LIVER FIBROSIS IS ASSOCIATED WITH

INCREASED OXIDATIVE STRESS IN HIV/HCV

CO-INFECTED ADULTS

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.1-6

1. Kim A, et al., 2009. 2. Rotman Y et al., 2009. 3. Pascual-Pareja J et al., 20094. Sulkowski MS. 2003. 5. Monga HK et al., 2001. 6. 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.10

7. Yadav D et al, 2002 8. Choi J & Ou J, 2006 9. Lin W et al,. 201110. 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.8

11. Baum MK et al., 2011 12. 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 activity18 & fibrosis staging.17,20

MDA is inversely correlated with hepatic and plasma reduced glutathione.21

17. De Maria N et al.,1996. 18. Ko WS et al., 2005. 19. Malvy DJM, et al. 1994 .20. Serejo F, et al., 2003. 21. Barbaro G, et al., 1999. 22. 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.23

22. Akerboom TP et al., 1981. 23. 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 stress26-29 & is a risk factor for hepatocellular carcinoma.30

24. Fromenty B et al., 1997. 25. Dagon T et al., 2002. 26. Gerschenson M et al., 2009. 27. Aukurst P et al., 1999. 28. Wu G et al., 2004. 29. Barbaro G et al., 1999. 30. 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. 1.344±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).30

30. Baum MK et al., 2011

Preliminary Study

Significantly increased glutathione peroxidase concentration as liver disease advanced, as measured by APRI (β=50.00118; p=0.0082) and FIB-4 (β=0.0029; p=0.0177).

Vitamin A concentration significantly decreased (β=0.00581, p=0.0417) as APRI increased.30

30. 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 CriteriaHBV diseaseEnd stage liver diseasePregnancy Chronic Inflammatory DiseasesMorbid obesity <40 years old>60 years oldUncontrolled 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 = cirrhosis31

Liver activity A0 = no histological activity A1 = mild activity A2 = moderate activity A3 = severe activity32

31. The French METAVIR Cooperative Study Group. 1994.32. Bedossa P, et al., 1996.

ResultsTable1: Characteristics of the Population

Characteristic N=136

Age, (years±SD) 48.58±5.64

Gender % (N) Male Female

61.03 (83)38.97 (53)

Race/Ethnicity % (N) White Non-Hispanic White Hispanic Black Non-Hispanic Black Hispanic American Indian Other

3.36 (4)23.53 (28)62.18 (74)

5.04 (6)1.68 (2)4.28 (5)

BMI (kg/m2±SD) 27.88±5.48

CD4 Cell Count (cells/µL±SD) 532.59±329.97

HIV Viral Load (copies/mL±SD 2,722.97±11,853.44

HCV Viral Load (copies /mL±SD(HIV/HCV co-infected, N=35)

9,286,462.35±12,057,910.8440.35% genotype 1a59.65% genotype 1b

Results

Table 2: Frequencies of Liver Fibrosis and Activity Scores

Liver Fibrosis Score %(N)

01234

3.5039.29 32.14 21.43 3.57

Liver Activity Score %(N)

A0A1A2A3

3.57 21.4350.0025.00

Results (N=136)

Variable HIV Mono-InfectedMean±SD

HIV/HCV Co-InfectedMean±SD

p-Value

MDA (µM) 0.586±0.019 0.587±0.173 0.978

8-oxo-dG (∆Ct) 0.743±0.374 1.072±0.524 0.005*

Reduced Glutathione Concentration (µM)

758.40±177.30 711.00±208.40 0.279

Reduced Glutathione/Total Glutathione Ratio (%)

77.59±5.98 75.79±7.89 0.238

Oxidized Glutathione Concentration (µM)

215.80±65.65 217.50±76.00 0.917

Oxidized Glutathione/Total Glutathione Ratio (%)

22.45±6.09 24.21±7.89 0.2558

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-o

xo-d

G (

∆C

t) p=0.05β=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, 33

31. Leonardo A, et al, 2004 32. Moriya K, et al, 2001 33. 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, 2005. 35. Gabbay E, et al., 200736. 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 # R01DA023405 & D-CFAR, University of Miami

The project was supported by Award Number R01DA023405 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.