Liver Fibrosis is Associated with Increased Oxidative Stress

Report
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.
4. Sulkowski MS. 2003.
2. Rotman Y et al., 2009.
5. Monga HK et al., 2001.
3. Pascual-Pareja J et al., 2009
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
10. Korenaga M. et al., 2005
8. Choi J & Ou J, 2006
9. Lin W et al,. 2011
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.
20. Serejo F, et al., 2003.
18. Ko WS et al., 2005.
21. Barbaro G, et al., 1999.
19. Malvy DJM, et al. 1994 .
22. Fromenty B et al., 1997.
Background

Glutathione is the most abundant nonenzymatic 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 mtspecific 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.
27. Aukurst P et al., 1999.
30. Chuma M et al, 2008.
25. Dagon T et al., 2002.
28. Wu G et al., 2004.
26. Gerschenson M et al., 2009.
29. Barbaro G et al., 1999.
Preliminary Study

HIV/HCV-co-infected compared to HIVmono-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 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 = 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.
Results
Table1: 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±12,057,910.84
40.35% genotype 1a
59.65% genotype 1b
Results

Table 2: Frequencies of Liver Fibrosis
and Activity Scores
Liver Fibrosis Score
0
1
2
3
4
%(N)
3.50
39.29
32.14
21.43
3.57
Liver Activity Score
A0
A1
A2
A3
%(N)
3.57
21.43
50.00
25.00
Results (N=136)
Table 3: Difference In Mean Measures Of Oxidative Stress Between
HIV Mono-Infected And HV/HCV Co-Infected Participants
Variable
HIV Mono-Infected
Mean±SD
HIV/HCV Co-Infected
Mean±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*
758.40±177.30
711.00±208.40
0.279
77.59±5.98
75.79±7.89
0.238
215.80±65.65
217.50±76.00
0.917
22.45±6.09
24.21±7.89
0.2558
Reduced Glutathione
Concentration (µM)
Reduced Glutathione/Total
Glutathione Ratio (%)
Oxidized Glutathione
Concentration (µM)
Oxidized Glutathione/Total
Glutathione Ratio (%)
*Statistically significant, p<0.05
Results
8-oxo-dG (∆Ct)
•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
p=0.05
β=0.391
Fibrosis
• The
grade of inflammation was positively and strongly
associated with oxidized (GSSG) glutathione
(β=0.0034, p=0.002).
Results
Relationship between HCV Viral
Load and 8-oxo-dG
8-oxo-dG (∆Ct)

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.
Results
Relationship between HCV
Viral Load and MDA

Using linear regression,
HCV viral load was
positively associated
with MDA (β=3.97,
p=0.049) after
controlling for BMI.
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.
36. Nakamura M, et al, 2010
35. Gabbay E, et al., 2007
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.

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