Health and Primary Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Lin Zhang
- School of Population Medicine and Public Health, Peking Union Medical College and The Chinese Academy of Medical Sciences, Beijing, China; Centre of Cancer Research, Victorian Comprehensive Cancer Centre, Melbourne, Victoria, Australia
| | - Wanghong Xu
- Department of Epidemiology, School of Public Health, Fudan University, Shanghai, China
| | - Zhi-Jie Zheng
- Department of Global Health, School of Public Health, Peking University, Beijing, China
| | - Peter Ka-Fung Chiu
- S.H. Ho Urology Centre, Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; European Association of Urology - Young Academic Urologists (EAU-YAU), Arnhem, The Netherlands
| | - Nikhil Vasdev
- Department of Urology, Hertfordshire and Bedfordshire Urological Cancer Centre, Lister Hospital Stevenage, School of Medicine and Life Sciences, University of Hertfordshire, Hatfield, United Kingdom
| | - Dmitry Enikeev
- Institute for Urology and Reproductive Health, Sechenov University, Moscow, Russia
| | - Shahrokh F Shariat
- Institute for Urology and Reproductive Health, Sechenov University, Moscow, Russia; Department of Urology, Medical University of Vienna, Vienna, Austria; Department of Urology, Weill Cornell Medical College, New York, NY; Department of Urology, University of Texas Southwestern, Dallas, TX; Division of Urology, Department of Special Surgery, Jordan University Hospital, The University of Jordan, Amman, Jordan; Department of Urology, 2nd Faculty of Medicine, Hospital Motol, Charles University, Prague, Czech Republic
| | - Chi-Fai Ng
- S.H. Ho Urology Centre, Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Jeremy Yuen-Chun Teoh
- S.H. Ho Urology Centre, Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; European Association of Urology - Young Academic Urologists (EAU-YAU), Arnhem, The Netherlands; Office of Global Engagement, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
| | - Martin C S Wong
- Jockey Club School of Public Health and Primary Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; Centre for Health Education and Health Promotion, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; School of Population Medicine and Public Health, Peking Union Medical College and The Chinese Academy of Medical Sciences, Beijing, China; Department of Global Health, School of Public Health, Peking University, Beijing, China.
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Attal N, Marrero E, Thompson KJ, McKillop IH. Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis. Alcohol Clin Exp Res 2022; 46:928-940. [PMID: 35403271 DOI: 10.1111/acer.14828] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Revised: 03/31/2022] [Accepted: 04/04/2022] [Indexed: 12/31/2022]
Abstract
BACKGROUND Hepatic steatosis is an early pathology of alcohol-associated liver disease (ALD). Fatty acid-binding protein-4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. METHODS Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1-inhibitor; CMZ) and/or EX-527 (a sirtuin-1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4 mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], β-catenin, peroxisome proliferator-activated receptor-α [PPARα], and lipin-1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. RESULTS CYP2E1-dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased β-catenin, PPARα, and lipin-1α protein levels in CYP2E1-expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1 mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1-expressing cells. Pharmacological inhibition of β-catenin, PPARα, or lipin-1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. CONCLUSION CYP2E1-dependent ethanol metabolism inhibits SIRT1-FOXO1 signaling, which leads to increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.
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Affiliation(s)
- Neha Attal
- Department of Surgery, Carolinas Medical Center, Atrium Health, Charlotte, North Carolina, USA
| | - Emilio Marrero
- Department of Surgery, Carolinas Medical Center, Atrium Health, Charlotte, North Carolina, USA
| | - Kyle J Thompson
- Department of Surgery, Carolinas Medical Center, Atrium Health, Charlotte, North Carolina, USA
| | - Iain H McKillop
- Department of Surgery, Carolinas Medical Center, Atrium Health, Charlotte, North Carolina, USA
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Casey CA, Macke AJ, Gough RR, Pachikov AN, Morris ME, Thomes PG, Kubik JL, Holzapfel MS, Petrosyan A. Alcohol-Induced Liver Injury: Down-regulation and Redistribution of Rab3D Results in Atypical Protein Trafficking. Hepatol Commun 2022; 6:374-388. [PMID: 34494400 PMCID: PMC8793998 DOI: 10.1002/hep4.1811] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 08/05/2021] [Accepted: 08/05/2021] [Indexed: 12/17/2022] Open
Abstract
Previous work from our laboratories has identified multiple defects in endocytosis, protein trafficking, and secretion, along with altered Golgi function after alcohol administration. Manifestation of alcohol-associated liver disease (ALD) is associated with an aberrant function of several hepatic proteins, including asialoglycoprotein receptor (ASGP-R), their atypical distribution at the plasma membrane (PM), and secretion of their abnormally glycosylated forms into the bloodstream, but trafficking mechanism is unknown. Here we report that a small GTPase, Rab3D, known to be involved in exocytosis, secretion, and vesicle trafficking, shows ethanol (EtOH)-impaired function, which plays an important role in Golgi disorganization. We used multiple approaches and cellular/animal models of ALD, along with Rab3D knockout (KO) mice and human tissue from patients with ALD. We found that Rab3D resides primarily in trans- and cis-faces of Golgi; however, EtOH treatment results in Rab3D redistribution from trans-Golgi to cis-medial-Golgi. Cells lacking Rab3D demonstrate enlargement of Golgi, especially its distal compartments. We identified that Rab3D is required for coat protein I (COPI) vesiculation in Golgi, and conversely, COPI is critical for intra-Golgi distribution of Rab3D. Rab3D/COPI association was altered not only in the liver of patients with ALD but also in the donors consuming alcohol without steatosis. In Rab3D KO mice, hepatocytes experience endoplasmic reticulum (ER) stress, and EtOH administration activates apoptosis. Notably, in these cells, ASGP-R, despite incomplete glycosylation, can still reach cell surface through ER-PM junctions. This mimics the effects seen with EtOH-induced liver injury. Conclusion: We revealed that down-regulation of Rab3D contributes significantly to EtOH-induced Golgi disorganization, and abnormally glycosylated ASGP-R is excreted through ER-PM connections, bypassing canonical (ER→Golgi→PM) anterograde transportation. This suggests that ER-PM sites may be a therapeutic target for ALD.
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Affiliation(s)
- Carol A. Casey
- Department of Research ServiceOmaha Western Iowa Health Care System, VA ServiceOmahaNEUSA
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Amanda J. Macke
- Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Ryan R. Gough
- Department of Research ServiceOmaha Western Iowa Health Care System, VA ServiceOmahaNEUSA
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNEUSA
- Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Artem N. Pachikov
- Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaNEUSA
- The Fred and Pamela Buffett Cancer CenterOmahaNEUSA
| | - Mary E. Morris
- Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Paul G. Thomes
- Department of Research ServiceOmaha Western Iowa Health Care System, VA ServiceOmahaNEUSA
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Jacy L. Kubik
- Department of Research ServiceOmaha Western Iowa Health Care System, VA ServiceOmahaNEUSA
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Melissa S. Holzapfel
- Department of Pathology and MicrobiologyUniversity of Nebraska Medical CenterOmahaNEUSA
| | - Armen Petrosyan
- Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaNEUSA
- The Fred and Pamela Buffett Cancer CenterOmahaNEUSA
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The Multifaceted Role of Flavonoids in Cancer Therapy: Leveraging Autophagy with a Double-Edged Sword. Antioxidants (Basel) 2021; 10:antiox10071138. [PMID: 34356371 PMCID: PMC8301186 DOI: 10.3390/antiox10071138] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 07/05/2021] [Accepted: 07/15/2021] [Indexed: 02/05/2023] Open
Abstract
Flavonoids are considered as pleiotropic, safe, and readily obtainable molecules. A large number of recent studies have proposed that flavonoids have potential in the treatment of tumors by the modulation of autophagy. In many cases, flavonoids suppress cancer by stimulating excessive autophagy or impairing autophagy flux especially in apoptosis-resistant cancer cells. However, the anti-cancer activity of flavonoids may be attenuated due to the simultaneous induction of protective autophagy. Notably, flavonoids-triggered protective autophagy is becoming a trend for preventing cancer in the clinical setting or for protecting patients from conventional therapeutic side effects in normal tissues. In this review, focusing on the underlying autophagic mechanisms of flavonoids, we hope to provide a new perspective for clinical application of flavonoids in cancer therapy. In addition, we highlight new research ideas for the development of new dosage forms of flavonoids to improve their various pharmacological effects, establishing flavonoids as ideal candidates for cancer prevention and therapy in the clinic.
