Nevirapine induced mitochondrial dysfunction in HepG2 cells

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor frequently used in combination with other antiretroviral agents for highly active antiretroviral therapy (HAART) of patients infected with the human immunodeficiency virus type 1 (HIV-1). However NVP can cause serious, life-threatening complications. Hepatotoxicity is one of the most severe adverse effects, particularly in HIV patients with chronic hepatitis C virus co-infection as these patients can develop liver toxicity after a relatively short course of treatment. However, the mechanism of NVP-associated hepatotoxicity remains unclear. This study sought to investigate the effect of NVP on protein expression in liver cells using a proteomic approach. HepG2 cells were treated or not treated with NVP and proteins were subsequently resolved by two-dimensional gel electrophoresis. A total of 33 differentially regulated proteins were identified, of which nearly 40% (13/33) were mitochondrial proteins. While no obvious differences were observed between NVP treated and untreated cells after staining mitochondria with mitotracker, RT-PCR expression analysis of three mitochondrially encoded genes showed all were significantly up-regulated in NVP treated cells. Mitochondrial dysfunction was observed in response to treatment even with slightly sub-optimal therapeutic treatment concentrations of NVP. This study shows that NVP induces mitochondrial dysregulation in HepG2 cells.

[1]  I. Harris,et al.  A Systematic Review and Meta-Analysis of Randomized Controlled Trials , 2010 .

[2]  Joseph D. Bauman,et al.  HIV-1 reverse transcriptase complex with DNA and nevirapine reveals nonnucleoside inhibition mechanism , 2012, Nature Structural &Molecular Biology.

[3]  S. Fucharoen,et al.  Mitochondrial Changes in β0-Thalassemia/Hb E Disease , 2016, PloS one.

[4]  M. Forte,et al.  VDAC Channels , 2001, IUBMB life.

[5]  R. Pollard,et al.  Safety profile of nevirapine, a nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus infection. , 1998, Clinical therapeutics.

[6]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[7]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[8]  Wei-Shau Hu,et al.  HIV-1 reverse transcription. , 2012, Cold Spring Harbor perspectives in medicine.

[9]  V. Shoshan-Barmatz,et al.  Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. , 2001, The Biochemical journal.

[10]  D. Stuart,et al.  Structure of HIV-2 reverse transcriptase at 2.35-Å resolution and the mechanism of resistance to non-nucleoside inhibitors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Uetrecht,et al.  Demonstration of the metabolic pathway responsible for nevirapine-induced skin rash. , 2008, Chemical research in toxicology.

[12]  Jennifer R Stevens,et al.  Mother to child transmission of HIV: what works and how much is enough? , 2014, Journal of Infection.

[13]  Alfons Lawen,et al.  Voltage‐dependent anion‐selective channel (VDAC) in the plasma membrane , 2010, FEBS letters.

[14]  E. White,et al.  Comparison of the effect of Carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human polymerases. , 1989, Biochemical and biophysical research communications.

[15]  K. Tilmant,et al.  Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins , 2012, Cell Biology and Toxicology.

[16]  U. Walker,et al.  Mitochondrial Toxicity of Nucleoside Analogues in Primary Human Lymphocytes , 2005, Antiviral therapy.

[17]  Edward J. Mills,et al.  Adverse events associated with nevirapine and efavirenz-based first-line antiretroviral therapy: a systematic review and meta-analysis , 2013, AIDS.

[18]  J. Uetrecht,et al.  Bioactivation of Nevirapine to a Reactive Quinone Methide: Implications for Liver Injury , 2012, Chemical research in toxicology.

[19]  E. Moran,et al.  Antiviral drug allergy. , 2014, Immunology and allergy clinics of North America.

[20]  F. Beland,et al.  Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine. , 2013, The Journal of toxicological sciences.

[21]  J. Nunnari,et al.  Mitochondrial form and function , 2014, Nature.

[22]  S. Fucharoen,et al.  Increased oxidative metabolism is associated with erythroid precursor expansion in β0-thalassaemia/Hb E disease. , 2011, Blood cells, molecules & diseases.

[23]  J. Adams,et al.  Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor. , 1990, Science.

[24]  Brad T. Sherman,et al.  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists , 2008, Nucleic acids research.

