Jayaweera DT, Campa A, Casillas VJ, Martinez SS, Shin DH, Li Y, Young S, Baum MK

Background  Patients co-infected with HIV/HCV compared to those infected with HCV alone have: Higher levels of HCV-RNA. An accelerated progression to liver disease. More rapid development of liver fibrosis and cirrhosis. Greater morbidity and mortality Kim A, et al., Rotman Y et al., Pascual-Pareja J et al., Sulkowski MS Monga HK et al., Maciás et al., 2009.

Background  HCV infection characterized by oxidative stress generated by chronic inflammation. 7,8  Limited data available on HIV/HCV co-infection.  HIV and HCV independently regulate hepatic fibrosis progression through generation of reactive oxygen species (ROS). 9  HCV core protein localizes to mitochondria and its expression causes inhibition of electron transport, ROS production & glutathione Yadav D et al, Choi J & Ou J, Lin W et al, Korenaga M. et al., 2005

Background  As HCV disease progresses: Lipid peroxidation products are and glutathione is in serum, PBMC & liver specimens. 11,12 Antioxidant micronutrients are depleted in serum and liver biopsy specimens. 8 Decreased liver micronutrient antioxidant levels are associated with increased liver fibrosis Baum MK et al., Mahmood S et al 2004.

Background  Oxidative stress is determined by levels of malondialdehyde (MDA), glutathione, 17,18 & the extent of mtDNA damage. 22  MDA, an index of lipid peroxidation, is in liver, serum, erythrocytes and PBMC in liver diseases, 19 correlates with HCV disease activity 18 & fibrosis staging. 17,20  MDA is inversely correlated with hepatic and plasma reduced glutathione De Maria N et al., Ko WS et al., Malvy DJM, et al Serejo F, et al., Barbaro G, et al., Fromenty B et al., 1997.

Background  Glutathione is the most abundant non- enzymatic antioxidant in cells. 22  Reduced glutathione (GSH) determines the extent of oxidative capacity.  Oxidized glutathione (GSSG) with severity of HCV, indicating increased oxidative stress & decreased antioxidative capacity Akerboom TP et al., Wu G et al., 2004.

Background  Oxidative damage of mtDNA leads to base modification, misreading during replication, and point mutations. 24  The most common form of mtDNA oxidative damage, is increase in the amount of mt- specific 8-oxo-dG. 25  Mt-specific 8-oxo-dG strongly correlates with markers of systemic oxidative stress & is a risk factor for hepatocellular carcinoma Fromenty B et al., Dagon T et al., Gerschenson M et al., Aukurst P et al., Wu G et al., Barbaro G et al., Chuma M et al, 2008.

Preliminary Study  HIV/HCV-co-infected compared to HIV- mono-infected participants had: Higher plasma MDA (1.897±0.835 vs ±0.223 nmol/mL, p=0.006). Lower plasma antioxidant concentrations: ○ Vitamin A (39.5±14.1 vs. 52.4±16.2 mg/dL, p=0.0004). ○ Vitamin E (8.29±2.1 vs. 9.89±4.5 mg/mL, p=0.043). ○ Zinc (0.61±0.14 vs. 0.67±0.15mg/L, p=0.016) Baum MK et al., 2011

Preliminary Study  Significantly increased glutathione peroxidase concentration as liver disease advanced, as measured by APRI (β= ; p=0.0082) and FIB-4 (β=0.0029; p=0.0177).  Vitamin A concentration significantly decreased (β= , p=0.0417) as APRI increased Baum MK et al., 2011

Objective  To assess the association between oxidative stress and liver fibrosis in HIV/HCV co-infected adults.  To compare markers of oxidative stress between HIV/HCV co-infected and HIV mono-infected adults.

Methods  Study Design: Cross-sectional  Cohorts: -35 HIV/HCV co-infected patients who had recently undergone a liver biopsy, & -101 HIV mono-infected patients  Exclusion Criteria HBV disease End stage liver disease Pregnancy Chronic Inflammatory Diseases Morbid obesity <40 years old >60 years old Uncontrolled viral load

Methods  Blood was collected for measures of oxidative stress, HIV and HCV disease progression: ○ Mitochondrial specific 8-hydroxy- deoxyguanosine (mt-specific 8-oxo-dG) ○ Malondialdehyde (MDA) ○ Reduced, oxidized and total glutathione ○ CD4 cell count and HIV viral load ○ HCV genotype and HCV viral load

Methods  Liver fibrosis and inflammation were assessed with liver biopsy and scored using the Metavir Liver Fibrosis Scale.  Liver fibrosis (0 to 4) 0 = no fibrosis 1 = portal fibrosis without septa 2 = few septa 3 = numerous septa without cirrhosis 4 = cirrhosis 31  Liver activity A0 = no histological activity A1 = mild activity A2 = moderate activity A3 = severe activity The French METAVIR Cooperative Study Group Bedossa P, et al., 1996.