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Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2020; 2020:2067959. [PMID: 32685086 PMCID: PMC7336232 DOI: 10.1155/2020/2067959] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Revised: 05/20/2020] [Accepted: 06/08/2020] [Indexed: 02/06/2023]
Abstract
Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.
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Janeiro C, Santos R, Teixeira J, Mesquita P, Eça R, Vale L. In the Beginning ... It was Already the End. GE PORTUGUESE JOURNAL OF GASTROENTEROLOGY 2020; 27:43-46. [PMID: 31970240 PMCID: PMC6959100 DOI: 10.1159/000499979] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Accepted: 02/26/2019] [Indexed: 06/10/2023]
Abstract
Sometimes, the presentation of some diseases can be fulminating. The authors present the case of a 51-year- old male brought to the emergency department visibly drunk and complaining of abdominal pain. Immediately, the diagnosis of hemorrhagic shock due to an accentuated drop of the hemoglobin level was made. After stabilization, he underwent a computed tomography of the abdomen, revealing a hepatocellular carcinoma with rupture of the Glisson capsule and massive intraperitoneal hemorrhage. With this case, the authors want to bring attention to a rare first presentation of hepatocellular carcinoma with a catastrophic result.
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Affiliation(s)
| | | | | | | | | | - Luis Vale
- *Luis Vale, Serviço de Medicina 2.1, Hospital de Santo António dos Capuchos, Alameda de Santo António dos Capuchos, PT–1169-050 Lisbon (Portugal), E-Mail
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Abou-Alfa GK, Jarnagin W, El Dika I, D'Angelica M, Lowery M, Brown K, Ludwig E, Kemeny N, Covey A, Crane CH, Harding J, Shia J, O'Reilly EM. Liver and Bile Duct Cancer. ABELOFF'S CLINICAL ONCOLOGY 2020:1314-1341.e11. [DOI: 10.1016/b978-0-323-47674-4.00077-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Hernández-Muñoz R, Lucinda Contreras-Zentella M. Involvement of cell oxidant status and redox state in the increased non-enzymatic ethanol oxidation by the regenerating rat liver. Biochem Pharmacol 2019; 161:63-72. [PMID: 30625299 DOI: 10.1016/j.bcp.2019.01.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Accepted: 01/02/2019] [Indexed: 12/20/2022]
Abstract
Ethanol administration is capable of inhibiting or delaying the partial hepatectomy (PH)-induced liver regeneration, probably altering liver metabolism by means of its oxidative metabolism. Since the regenerating liver has increased capacity for oxidizing ethanol, the present study was aimed to address the contribution of the ethanol-oxidizing metabolic pathways in the regenerating liver cells. Isolated hepatocytes were prepared from control livers and from animals subjected to two-thirds PH. In both preparations, ethanol oxidation was largely increased by incubation with glucose and was highly sensitive to inhibitors of ethanol-oxidizing enzymatic pathways (alcohol dehydrogenase, catalase and cytochrome P-4502E1 activities). The latter led to a total blockade of ethanol disposal by control hepatocytes, while liver cells from PH-rats only showed an early 70-75% inhibition of ethanol catabolism with the inhibitors used. In regenerating hepatocytes, the enhanced ethanol oxidation was blocked by scavengers of reactive oxygen species, an effect that correlated with enhanced cytoplasmic lipid peroxidation by-products. Both cell preparations showed similar sensitivity to inhibitors for the malate-aspartate shuttle and mitochondrial electron transport chain; the shift of the cytoplasmic redox state was also quite similar after ethanol oxidation. A more oxidized mitochondrial redox state was found in hepatocytes from PH-rats and more shifted to the reduced state during ethanol oxidation this effect was not abolished by inhibiting alcohol dehydrogenase activity. In conclusion, data clearly show that an important fraction of ethanol is metabolized through a non-enzymatic-mediated oxidative event, which could largely contribute to the deleterious effect of ethanol on the proliferating liver.
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Affiliation(s)
- Rolando Hernández-Muñoz
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Apdo. Postal 70-245, Mexico City 04510, Mexico.
| | - Martha Lucinda Contreras-Zentella
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Apdo. Postal 70-245, Mexico City 04510, Mexico
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Giantin Is Required for Post-Alcohol Recovery of Golgi in Liver Cells. Biomolecules 2018; 8:biom8040150. [PMID: 30453527 PMCID: PMC6316505 DOI: 10.3390/biom8040150] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 11/09/2018] [Accepted: 11/12/2018] [Indexed: 12/17/2022] Open
Abstract
In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Perinuclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber–DeCarli diet)). For recovery, EtOH was removed and replenished with control medium (48 h for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.
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Naghdi S, Slovinsky WS, Madesh M, Rubin E, Hajnóczky G. Mitochondrial fusion and Bid-mediated mitochondrial apoptosis are perturbed by alcohol with distinct dependence on its metabolism. Cell Death Dis 2018; 9:1028. [PMID: 30301883 PMCID: PMC6177459 DOI: 10.1038/s41419-018-1070-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Revised: 06/29/2018] [Accepted: 08/20/2018] [Indexed: 12/11/2022]
Abstract
Environmental stressors like ethanol (EtOH) commonly target mitochondria to influence the cell’s fate. Recent literature supports that chronic EtOH exposure suppresses mitochondrial dynamics, central to quality control, and sensitizes mitochondrial permeability transition pore opening to promote cell death. EtOH-induced tissue injury is primarily attributed to its toxic metabolic products but alcoholism also impairs tissues that poorly metabolize EtOH. We embarked on studies to determine the respective roles of EtOH and its metabolites in mitochondrial fusion and tBid-induced mitochondrial apoptosis. We used HepG2 cells that do not metabolize EtOH and its engineered clone that expresses EtOH-metabolizing Cytochrome P450 E2 and alcohol dehydrogenase (VL-17A cells). We found that fusion impairment by prolonged EtOH exposure was prominent in VL-17A cells, probably owing to reactive oxygen species increase in the mitochondrial matrix. There was no change in fusion protein abundance, mitochondrial membrane potential or Ca2+ uptake. By contrast, prolonged EtOH exposure promoted tBid-induced outer mitochondrial membrane permeabilization and cell death only in HepG2 cells, owing to enhanced Bak oligomerization. Thus, mitochondrial fusion inhibition by EtOH is dependent on its metabolites, whereas sensitization to tBid-induced death is mediated by EtOH itself. This difference is of pathophysiological relevance because of the tissue-specific differences in EtOH metabolism.