[25]  Hiroaki Kitano,et al.  The PANTHER database of protein families, subfamilies, functions and pathways , 2004, Nucleic Acids Res..

[26]  M. Colombini,et al.  ATP Flux Is Controlled by a Voltage-gated Channel from the Mitochondrial Outer Membrane* , 1996, The Journal of Biological Chemistry.

[27]  M. Delgado-Rodríguez,et al.  Systematic review and meta-analysis. , 2017, Medicina intensiva.

[28]  P. Chinnery,et al.  HIV Treatment and Associated Mitochondrial Pathology , 2014, Toxicologic pathology.

[29]  C. Thepparit,et al.  Nevirapine induces apoptosis in liver (HepG2) cells. , 2016, Asian Pacific journal of tropical medicine.

[30]  N. Pfanner,et al.  The Mitochondrial Proteome: From Inventory to Function , 2008, Cell.

[31]  Davide Heller,et al.  STRING v10: protein–protein interaction networks, integrated over the tree of life , 2014, Nucleic Acids Res..

[32]  S. Fucharoen,et al.  Proteomic analysis of hemoglobin H-constant spring (Hb H-CS) erythroblasts. , 2012, Blood cells, molecules & diseases.

[33]  E. Kohli,et al.  Usefulness of Therapeutic Drug Monitoring of Antiretrovirals in Routine Clinical Practice , 2004, HIV clinical trials.

[34]  A. Weissbach,et al.  DNA polymerase of mitochondria is a gamma-polymerase. , 1977, The Journal of biological chemistry.

[35]  G. Rutherford,et al.  Efavirenz or nevirapine in three‐drug combination therapy with two nucleoside or nucleotide‐reverse transcriptase inhibitors for initial treatment of HIV infection in antiretroviral‐naïve individuals , 2016, The Cochrane database of systematic reviews.

[36]  A. Chuturgoon,et al.  Lymphocyte Mitochondrial Depolarization and Apoptosis in HIV-1-Infected HAART Patients , 2008, Journal of acquired immune deficiency syndromes.

[37]  J. Esplugues,et al.  Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells , 2010, Hepatology.

[38]  Ò. Miró,et al.  Mitochondrial Dna Depletion and Respiratory Chain Enzyme Deficiencies are Present in Peripheral Blood Mononuclear Cells of HIV-Infected Patients with Haart-Related Lipodystrophy , 2002, Antiviral therapy.

[39]  Yuan Chen,et al.  Metabolic Activation of Nevirapine in Human Liver Microsomes: Dehydrogenation and Inactivation of Cytochrome P450 3A4 , 2009, Drug Metabolism and Disposition.

[40]  V. De Pinto,et al.  VDAC, a multi-functional mitochondrial protein regulating cell life and death. , 2010, Molecular aspects of medicine.

[41]  V. Shoshan-Barmatz,et al.  Calcium Binding and Translocation by VDAC: a possible regulatory mechanism in mitochondrial function , 2000 .

[42]  J. L. Martin,et al.  Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis , 1994, Antimicrobial Agents and Chemotherapy.

[43]  P. Kawalec,et al.  Nevirapine-Based Regimens in HIV-Infected Antiretroviral-Naive Patients: Systematic Review and Meta-Analysis of Randomized Controlled Trials , 2013, PloS one.

[44]  A. Badley,et al.  Induction of Apoptosis by a Nonnucleoside Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor , 2002, Antimicrobial Agents and Chemotherapy.

[45]  N. Vermeulen,et al.  Different Reactive Metabolites of Nevirapine Require Distinct Glutathione S-Transferase Isoforms for Bioinactivation. , 2016, Chemical Research in Toxicology.

[46]  M. Campbell,et al.  PANTHER: a library of protein families and subfamilies indexed by function. , 2003, Genome research.

[47]  R H Levy,et al.  Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[48]  A. Messina,et al.  Characterization of human VDAC isoforms: a peculiar function for VDAC3? , 2010, Biochimica et biophysica acta.

[49]  Leming Shi,et al.  Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human Hepatocytes , 2011, Drug Metabolism And Disposition.

[50]  M. Wainberg,et al.  Resistance to reverse transcriptase inhibitors used in the treatment and prevention of HIV-1 infection. , 2015, Future microbiology.