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Mitochondrial DNA depletion in liver tissue of patients infected with hepatitis C virus: contributing effect of HIV infection?
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HIV Medicine
March 2005
J Bauerle 1 , M Laguno 2 , S Mauss 3 , J Mallolas 2 , J Murillas 2 , R Miquel 2 , G Schmutz 3 , B Setzer 1 , JM Gatell 2 and UA Walker 1
1Department of Clinical Immunology, Medizinische Universitatsklinik, Freiburg, Germany, 2Hospital Clinic, Barcelona, Spain, and 3Center for HIV and Hepatogastroenterology, DŸsseldorf, Germany
"....The major finding from these cross-sectional mtDNA-measurements in the liver was an association of HCV infection with mtDNA decline, on the one hand, and lack of a marked additional effect of coinfection with HIV, on the other hand. However, we identified a weak trend towards lower mtDNA levels in HIV/HCV-coinfected patients...."
ABSTRACT
Objectives: It has been suggested that chronic hepatitis C virus (HCV) infection depletes mitochondrial DNA (mtDNA) in the liver. Because decreased mtDNA levels were also found in humans infected with HIV, we investigated whether HIV may have aggravated hepatic mtDNA depletion in individuals with HCV infection.
Methods: In this cross-sectional study, liver biopsies were performed in a total of 40 individuals prior to any antiviral therapy. The individuals were recruited from the Hospital Clinic, Barcelona and the HIV Centre, DŸsseldorf. Seventeen patients were negative for HIV and HCV and were biopsied for liver enzyme elevation of unknown cause (controls), 14 individuals had chronic HCV but no HIV infection, and nine subjects were coinfected with both viruses. mtDNA and liver histology were centrally assessed.
Results: The groups did not differ with respect to age, gender, liver function tests and HCV viral load, where applicable. mtDNA levels were decreased by 19% in the HCV-monoinfected group (P=0.03) and by 27% in the HIV/HCV-coinfected subjects (P=0.02) compared to controls. The mtDNA content, however, did not differ between individuals with HCV monoinfection and HCV/HIV coinfection (P=0.75). The degrees of liver fibrosis, inflammatory activity or steatosis did not correlate with mtDNA content.
Liver mtDNA
Although mtDNA levels were not normally distributed in groups A and B, the mean mtDNA/nDNA ratio for group A was set at 100% for easier comparison of the quantitative mtDNA results. Compared to controls, mtDNA levels were reduced significantly in HIV-negative patients with chronic HCV infection (81%; range 44-168%; P=0.03), and in HIV/HCV-coinfected patients (73%; range 51-106%; P=0.02). Importantly, however, mtDNA levels did not differ between HCV-monoinfected and HCV/HIV-coinfected patients (P=0.75).
Of all the HCV-infected individuals studied, those carrying HCV genotype 1 (n=15) had higher liver mtDNA levels (82%; range 44-168%) than those infected with other HCV genotypes (70%; range 45-106%; n=8; P=0.38), although this difference was not statistically significant. Furthermore, no association between mtDNA depletion and HCV genotype 1 could be detected after stratification of HCV-positive patients into HIV-positive (P=0.67) and HIV-negative (P=0.30) subgroups. There was no correlation between mtDNA levels and any histological parameter. mtDNA deletions were not observed.
Conclusions: Liver mtDNA content is reduced in both HCV-monoinfected and HIV/HCV-coinfected patients. Under the limitations of our study, we could demonstrate only a slight trend towards more pronounced mtDNA depletion in HIV/HCV-coinfected subjects.
AUTHOR DISCUSSION
The results of our study may be limited by several factors. It is important to note that the HIV/HCV-coinfected group differed from its HIV-negative counterparts in a lower percentage of HCV genotype 1. Genotype 1 was previously associated with mtDNA depletion [9], and an uneven distribution of this genotype could have obscured an effect of HIV infection. In a separate analysis, however, we did not identify an association between mtDNA depletion and HCV genotype. This could have been due to the small study population and the uneven distribution of genotypes in groups B and C.
Individuals suffering from HIV/HCV coinfection had a significantly longer duration of HCV seropositivity than HCV-monoinfected patients, despite similar levels of hepatic mtDNA, indicating that the time of HCV infection may not be a major factor in the mtDNA decline.