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Affiliation(s)
- Shamim Naghdi
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - William S Slovinsky
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Muniswamy Madesh
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Emanuel Rubin
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - György Hajnóczky
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
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Zhou C, Zhang W, Chen W, Yin Y, Atyah M, Liu S, Guo L, Shi Y, Ye Q, Dong Q, Ren N. Integrated Analysis of Copy Number Variations and Gene Expression Profiling in Hepatocellular carcinoma. Sci Rep 2017; 7:10570. [PMID: 28874807 PMCID: PMC5585301 DOI: 10.1038/s41598-017-11029-y] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Accepted: 08/18/2017] [Indexed: 01/23/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the top three cancer killers worldwide. To identify CNV-driven differentially expressed genes (DEGs) in HBV related HCC, this study integrated analysis of copy number variations (CNVs) and gene expression profiling. Significant genes in regions of CNVs were overlapped with those obtained from the expression profiling. 93 CNV-driven genes exhibiting increased expression in the duplicated regions and 45 showing decreased expression in the deleted regions were obtained, which duplications and deletions were mainly documented at chromosome 1 and 4. Functional and pathway enrichment analyses were performed using DAVID and KOBAS, respectively. They were mainly enriched in metabolic process and cell cycle. Protein-protein interaction (PPI) network was constructed by Cytoscape, then four hub genes were identified. Following, survival analyses indicated that only high NPM1 expression was significantly and independently associated with worse survival and increased recurrence in HCC patients. Moreover, this correlation remained significant in patients with early stage of HCC. In addition, we showed that NPM1 was overexpressed in HCC cells and in HCC versus adjacent non-tumor tissues. In conclusion, these results showed that integrated analysis of genomic and expression profiling might provide a powerful potential for identifying CNV-driven genes in HBV related HCC pathogenesis.
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Affiliation(s)
- Chenhao Zhou
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Wentao Zhang
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Wanyong Chen
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Yirui Yin
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Manar Atyah
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Shuang Liu
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Lei Guo
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Yi Shi
- Biomedical Research Centre, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Qinghai Ye
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China
| | - Qiongzhu Dong
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Zhongshan Hospital, Fudan University, Shanghai, China.
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
| | - Ning Ren
- Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China.
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Zhongshan Hospital, Fudan University, Shanghai, China.
- Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China.
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Cho YE, Mezey E, Hardwick JP, Salem N, Clemens DL, Song BJ. Increased ethanol-inducible cytochrome P450-2E1 and cytochrome P450 isoforms in exosomes of alcohol-exposed rodents and patients with alcoholism through oxidative and endoplasmic reticulum stress. Hepatol Commun 2017; 1:675-690. [PMID: 29404485 PMCID: PMC5721437 DOI: 10.1002/hep4.1066] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Revised: 05/25/2017] [Accepted: 06/06/2017] [Indexed: 12/17/2022] Open
Abstract
This study investigated the role of ethanol‐inducible cytochrome P450‐2E1 (CYP2E1) in enhancing CYP2E1 and other P450 proteins in extracellular vesicles (EVs) from alcohol‐exposed rodents and human patients with alcoholism and their effects on oxidative hepatocyte injury. Female Fischer rats and wild‐type or Cyp2e1‐null mice were exposed to three oral doses of binge ethanol or dextrose control at 12‐hour intervals. Plasma EV and hepatic proteins from alcohol‐exposed rodents, patients with alcoholism, and their respective controls were isolated and characterized. The number of EVs and the amounts of EV CYP2E1, CYP2A, CYP1A1/2, and CYP4B proteins were markedly elevated in both patients with alcoholism and alcohol‐exposed rats and mice. The number of EVs and EV P450 proteins were significantly reduced in ethanol‐exposed rats fed a diet containing polyunsaturated fatty acids. The increased number of EVs and EV CYP2E1 and other P450 isoforms in alcohol‐exposed wild types were significantly reduced in the corresponding Cyp2e1‐null mice. EV CYP2E1 amounts depended on increased oxidative and endoplasmic reticulum (ER) stress because their levels were decreased by cotreatment with the antioxidant N‐acetylcysteine or the CYP2E1 inhibitor chlormethiazole but increased by ER stress‐inducer thapsigargin, which was blocked by 4‐phenylbutyric acid. Furthermore, cell death rates were elevated when primary hepatocytes or human hepatoma cells were exposed to EVs from alcohol‐exposed rodents and patients with alcoholism, demonstrating that EVs from alcohol‐exposed rats and patients with alcoholism are functional and can promote cell death by activating the apoptosis signaling pathway, including phospho‐c‐Jun N‐terminal kinase, proapoptotic Bax, and activated caspase‐3. Conclusion: CYP2E1 has an important role in elevating EV CYP2E1 and other P450 isoforms through increased oxidative and ER stress. Elevated EV‐CYP2E1 detected after withdrawal from alcohol or exposure to the CYP2E1 inducer pyrazole can be a potential biomarker for liver injury. (Hepatology Communications 2017;1:675–690)
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Affiliation(s)
- Young-Eun Cho
- Section of Molecular Pharmacology and Toxicology Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health Bethesda MD
| | - Esteban Mezey
- Department of Medicine The Johns Hopkins University School of Medicine Baltimore MD
| | - James P Hardwick
- Department of Integrative Medical Sciences College of Medicine, Northeast Ohio Medical University Rootstown OH
| | - Norman Salem
- Section of Molecular Pharmacology and Toxicology Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health Bethesda MD
| | - Dahn L Clemens
- Department of Internal Medicine University of Nebraska Medical Center Omaha NE
| | - Byoung-Joon Song
- Section of Molecular Pharmacology and Toxicology Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health Bethesda MD
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14
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Schott MB, Rasineni K, Weller SG, Schulze RJ, Sletten AC, Casey CA, McNiven MA. β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure. J Biol Chem 2017; 292:11815-11828. [PMID: 28515323 DOI: 10.1074/jbc.m117.777748] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2017] [Revised: 05/05/2017] [Indexed: 12/21/2022] Open
Abstract
In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.
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Affiliation(s)
- Micah B Schott
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905
| | - Karuna Rasineni
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198
| | - Shaun G Weller
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905
| | - Ryan J Schulze
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905
| | - Arthur C Sletten
- Division of Gastroenterology & Hepatology, Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905
| | - Carol A Casey
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska Western Iowa Health Care System Research Service, Omaha, Nebraska 68105
| | - Mark A McNiven
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
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15
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Schulze RJ, Rasineni K, Weller SG, Schott MB, Schroeder B, Casey CA, McNiven MA. Ethanol exposure inhibits hepatocyte lipophagy by inactivating the small guanosine triphosphatase Rab7. Hepatol Commun 2017; 1:140-152. [PMID: 29404450 PMCID: PMC5721426 DOI: 10.1002/hep4.1021] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/24/2016] [Accepted: 01/29/2017] [Indexed: 12/20/2022] Open
Abstract
Alcohol consumption is a well-established risk factor for the onset and progression of fatty liver disease. An estimated 90% of heavy drinkers are thought to develop significant liver steatosis. For these reasons, an increased understanding of the molecular basis for alcohol-induced hepatic steatosis is important. It has become clear that autophagy, a catabolic process of intracellular degradation and recycling, plays a key role in hepatic lipid metabolism. We have shown that Rab7, a small guanosine triphosphatase known to regulate membrane trafficking, acts as a key orchestrator of hepatocellular lipophagy, a selective form of autophagy in which lipid droplets (LDs) are specifically targeted for turnover by the autophagic machinery. Nutrient starvation results in Rab7 activation on the surface of the LD and lysosomal compartments, resulting in the mobilization of triglycerides stored within the LDs for energy production. Here, we examine whether the steatotic effects of alcohol exposure are a result of perturbations to the Rab7-mediated lipophagic pathway. Rats chronically fed an ethanol-containing diet accumulated significantly higher levels of fat in their hepatocytes. Interestingly, hepatocytes isolated from these ethanol-fed rats contained juxtanuclear lysosomes that exhibited impaired motility. These changes are similar to those we observed in Rab7-depleted hepatocytes. Consistent with these defects in the lysosomal compartment, we observed a marked 80% reduction in Rab7 activity in cultured hepatocytes as well as a complete block in starvation-induced Rab7 activation in primary hepatocytes isolated from chronic ethanol-fed animals. Conclusion: A mechanism is supported whereby ethanol exposure inhibits Rab7 activity, resulting in the impaired transport, targeting, and fusion of the autophagic machinery with LDs, leading to an accumulation of hepatocellular lipids and hepatic steatosis. (Hepatology Communications 2017;1:140-152).