Results Table1: Characteristics of the Population CharacteristicN=136 Age, (years±SD)48.58±5.64 Gender % (N) Male Female (83) (53) Race/Ethnicity % (N) White Non-Hispanic White Hispanic Black Non-Hispanic Black Hispanic American Indian Other 3.36 (4) (28) (74) 5.04 (6) 1.68 (2) 4.28 (5) BMI (kg/m 2 ±SD)27.88±5.48 CD4 Cell Count (cells/µL±SD)532.59± HIV Viral Load (copies/mL±SD2,722.97±11, HCV Viral Load (copies /mL±SD (HIV/HCV co-infected, N=35) 9,286,462.35±12,057, % genotype 1a 59.65% genotype 1b

Results  Table 2: Frequencies of Liver Fibrosis and Activity Scores Liver Fibrosis Score%(N) Liver Activity Score%(N) A0 A1 A2 A

Results (N=136) VariableHIV Mono-Infected Mean±SD HIV/HCV Co-Infected Mean±SD p-Value MDA (µM)0.586± ± oxo-dG (∆Ct)0.743± ± * Reduced Glutathione Concentration (µM) ± ± Reduced Glutathione/Total Glutathione Ratio (%) 77.59± ± Oxidized Glutathione Concentration (µM) ± ± Oxidized Glutathione/Total Glutathione Ratio (%) 22.45± ± Table 3: Difference In Mean Measures Of Oxidative Stress Between HIV Mono-Infected And HV/HCV Co-Infected Participants *Statistically significant, p<0.05

Results Low Fibrosis scores were significantly associated with lower levels of 8-oxo-dG (β=0.391, p=0.05,) than high fibrosis scores in HIV/HCV Co-Infection Fibrosis 8-oxo-dG (∆Ct) p=0.05 β=0.391 The grade of inflammation was positively and strongly associated with oxidized (GSSG) glutathione (β=0.0034, p=0.002).

Results  Using linear regression, HCV viral load was positively associated with 8-oxo-dG (β=1.18, p=0.042) after controlling for age, race, and BMI. Relationship between HCV Viral Load and 8-oxo-dG 8-oxo-dG (∆Ct)

Results  Using linear regression, HCV viral load was positively associated with MDA (β=3.97, p=0.049) after controlling for BMI. Relationship between HCV Viral Load and MDA

Discussion/Conclusions  HIV/HCV co-infection in our study was associated with pro-oxidative environment; higher oxidative stress correlated with higher fibrosis & inflammation scores, and higher HCV viral load.  Oxidative stress induces proliferation of hepatic stellate cells, TGF-β and collagen synthesis. 9, 31, 33  Oxidative stress participates in the development of progression to fibrosis in patients with Hepatitis C. 31, 32, Leonardo A, et al, Moriya K, et al, Choi J & Ou JH, 2006

Discussion/ Conclusions  Evidence suggests that antioxidants effectively attenuate the oxidative liver injury, & improve inflammation & fibrosis progression in HCV: Normalization of liver enzymes, histological improvement & reduction of HCV viral load was observed in a significant proportion of patients with chronic HCV using a combination antioxidant therapy. 34,35 However, antioxidant resveratrol enhanced the HCV replication in vitro. 36  Strategies to limit HIV/HCV induction of oxidative stress are warranted to slow hepatic fibrosis. Antioxidants need to be tested for their effects on HCV replication. 34. Melhem A, et al, Gabbay E, et al., Nakamura M, et al, 2010

Collaborators  Florida International University Marianna K Baum, PhD Adriana Campa, PhD Dong-Ho Shin, PhD Sabrina Sales Martinez, MS Yinghui Li, MS  University of Miami Dushyantha T Jayaweera, MD Victor J Casillas, MD Funded by NIDA grant # R01DA & D-CFAR, University of Miami The project was supported by Award Number R01DA from the National Institute On Drug Abuse. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Drug Abuse or the National Institutes of Health.