The degree of liver fibrosis was similar in all groups, suggesting that the low mtDNA levels in the HCV-positive groups were not a nonspecific effect of fibrosis. Subjects with HCV monoinfection differed from controls in their mtDNA levels, despite similar inflammatory activity, indicating that nonspecific liver inflammation per se is not a main determinant of mtDNA depletion.
For ethical reasons, we were unable to obtain liver biopsies from healthy subjects. Ten individuals within the control group had alcoholic or nonalcoholic steatohepatitis, two conditions which may also produce low hepatic mtDNA [23,24]. Furthermore, two control subjects suffered from primary biliary cirrhosis, a condition that is associated with circulating antimitochondrial antibodies. This raises the possibility that we might have detected an even greater influence of HCV infection on mtDNA levels, if we had been able to obtain liver tissue from healthy persons without any liver pathology.
Our study is also limited by the fact that we did not have a control group with HIV monoinfection. Therefore, we were not able to demonstrate the mitochondrial toxicity of HIV itself. Additionally, the small size of our study population may explain both the lack of a difference in mtDNA levels between HCV monoinfection and HIV/HCV coinfection and the uneven distribution of mtDNA levels.
Hepatic mtDNA deletions were previously detected using sensitive polymerase chain reaction (PCR) techniques in transgenic mice expressing the HCV core antigen and in humans with HCV-related liver disease [25,26]. We failed to detect any large-scale mtDNA rearrangement by Southern blot, suggesting that these qualitative mtDNA alterations are not present at high levels of heteroplasmy.
In summary, we confirmed low hepatic mtDNA levels attributable to chronic HCV infection, but failed to identify a marked additional effect of HCV genotype or HIV on hepatic mtDNA content. It is possible that the HCV-related mtDNA depletion renders the liver more vulnerable to alcohol [27] or some antiretrovirals [28].
INTRODUCTION
The management of chronic hepatitis C virus (HCV) infection has become a challenge in HIV-infected individuals, as HCV-related complications are a growing cause of mortality in this population [1-3].
HIV-infected persons are usually treated with a combination of antiretrovirals, most of which carry a risk of liver toxicity [3-5]. Most of the side effects of nucleoside reverse transcriptase inhibitors (NRTIs) are related to the fact that at clinically relevant concentrations they inhibit polymerase-gamma, the enzyme responsible for the replication of mitochondrial DNA (mtDNA) [6,7]. Toxic effects of NRTIs on liver mitochondria have been associated with elevated transaminases and cases of steatohepatitis and lactic acidosis [8].
HCV infection (and HCV genotype 1 in particular) has also been found to decrease hepatic mtDNA [9]. Increased formation of reactive oxygen species (ROS) is thought to be an important factor in the HCV-mediated mtDNA decline, although the precise mechanism is not clear [10,11]. Both structural and nonstructural HCV proteins generate ROS in the absence of inflammation, but inflammatory cells may also contribute nonspecifically [12,13]. Consistent with the importance of oxygen radicals, antioxidant systems were found to be reduced in HCV-infected patients [9].
HIV infection is similarly associated with increased oxidative stress, weak antioxidant defenses, mitochondrial dysfunction and mtDNA depletion [14-17], and may therefore aggravate any mitochondrial damage in persons with chronic hepatitis C.
Our aim was to ascertain that HCV decreases mtDNA levels in liver and to investigate whether HIV status has an additive effect on HCV-triggered mtDNA depletion.
Results
Demographics, virology and immunology
Forty individuals participated in the study: 17 patients had neither HCV nor HIV infection and served as controls (group A). These patients suffered from primary biliary cirrhosis (n=2), autoimmune hepatitis (n=1), or alcoholic (n=2) or nonalcoholic steatohepatitis (n=8). In four subjects an exact diagnostic classification of confirmed liver pathology was not possible.
Fourteen patients had chronic HCV infection but no HIV infection (group B), and nine subjects were infected with both HIV and HCV (group C); their mean HIV viral load was 3.2 log HIV-1 RNA copies/mL (range 2.3-4.4 copies/mL) and the mean CD4 count was 609 cells/muL (range 454-800 cells/muL).