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Affiliation(s)
- Ryan J. Schulze
- Department of Biochemistry and Molecular Biology and the Center for Digestive DiseasesMayo ClinicRochesterMN
| | - Karuna Rasineni
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNE
| | - Shaun G. Weller
- Department of Biochemistry and Molecular Biology and the Center for Digestive DiseasesMayo ClinicRochesterMN
| | - Micah B. Schott
- Department of Biochemistry and Molecular Biology and the Center for Digestive DiseasesMayo ClinicRochesterMN
| | - Barbara Schroeder
- Department of Biochemistry and Molecular Biology and the Center for Digestive DiseasesMayo ClinicRochesterMN
- Present address:
Helmholtz Zentrum München, Institute of Biological and Medical ImagingNeuherbergGermany
| | - Carol A. Casey
- Department of Internal MedicineUniversity of Nebraska Medical CenterOmahaNE
- Research Service, VA Nebraska‐Western Iowa Health Care SystemOmahaNE
| | - Mark A. McNiven
- Department of Biochemistry and Molecular Biology and the Center for Digestive DiseasesMayo ClinicRochesterMN
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16
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Casey CA, Bhat G, Holzapfel MS, Petrosyan A. Study of Ethanol-Induced Golgi Disorganization Reveals the Potential Mechanism of Alcohol-Impaired N-Glycosylation. Alcohol Clin Exp Res 2016; 40:2573-2590. [PMID: 27748959 PMCID: PMC5133184 DOI: 10.1111/acer.13247] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Accepted: 09/20/2016] [Indexed: 01/18/2023]
Abstract
BACKGROUND It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. METHODS HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM). RESULTS First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive. CONCLUSIONS Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.
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Affiliation(s)
- Carol A. Casey
- Department of Internal Medicine, University of Nebraska Medical Center, and the Fred and Pamela Buffett Cancer Center, Omaha, NE, USA
| | - Ganapati Bhat
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, and the Fred and Pamela Buffett Cancer Center, Omaha, NE, USA
| | - Melissa S. Holzapfel
- Department of Pathology and Microbiology, University of Nebraska Medical Center, and the Fred and Pamela Buffett Cancer Center, Omaha, NE, USA
| | - Armen Petrosyan
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, and the Fred and Pamela Buffett Cancer Center, Omaha, NE, USA
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17
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Petrosyan A, Casey CA, Cheng PW. The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization. Sci Rep 2016; 6:31962. [PMID: 27535804 PMCID: PMC4989220 DOI: 10.1038/srep31962] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 07/27/2016] [Indexed: 12/12/2022] Open
Abstract
Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a "tug-of-war". However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell.
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Affiliation(s)
- Armen Petrosyan
- Department of Biochemistry and Molecular Biology, College of Medicine, the Fred and Pamela Buffett Cancer Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Carol A Casey
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA.,Nebraska Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Pi-Wan Cheng
- Department of Biochemistry and Molecular Biology, College of Medicine, the Fred and Pamela Buffett Cancer Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA.,Nebraska Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
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18
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Kade S, Herzog N, Schmidtke KU, Küpper JH. Chronic ethanol treatment depletes glutathione regeneration capacity in hepatoma cell line HepG2. ACTA ACUST UNITED AC 2016. [DOI: 10.3233/jcb-15019] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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Scheer MA, Schneider KJ, Finnigan RL, Maloney EP, Wells MA, Clemens DL. The Involvement of Acetaldehyde in Ethanol-Induced Cell Cycle Impairment. Biomolecules 2016; 6:biom6020017. [PMID: 27043646 PMCID: PMC4919912 DOI: 10.3390/biom6020017] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Revised: 03/09/2016] [Accepted: 03/24/2016] [Indexed: 01/15/2023] Open
Abstract
Background: Hepatocytes metabolize the vast majority of ingested ethanol. This metabolic activity results in hepatic toxicity and impairs the ability of hepatocytes to replicate. Previous work by our group has shown that ethanol metabolism results in a G2/M cell cycle arrest. The intent of these studies was to discern the roles of acetaldehyde and reactive oxygen, two of the major by-products of ethanol metabolism, in the G2/M cell cycle arrest. Methods: To investigate the role of ethanol metabolites in the cell cycle arrest, VA-13 and VL-17A cells were used. These are recombinant Hep G2 cells that express alcohol dehydrogenase or alcohol dehydrogenase and cytochrome P450 2E1, respectively. Cells were cultured with or without ethanol, lacking or containing the antioxidants N-acetylcysteine (NAC) or trolox, for three days. Cellular accumulation was monitored by the DNA content of the cultures. The accumulation of the cyclin-dependent kinase, Cdc2 in the inactive phosphorylated form (p-Cdc2) and the cyclin-dependent kinase inhibitor p21 were determined by immunoblot analysis. Results: Cultures maintained in the presence of ethanol demonstrated a G2/M cell cycle arrest that was associated with a reduction in DNA content and increased levels of p-Cdc2 and p21, compared with cells cultured in its absence. Inclusion of antioxidants in the ethanol containing media was unable to rescue the cells from the cell cycle arrest or these ethanol metabolism-mediated effects. Additionally, culturing the cells in the presence of acetaldehyde alone resulted in increased levels of p-Cdc2 and p21. Conclusions: Acetaldehyde produced during ethanol oxidation has a major role in the ethanol metabolism-mediated G2/M cell cycle arrest, and the concurrent accumulation of p21 and p-Cdc2. Although reactive oxygen species are thought to have a significant role in ethanol-induced hepatocellular damage, they may have a less important role in the inability of hepatocytes to replace dead or damaged cells.
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Affiliation(s)
- Marc A Scheer
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
| | - Katrina J Schneider
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
- Nebraska and Western Iowa Veterans Administration Medical Center, University of Nebraska Medical Center, Omaha, NE 68105, USA.
| | - Rochelle L Finnigan
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
| | - Eamon P Maloney
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
| | - Mark A Wells
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
| | - Dahn L Clemens
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105, USA.
- Nebraska and Western Iowa Veterans Administration Medical Center, University of Nebraska Medical Center, Omaha, NE 68105, USA.
- Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE 68105, USA.
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20
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Verma VK, Li H, Wang R, Hirsova P, Mushref M, Liu Y, Cao S, Contreras PC, Malhi H, Kamath PS, Gores GJ, Shah VH. Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles. J Hepatol 2016; 64:651-60. [PMID: 26632633 PMCID: PMC4761285 DOI: 10.1016/j.jhep.2015.11.020] [Citation(s) in RCA: 166] [Impact Index Per Article: 18.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 10/26/2015] [Accepted: 11/09/2015] [Indexed: 12/20/2022]
Abstract
BACKGROUND & AIMS The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.
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Affiliation(s)
- Vikas K Verma
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Haiyang Li
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou, China
| | - Ruisi Wang
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Petra Hirsova
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Malek Mushref
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Yaming Liu
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; First Hospital of Jilin University, China
| | - Sheng Cao
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | | | - Harmeet Malhi
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Patrick S Kamath
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Gregory J Gores
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Vijay H Shah
- Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
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Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2015; 2016:9209825. [PMID: 26788255 PMCID: PMC4691636 DOI: 10.1155/2016/9209825] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Accepted: 09/15/2015] [Indexed: 12/15/2022]
Abstract
Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.
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22
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Petrosyan A, Cheng PW, Clemens DL, Casey CA. Downregulation of the small GTPase SAR1A: a key event underlying alcohol-induced Golgi fragmentation in hepatocytes. Sci Rep 2015; 5:17127. [PMID: 26607390 PMCID: PMC4660820 DOI: 10.1038/srep17127] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Accepted: 10/26/2015] [Indexed: 12/14/2022] Open
Abstract
The hepatic asialoglycoprotein receptor (ASGP-R) is posttranslationally modified in the Golgi en route to the plasma membrane, where it mediates clearance of desialylated serum glycoproteins. It is known that content of plasma membrane-associated ASGP-R is decreased after ethanol exposure, although the mechanisms remain elusive. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein giantin. We hypothesize that ethanol-impaired giantin function may be related to altered trafficking of ASGP-R. Here we report that in HepG2 cells expressing alcohol dehydrogenase and hepatocytes of ethanol-fed rats, ethanol metabolism results in Golgi disorganization. This process is initiated by dysfunction of SAR1A GTPase followed by altered COPII vesicle formation and impaired Golgi delivery of the protein disulfide isomerase A3 (PDIA3), an enzyme that catalyzes giantin dimerization. Additionally, we show that SAR1A gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, arresting PDIA3 in the endoplasmic reticulum (ER) and large-scale alterations in Golgi architecture. Ethanol-induced Golgi fission has no effect on ER-to-Golgi transportation of ASGP-R, however, it results in its deposition in cis-medial-, but not trans-Golgi. Thus, alcohol-induced deficiency in COPII vesicle formation predetermines Golgi fragmentation which, in turn, compromises the Golgi-to-plasma membrane transportation of ASGP-R.
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Affiliation(s)
- Armen Petrosyan
- Department of Biochemistry and Molecular Biology, College of Medicine, Omaha, NE, USA
| | - Pi-Wan Cheng
- Department of Biochemistry and Molecular Biology, College of Medicine, Omaha, NE, USA
- Nebraska Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Dahn L. Clemens
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA
- Nebraska Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
| | - Carol A. Casey
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA
- Nebraska Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA
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Welti M, Hülsmeier AJ. Ethanol-induced impairment in the biosynthesis of N-linked glycosylation. J Cell Biochem 2014; 115:754-62. [PMID: 24243557 DOI: 10.1002/jcb.24713] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2013] [Accepted: 11/07/2013] [Indexed: 12/20/2022]
Abstract
Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.
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Affiliation(s)
- Michael Welti
- Institute of Physiology, University of Zürich, Zürich, Switzerland
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Swaminathan K, Kumar SM, Clemens DL, Dey A. Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells. Biochim Biophys Acta Gen Subj 2013; 1830:4407-16. [DOI: 10.1016/j.bbagen.2013.05.022] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2013] [Revised: 05/13/2013] [Accepted: 05/17/2013] [Indexed: 01/22/2023]
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Sawada N, Inoue M, Iwasaki M, Sasazuki S, Yamaji T, Shimazu T, Tsugane S. Alcohol and smoking and subsequent risk of prostate cancer in Japanese men: the Japan Public Health Center-based prospective study. Int J Cancer 2013; 134:971-8. [PMID: 23929133 DOI: 10.1002/ijc.28423] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2013] [Accepted: 07/22/2013] [Indexed: 11/12/2022]
Abstract
Although alcohol and smoking have not been established as risk factors for prostate cancer, they are important risk factors for other human cancers and potentially major avoidable factors. Alcohol drinkers and smokers might be less likely to get screening, which might lead to attenuation of the positive association. Here, we investigated the association of alcohol drinking and smoking and prostate cancer according to stage, as well as prostate cancer detected by subjective symptoms, in a large prospective study among Japanese men. The Japan Public Health Center-based prospective study (JPHC study) was established in 1990 for Cohort I and in 1993 for Cohort II. Subjects were 48,218 men aged 40-69 years who completed a questionnaire, which included their alcohol and smoking habits at baseline, and who were followed until the end of 2010. During 16 years of follow-up, 913 men were newly diagnosed with prostate cancer; of whom 248 had advanced cases, 635 were organ-localized and 30 were of an undetermined stage. Alcohol consumption was dose-dependently associated with advanced prostate cancer [nondrinkers: reference, 0-150 g/week: hazard ratio (HR) = 1.23, 95% confidence interval (CI) = 0.83-1.82; 150-300 g/week: HR = 1.51, 95% CI = 1.04-2.19; ≥ 300 g/week: HR = 1.41, 95% CI = 0.97-2.05, p for trend = 0.02]. The positive association was not substantially changed among cancers detected by subjective symptoms. Smoking was inversely associated with prostate cancer among total subjects, but tended to increase the risk of advanced prostate cancer detected by subjective symptoms. In conclusion, abstinence from alcohol and prohibition of smoking might be important factors in the prevention of advanced prostate cancer.
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Affiliation(s)
- Norie Sawada
- Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo, Japan
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26
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Elk1 and AP-1 sites in the TBP promoter mediate alcohol-induced deregulation of Pol III-dependent genes. Gene 2013; 526:54-60. [PMID: 23454483 DOI: 10.1016/j.gene.2013.02.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2012] [Revised: 02/05/2013] [Accepted: 02/08/2013] [Indexed: 01/22/2023]
Abstract
The major risk factors for hepatocellular carcinoma (HCC) are chronic liver diseases that include hepatitis B, hepatitis C, alcoholic liver disease and non-alcoholic steatohepatitis. However, the mechanisms of alcohol-associated HCC remain to be elucidated. The products of RNA Pol III (RNA polymerase III) dependent genes are elevated in both transformation cells and tumor cells. TBP (TATA-box binding protein) is a central transcription factor, which regulates Pol I, Pol II and Pol III gene activity. Our studies have demonstrated that alcohol increases TBP expression and Pol III gene transcription to promote liver tumor formation. We continue to investigate how ethanol mediates TBP expression. Here, we report that ethanol induces TBP promoter activity and the induction is ethanol dose dependent. Blocking the JNK1 pathway by a chemical inhibitor and siRNA reduces this ethanol-induced activity. Furthermore, mutating G>A at a -46 bp Elk1 binding site of the TBP promoter or mutating AP-1 binding site at -37 bp (A>G) and -38 bp (C>T) reduces the TBP promoter activity. Mutation of both Elk1 and AP-1 binding sites dramatically represses this induction. Together, these studies demonstrate that, for the first time, alcohol increases Pol III gene transcription through a response element, which is composed of the overlapping Elk1 and AP-1 binding sites of the TBP promoter and affected by alcohol. It suggests that these binding sites may play a critical role in alcohol-induced deregulation of Pol III genes in liver tumor development.
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Thomes PG, Osna NA, Davis JS, Donohue TM. Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1. Int J Biochem Cell Biol 2013; 45:454-463. [PMID: 23103837 PMCID: PMC3549023 DOI: 10.1016/j.biocel.2012.10.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2012] [Revised: 10/04/2012] [Accepted: 10/10/2012] [Indexed: 02/08/2023]
Abstract
Recent studies have shown that the transcription factor early growth response-1 (Egr-1) regulates ethanol-induced fatty liver. However, the mechanism(s) through which ethanol oxidation controls Egr-1 is unknown. Here, using recombinant hepatoma (HepG2; VL-17A) cells that metabolize ethanol, we show that alcohol dehydrogenase catalysis of ethanol oxidation and subsequent acetaldehyde production controls Egr-1 expression. Further, the induction of Egr-1 enhances expression of other steatosis-related genes, resulting in triglyceride accumulation. Ethanol exposure increased Egr-1 promoter activity, messenger RNA and Egr-1 protein levels in VL-17A cells. Elevated Egr-1 protein was sustained by an ethanol-induced decrease in proteasome activity, thereby stabilizing the Egr-1 protein. Egr-1 induction depended on ethanol oxidation, as it was prevented when ethanol oxidation was blocked. Ethanol exposure induced Egr-1 and triglyceride accumulation only in alcohol dehydrogenase-expressing cells that produced acetaldehyde. Such induction did not occur in parental, non-metabolizing HepG2 cells or in cells that express only cytochrome P450 2E1. However, direct exposure of HepG2 cells to acetaldehyde induced both Egr-1 protein and triglycerides. Egr-1 over-expression elevated triglyceride levels, which were augmented by ethanol exposure. However, these triglyceride levels did not exceed those in ethanol-exposed cells that had normal Egr-1 expression. Conversely, Egr-1 knockdown by siRNA only partially blocked ethanol-induced triglyceride accumulation and was associated not only with lower Egr-1 expression but also attenuation of SREBP1c and TNF-α mRNAs. Double knockdown of both Egr-1 and SREBP-1c abolished ethanol-elicited steatosis. Collectively, our findings provide important new insights into the temporal regulation by ethanol oxidation of Egr-1 and cellular steatosis.