The demographic and virological data are presented in Table 1. No statistical differences were found between groups with respect to age, sex, liver values or HCV viral load (where applicable). HCV monoinfected individuals, however, had a shorter time of known HCV infection than their HIV-coinfected counterparts (3.6 vs. 17.9 years, respectively; P=0.002) and a higher percentage of HCV genotype 1 infections (86% vs. 33%, respectively; P=0.006).
Liver histology showed higher (P=0.02) histological degrees of inflammatory activity in individuals positive for both HCV and HIV, compared to controls (group A). No other differences between groups were identified with regard to liver fibrosis, inflammation, or macro- or microvesicular steatosis.
Patients and methods
Human subjects
After ethics committee approval, patients were recruited at the Hospital Clinic, Barcelona, Spain and the HIV Centre, DŸsseldorf, Germany from February 2001 until October 2002. Consecutive patients were enrolled, if they granted informed consent. Patients had to be aged 18 years or older and negative for hepatitis B surface antigen (HbsAg). Pregnant or lactating females were also excluded, as were patients who had a hepatocellular carcinoma, consumed intravenous or inhaled drugs, drank alcohol excessively (>80 g ethanol/day), or suffered from poorly controlled diabetes mellitus, end-stage renal disease or severe respiratory disease.
Group A consisted of individuals who had negative HIV- and HCV-antibody tests, who had been suspected of having diffuse liver pathology as a result of elevated liver enzymes and/or imaging data, and who had had such liver pathology bioptically confirmed.
Group B patients had a chronic HCV infection, confirmed by a positive HCV-antibody enzyme-linked immunosorbent assay (ELISA) and positive HCV RNA measurements (>500 IU/mL). Patients in this group had to meet further inclusion criteria, such as no interferon or ribavirin pretreatment and absence of other liver diseases (autoimmune hepatitis, primary biliary cirrhosis, alpha-1 antitrypsin deficiency, haemochromatosis and Wilson's disease).
Group C subjects were HIV/HCV-coinfected individuals na•ve to antiretroviral treatment who otherwise met all the entry criteria of group B.
Liver histology
Percutaneous liver biopsies were performed using a Mengini (16-gauge) needle. The largest aliquot of each biopsy cylinder (>=20 mm) was embedded in paraffin and centrally assessed by a single pathologist, who was blinded to the clinical and laboratory information. The degrees of liver fibrosis and necroinflammatory activity were scored on haematoxylin-eosin and Masson's trichrome stains according to Scheuer's classification [18]. The percentage of hepatocytes displaying signs of macro- or microvesicular steatosis was also scored.
Quantification of mtDNA
The second aliquot of the liver biopsy (4 mm) was immediately frozen and stored at -70¡C until shipment on dry ice for centralized and blinded mtDNA measurements by quantitative Southern blot, as previously described [19,20]. mtDNA and nuclear DNA (nDNA) were simultaneously detected with a digoxigenin-labelled probe. Signal intensities were densitometrically quantified using Scion-image tm (Scion Corporation, Frederick, MD). mtDNA was normalized for nDNA content by calculating the mtDNA/nDNA ratio. DNA from HepG2 hepatoma cells (ATCC HB-8065) and human fibroblasts was run on every blot to standardize for assay variations. The mtDNA/nDNA measurements were reliable, with an inter-run variation of 20%; large variations in the amount of DNA loaded on to the gel do not influence the result [20,21]. The Southern blot was also used to screen for large-scale mtDNA deletions.
Other measurements
Further evaluation included quantification of serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and total bilirubin. HCV and HIV viral loads (Cobas Amplicor HCV Monitor¨, version 2.0, and Cobas Amplicor HIV Monitor¨, version 1.5; Roche Diagnostics, Basel, Switzerland) as well as CD4 lymphocyte counts were measured at the time of biopsy. HCV genotype was determined as described elsewhere [22].
Statistics
The laboratory results and the demographic and clinical parameters for the groups were univariately compared by Fisher's exact test for categorical variables and an unpaired t-test or a single Wilcoxon Mann-Whitney test for continuous variables, as appropriate. Correlations were calculated using Pearson's product moment procedure. All statistical analyses were performed using the Sigma Stat for Windows software version 1.0 (Jandel Corporation, San Rafael, CA).
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