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Affiliation(s)
- Paul G. Thomes
- VA-Nebraska-Western Iowa Health Care System, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68105, USA
| | - Natalia A. Osna
- VA-Nebraska-Western Iowa Health Care System, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68105, USA
| | - John S. Davis
- VA-Nebraska-Western Iowa Health Care System, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- The Olson Center for Women’s Health, Department of Obstetrics and Gynecology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, 68105, USA
| | - Terrence M. Donohue
- VA-Nebraska-Western Iowa Health Care System, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68105, USA
- The Center for Environmental Toxicology, College of Public Health, University of Nebraska Medical Center, Omaha, NE, 68105, USA
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28
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Swaminathan K, Clemens DL, Dey A. Inhibition of CYP2E1 leads to decreased malondialdehyde-acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure. Life Sci 2013; 92:325-36. [PMID: 23352969 DOI: 10.1016/j.lfs.2012.12.014] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Revised: 11/23/2012] [Accepted: 12/17/2012] [Indexed: 12/26/2022]
Abstract
AIM Ethanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde-acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated. MAIN METHODS Chronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor - diallyl sulfide or ADH inhibitor - pyrazole or ADH and CYP2E1 inhibitor - 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis. KEY FINDINGS Inhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury. SIGNIFICANCE CYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.
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Affiliation(s)
- Kavitha Swaminathan
- Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai-600044, India
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29
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Swaminathan K, Kumar SM, Clemens DL, Dey A. Chronic ethanol and high glucose inducible CYP2E1 mediated oxidative stress leads to greater cellular injury in VL-17A cells: a potential mechanism for liver injury due to chronic alcohol consumption and hyperglycemia. Toxicol Res (Camb) 2013. [DOI: 10.1039/c3tx50016k] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
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30
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Thomes PG, Ehlers RA, Trambly CS, Clemens DL, Fox HS, Tuma DJ, Donohue TM. Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells. Autophagy 2013; 9:63-73. [PMID: 23090141 PMCID: PMC3542219 DOI: 10.4161/auto.22490] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH (+) /CYP2E1 (+) ) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.
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Affiliation(s)
- Paul G Thomes
- Liver Study Unit, Department of Veterans Affairs, VA Nebraska-Western Iowa Health Care System (NWIHCS), Omaha, NE, USA.
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31
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Gyamfi D, Everitt HE, Tewfik I, Clemens DL, Patel VB. Hepatic mitochondrial dysfunction induced by fatty acids and ethanol. Free Radic Biol Med 2012; 53:2131-2145. [PMID: 23010494 DOI: 10.1016/j.freeradbiomed.2012.09.024] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/02/2012] [Revised: 09/14/2012] [Accepted: 09/15/2012] [Indexed: 02/08/2023]
Abstract
Understanding the key aspects of the pathogenesis of alcoholic fatty liver disease particularly alterations to mitochondrial function remains to be resolved. The role of fatty acids in this regard requires further investigation due to their involvement in fatty liver disease and obesity. This study aimed to characterize the early effects of saturated and unsaturated fatty acids alone on liver mitochondrial function and during concomitant ethanol exposure using isolated liver mitochondria and VA-13 cells (Hep G2 cells that efficiently express alcohol dehydrogenase). Liver mitochondria or VA-13 cells were treated with increasing concentrations of palmitic or arachidonic acid (1 to 160 μM) for 24 h with or without 100 mM ethanol. The results showed that in isolated liver mitochondria both palmitic and arachidonic acids significantly reduced state 3 respiration in a concentration-dependent manner (P<0.001), implicating their ionophoric activities. Increased ROS production occurred in a dose-dependent manner especially in the presence of rotenone (complex I inhibitor), which was significantly more prominent in arachidonic acid at 80 μM (+970%, P<0.001) than palmitic acid (+40%, P<0.01). In VA-13 cells, ethanol alone and both fatty acids (40 μM) were able to decrease the mitochondrial membrane potential and cellular ATP levels and increase lipid formation. ROS production was significantly increased with arachidonic acid (+110%, P<0.001) exhibiting a greater effect than palmitic acid (+39%, P<0.05). While in the presence of ethanol, the drop in the mitochondrial membrane potential, cellular ATP levels, and increased lipid formation were further enhanced by both fatty acids, but with greater effect in the case of arachidonic acid, which also correlated with significant cytotoxicity (P<0.001). This study confirms the ability of fatty acids to promote mitochondrial injury in the development of alcoholic fatty liver disease.
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Affiliation(s)
- Daniel Gyamfi
- Department of Biomedical Sciences, School of Life Sciences, University of Westminster, 115 New Cavendish Street, London, W1W 6UW, UK
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Chandrasekaran K, Swaminathan K, Kumar SM, Clemens DL, Dey A. Increased oxidative stress and toxicity in ADH and CYP2E1 overexpressing human hepatoma VL-17A cells exposed to high glucose. Integr Biol (Camb) 2012; 4:550-63. [DOI: 10.1039/c2ib00155a] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Cyanamide potentiates the ethanol-induced impairment of receptor-mediated endocytosis in a recombinant hepatic cell line expressing alcohol dehydrogenase activity. Int J Hepatol 2012; 2012:954157. [PMID: 22518324 PMCID: PMC3296211 DOI: 10.1155/2012/954157] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2011] [Accepted: 11/16/2011] [Indexed: 11/17/2022] Open
Abstract
Ethanol administration has been shown to alter receptor-mediated endocytosis in the liver. We have developed a recombinant hepatic cell line stably transfected with murine alcohol dehydrogenase cDNA to serve as an in vitro model to investigate these ethanol-induced impairments. In the present study, transfected cells were maintained in the absence or presence of 25 mM ethanol for 7 days, and alterations in endocytosis by the asialoglycoprotein receptor were determined. The role of acetaldehyde in this dysfunction was also examined by inclusion of the aldehyde dehydrogenase inhibitor, cyanamide. Our results showed that ethanol metabolism impaired internalization of asialoorosomucoid, a ligand for the asialoglycoprotein receptor. The addition of cyanamide potentiated the ethanol-induced defect in internalization and also impaired degradation of the ligand in the presence of ethanol. These results indicate that the ethanol-induced impairment in endocytosis is exacerbated by the inhibition of aldehyde dehydrogenase, suggesting the involvement of acetaldehyde in this dysfunction.
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Clemens DL, Schneider KJM, Nuss RF. Ethanol metabolism activates cell cycle checkpoint kinase, Chk2. Alcohol 2011; 45:785-93. [PMID: 21924579 DOI: 10.1016/j.alcohol.2011.07.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2010] [Revised: 07/15/2011] [Accepted: 07/16/2011] [Indexed: 01/15/2023]
Abstract
Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of the regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrated that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury.
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Affiliation(s)
- Dahn L Clemens
- Research Service, Veterans Administration Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA.
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35
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Guan X, Rubin E, Anni H. An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography. Alcohol Clin Exp Res 2011; 36:398-405. [PMID: 21895715 DOI: 10.1111/j.1530-0277.2011.01612.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). METHODS We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH-DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. RESULTS Derivatization of acetaldehyde was performed at pH 4.0 with an 80-fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-minute chromatography program. Acetaldehyde detection was linear up to 80 μM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 μM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 μl) plasma sampling. CONCLUSIONS An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small-volume sampling of culture media and biological fluids.
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Affiliation(s)
- Xiangying Guan
- Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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Howarth DL, Vacaru AM, Tsedensodnom O, Mormone E, Nieto N, Costantini LM, Snapp EL, Sadler KC. Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes. Alcohol Clin Exp Res 2011; 36:14-23. [PMID: 21790674 DOI: 10.1111/j.1530-0277.2011.01602.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. METHODS HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). RESULTS VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. CONCLUSIONS Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.
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Affiliation(s)
- Deanna L Howarth
- Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA
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Choudhury M, Pandey RS, Clemens DL, Davis JW, Lim RW, Shukla SD. Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells. Alcohol 2011; 45:311-24. [PMID: 21367571 DOI: 10.1016/j.alcohol.2010.12.003] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2010] [Revised: 09/09/2010] [Accepted: 12/06/2010] [Indexed: 01/23/2023]
Abstract
We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2.
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Affiliation(s)
- Mahua Choudhury
- Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65212, USA
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38
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Abstract
Synthetic quorum-sensing systems in mammalian cells has enabled the implementation of time- and distance-dependent bioprocesses, as well as the design of synthetic ecosystems emulating clinically important host-parasite interactions. In this chapter, we provide a detailed protocol of the design of a mammalian cell-to-cell signaling device and its integration into a mammalian quorum-sensing system for cell density-induced expression product genes. Cell-to-cell signaling is based on a sender cell, metabolically engineered for expression of alcohol dehydrogenase converting ethanol into acetaldehyde, and a receiver cell line for the dose-dependent translation of the acetaldehyde concentration into transgene expression by an acetaldehyde-responsive promoter. This protocol can be adapted easily to various cell types and transgenes for the design of versatile mammalian cell-based quorum-sensing systems.
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Affiliation(s)
- Wilfried Weber
- Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland
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Zhong S, Machida K, Tsukamoto H, Johnson DL. Alcohol induces RNA polymerase III-dependent transcription through c-Jun by co-regulating TATA-binding protein (TBP) and Brf1 expression. J Biol Chem 2010; 286:2393-401. [PMID: 21106530 DOI: 10.1074/jbc.m110.192955] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.
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Affiliation(s)
- Shuping Zhong
- Department of Biochemistry and Molecular Biology and the Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033, USA.
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40
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Seronello S, Ito C, Wakita T, Choi J. Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+. J Biol Chem 2009; 285:845-54. [PMID: 19910460 DOI: 10.1074/jbc.m109.045740] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. CONCLUSION Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.
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Affiliation(s)
- Scott Seronello
- School of Natural Sciences, University of California, Merced, Atwater, California 95343, USA
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41
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Valdés-Arzate A, Luna A, Bucio L, Licona C, Clemens DL, Souza V, Hernandez E, Kershenobich D, Gutiérrez-Ruiz MC, Gómez-Quiroz LE. Hepatocyte growth factor protects hepatocytes against oxidative injury induced by ethanol metabolism. Free Radic Biol Med 2009; 47:424-30. [PMID: 19463946 DOI: 10.1016/j.freeradbiomed.2009.05.014] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2009] [Revised: 05/07/2009] [Accepted: 05/13/2009] [Indexed: 11/22/2022]
Abstract
Hepatocyte growth factor (HGF) is involved in many cellular responses, such as mitogenesis and apoptosis protection; however, its effect against oxidative injury induced by ethanol metabolism is not well understood. The aim of this work was to address the mechanism of HGF-induced protection against ethanol-generated oxidative stress damage in the human cell line VL-17A (cytochrome P450 2E1/alcohol dehydrogenase-transfected HepG2 cells). Cells were pretreated with 50 ng/ml HGF for 12 h and then treated with 100 mM ethanol for 0-48 h. Some parameters of oxidative damage were evaluated. We found that ethanol induced peroxide formation (3.3-fold) and oxidative damage as judged by lipid peroxidation (5.4-fold). Damage was prevented by HGF. To address the mechanisms of HGF-induced protection we investigated the cellular antioxidant system. We found that HGF increased the GSH/GSSG ratio, as well as SOD1, catalase, and gamma-glutamylcysteine synthetase expression. To explore the signaling pathways involved in this process, VL-17A cells were pretreated with inhibitors against PI3K, Akt, and NF-kappaB. We found that all treatments decreased the expression of the antioxidant enzymes, thus abrogating the HGF-induced protection against oxidative stress. Our results demonstrate that HGF protects cells from the oxidative damage induced by ethanol metabolism by a mechanism driven by NF-kappaB and PI3K/Akt signaling.
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Affiliation(s)
- Argelia Valdés-Arzate
- Departamento de Ciencias de la Salud, DCBS, Universidad Autónoma Metropolitana-Iztapalapa, 09340 México, DF, México
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42
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Osna NA, White RL, Thiele GM, Donohue TM. Ethanol metabolism alters major histocompatibility complex class I-restricted antigen presentation in liver cells. Hepatology 2009; 49:1308-1315. [PMID: 19195028 PMCID: PMC2670776 DOI: 10.1002/hep.22787] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
UNLABELLED The proteasome is a major enzyme that cleaves proteins for antigen presentation. Cleaved peptides traffic to the cell surface, where they are presented in the context of major histocompatibility complex (MHC) class I. Recognition of these complexes by cytotoxic T lymphocytes is crucial for elimination of cells bearing "nonself" proteins. Our previous studies revealed that ethanol suppresses proteasome function in ethanol-metabolizing liver cells. We hypothesized that proteasome suppression reduces the hydrolysis of antigenic peptides, thereby decreasing the presentation of the peptide MHC class I complexes on the cell surface. To test this we used the mouse hepatocyte cell line (CYP2E1/ADH-transfected HepB5 cells) or primary mouse hepatocytes, both derived from livers of C57Bl/6 mice, which present the ovalbumin peptide, SIINFEKL, complexed with H2Kb. To induce H2Kb expression, HepB5 cells were treated with interferon gamma (IFNgamma) and then exposed to ethanol. In these cells, ethanol metabolism decreased not only proteasome activity, but also hydrolysis of the C-extended peptide, SIINFEKL-TE, and the presentation of SIINFEKL-H2Kb complexes measured after the delivery of SIINFEKL-TE to cytoplasm. The suppressive effects of ethanol were, in part, attributed to ethanol-elicited impairment of IFNgamma signaling. However, in primary hepatocytes, even in the absence of IFNgamma, we observed a similar decline in proteasome activity and antigen presentation after ethanol exposure. CONCLUSION Proteasome function is directly suppressed by ethanol metabolism and indirectly by preventing the activating effects of IFNgamma. Ethanol-elicited reduction in proteasome activity contributes to the suppression of SIINFEKL-H2Kb presentation on the surface of liver cells.
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Affiliation(s)
- Natalia A Osna
- Liver Study Unit, Omaha Veterans Affairs (VA) Medical Center, Omaha, NE 68105, USA
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Klassen LW, Thiele GM, Duryee MJ, Schaffert CS, DeVeney AL, Hunter CD, Olinga P, Tuma DJ. An in vitro method of alcoholic liver injury using precision-cut liver slices from rats. Biochem Pharmacol 2008; 76:426-36. [PMID: 18599023 DOI: 10.1016/j.bcp.2008.05.012] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2008] [Revised: 05/07/2008] [Accepted: 05/08/2008] [Indexed: 12/24/2022]
Abstract
Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.
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Affiliation(s)
- Lynell W Klassen
- Experimental Immunology Laboratory, University of Nebraska Medical Center, Department of Internal Medicine, Section of Rheumatology, 983025 Nebraska Medical Center, Omaha, NE 68198-3025, USA
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44
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Szuster-Ciesielska A, Plewka K, Daniluk J, Kandefer-Szerszeń M. Zinc inhibits ethanol-induced HepG2 cell apoptosis. Toxicol Appl Pharmacol 2007; 229:1-9. [PMID: 18396304 DOI: 10.1016/j.taap.2007.11.019] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2007] [Revised: 10/29/2007] [Accepted: 11/18/2007] [Indexed: 01/21/2023]
Abstract
Alcohol consumption produces a variety of metabolic alterations in liver cells, associated with ethanol oxidation and with nonoxidative metabolism of ethanol, among others apoptosis of hepatocytes. As zinc is known as a potent antioxidant and an inhibitor of cell apoptosis, the aim of this paper was to investigate whether zinc supplementation could inhibit ethanol-induced HepG2 apoptosis, and whether this inhibition was connected with attenuation of oxidative stress and modulation of FasR/FasL system expression. The results indicated that zinc supplementation significantly inhibited ethanol-induced HepG2 cell apoptosis (measured by cytochrome c release from mitochondria and caspase-3 activation) by attenuation of reactive oxygen species (ROS) production, increase in the cellular level of GSH, inhibition of ethanol-induced sFasR and FasL overexpression and caspase-8 activation. These results indicate that zinc can inhibit ethanol-induced hepatocyte apoptosis by several independent mechanisms, among others by an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-9 activation.
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45
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Abstract
Ethanol is a hepatotoxin. It appears that the liver is the target of ethanol induced toxicity primarily because it is the major site of ethanol metabolism. Metabolism of ethanol results in a number of biochemical changes that are thought to mediate the toxicity associated with ethanol abuse. These include the production of acetaldehyde and reactive oxygen species, as well as an accumulation of nicotinamide adenine dinucleotide (NADH). These biochemical changes are associated with the accumulation of fat and mitochondrial dysfunction in the liver. If these changes are severe enough they can themselves cause hepatotoxicity, or they can sensitize the liver to more severe damage by other hepatotoxins. Whether liver damage is the result of ethanol metabolism or some other hepatotoxin, recovery of the liver from damage requires replacement of cells that have been destroyed. It is now apparent that ethanol metabolism not only causes hepatotoxicity but also impairs the replication of normal hepatocytes. This impairment has been shown to occur at both the G1/S, and the G2/M transitions of the cell cycle. These impairments may be the result of activation of the checkpoint kinases, which can mediate cell cycle arrest at both of these transitions. Conversely, because ethanol metabolism results in a number of biochemical changes, there may be a number of mechanisms by which ethanol metabolism impairs cellular replication. It is the goal of this article to review the mechanisms by which ethanol metabolism mediates impairment of hepatic replication.
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Affiliation(s)
- Dahn L Clemens
- Department of Internal Medicine, University of Nebraska Medical Center and Veterans Affairs Medical Center, Omaha Nebraska 68105, USA.
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46
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Weber W, Daoud-El Baba M, Fussenegger M. Synthetic ecosystems based on airborne inter- and intrakingdom communication. Proc Natl Acad Sci U S A 2007; 104:10435-40. [PMID: 17551014 PMCID: PMC1886003 DOI: 10.1073/pnas.0701382104] [Citation(s) in RCA: 146] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Intercellular communication within an organism, between populations, or across species and kingdoms forms the basis of many ecosystems in which organisms coexist through symbiotic, parasitic, or predator-prey relationships. Using multistep airborne communication and signal transduction, we present synthetic ecosystems within a mammalian cell population, in mice, or across species and kingdoms. Inter- and intrakingdom communication was enabled by using sender cells that produce volatile aldehydes, small vitamin-derived molecules, or antibiotics that diffuse, by gas or liquid phase, to receiver cells and induce the expression of specific target genes. Intercellular and cross-kingdom communication was shown to enable quorum sensing between and among mammalian cells, bacteria, yeast, and plants, resulting in precise spatiotemporal control of IFN-beta production. Interconnection of bacterial, yeast, and mammalian cell signaling enabled the construction of multistep signal transduction and processing networks as well as the design of synthetic ecosystems that mimic fundamental coexistence patterns in nature, including symbiosis, parasitism, and oscillating predator-prey interactions.
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Affiliation(s)
- Wilfried Weber
- *Institute for Chemical and Bioengineering, Eidgenössiche Technische Hochschule, HCI F 115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland; and
| | - Marie Daoud-El Baba
- Institut Universitaire de Technologie, Département Génie Biologique, F-69622 Villeurbanne Cedex, France
| | - Martin Fussenegger
- *Institute for Chemical and Bioengineering, Eidgenössiche Technische Hochschule, HCI F 115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland; and
- To whom correspondence should be addressed. E-mail:
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47
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Wu H, Cai P, Clemens DL, Jerrells TR, Ansari GAS, Kaphalia BS. Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism. Toxicol Appl Pharmacol 2006; 216:238-47. [PMID: 16806343 DOI: 10.1016/j.taap.2006.05.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2006] [Revised: 04/08/2006] [Accepted: 05/05/2006] [Indexed: 12/22/2022]
Abstract
Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.
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Affiliation(s)
- Hai Wu
- University of Texas Medical Branch, Department of Pathology, 3 118A Keiller Building, Galveston, TX 77555, USA
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Lakshman R, Cederbaum AI, Hoek JB, Konishi M, Koop D, Donohu TM. Use of CYP2E1-Transfected Human Liver Cell Lines in Elucidating the Actions of Ethanol. Alcohol Clin Exp Res 2006; 29:1726-34. [PMID: 16205373 DOI: 10.1097/01.alc.0000179379.03078.8f] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
This article represents the proceedings of a symposium at the 2004 RSA Meeting held in Vancouver, Canada. The chairs were Arthur I. Cederbaum and Raj Lakshman. The presentations were (1) ethanol regulates 2,6-sialyltransferase (2,6-ST) gene expression posttranscriptionally by the interaction of a cytosolic binding protein with 2,6-ST mRNA in CYP2E1- and ADH-transfected HepG2 cells, by Raj Lakshman; (2) nature versus nurture: HepG2-E47 cells as a tool to investigate mechanisms of ethanol-mediated potentiation of cell killing, by Jan B. Hoek; (3) ethanol up-regulates profibrogenic connective tissue growth factor gene expression in HepG2 cells via cytochrome P-450 2E1-mediated ethanol oxidation, by Masahiro Konishi; (4) role of calcium and calcium-activated enzymes in CYP2E1-dependent toxicity, by Arthur I Cederbaum; (5) the use of cell lines to characterize the role of CYP2E1 in the metabolism of farnesol, by Dennis Koop; and (6) studies with HepG2 cells that express the two major ethanol-metabolizing enzymes, by Terrence M. Donohue.
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Affiliation(s)
- Raj Lakshman
- Lipid Research Laboratory, VA Medical Center, and the Department of Biochemistry, Molecular Biology, and Medicine, George Washington University, Washington, DC 20422, USA.
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49
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Abstract
A causal association has been established between alcohol consumption and cancers of the oral cavity, pharynx, larynx, oesophagus, liver, colon, rectum, and, in women, breast; an association is suspected for cancers of the pancreas and lung. Evidence suggests that the effect of alcohol is modulated by polymorphisms in genes encoding enzymes for ethanol metabolism (eg, alcohol dehydrogenases, aldehyde dehydrogenases, and cytochrome P450 2E1), folate metabolism, and DNA repair. The mechanisms by which alcohol consumption exerts its carcinogenic effect have not been defined fully, although plausible events include: a genotoxic effect of acetaldehyde, the main metabolite of ethanol; increased oestrogen concentration, which is important for breast carcinogenesis; a role as solvent for tobacco carcinogens; production of reactive oxygen species and nitrogen species; and changes in folate metabolism. Alcohol consumption is increasing in many countries and is an important cause of cancer worldwide.
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Affiliation(s)
- Paolo Boffetta
- International Agency for Research on Cancer, Lyon, France.
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50
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Donohue TM, Osna NA, Clemens DL. Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity. Int J Biochem Cell Biol 2006; 38:92-101. [PMID: 16181800 DOI: 10.1016/j.biocel.2005.07.010] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2005] [Accepted: 07/27/2005] [Indexed: 02/08/2023]
Abstract
HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.
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