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Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy
[Research Letters]
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AIDS:Volume 22(10)19 June 2008p 1226-1229
Matsukura, Motoia; Chu, Fanny FSb; Au, Maya; Lu, Helena; Chen, Jennifera; Rietkerk, Sonjac; Barrios, Rolandod; Farley, John De; Montaner, Julio Sd; Montessori, Valentina Cd; Walker, David Ca,b; Cote, Helene CFa
aDepartment of Pathology and Laboratory Medicine, University of British Columbia, Canada
bThe James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Canada
cImmunodeficiency Clinic, St Paul's Hospital, Canada
dBC Centre for Excellence in HIV/AIDS, Providence Healthcare Research Institute, Canada
eDepartment of Healthcare and Epidemiology, University of British Columbia and Dr John Farley Inc, Vancouver, British Columbia, Canada.
Abstract
Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.
Today, up to 50% of HIV patients die from end-stage liver diseases [1]. Possible reasons for this include longer life expectancy and a decline in opportunistic infections, accompanied by an increase in underlying comorbid conditions, such as liver diseases and idiosyncratic hepatotoxic reactions [1-3]. This is especially relevant to the HIV/hepatitis C virus (HCV) coinfected population in whom liver injury is more rapid and prevalent [2,4], poor liver conditions decrease tolerance for highly active antiretroviral therapy (HAART) and end-stage liver diseases are the primary cause of death [5,6].
Nucleoside reverse transcriptase inhibitors (NRTIs) can cause mitochondrial DNA (mtDNA) depletion and possibly deletion and mutation, which may affect mitochondrial structural integrity and functions [7,8]. Such HAART-related mitochondrial toxicity may lead to hepatic steatosis, hyperlactatemia/lactic acidosis and liver failure. Although abnormal liver mitochondrial ultrastructure has been demonstrated in HIV/HCV coinfection [9,10], it is unclear what damage is caused by HIV or HCV infection versus NRTI-related mitochondrial toxicity. We hypothesized that HIV/HCV coinfected individuals currently receiving HAART would show greater liver mitochondrial damage than those not on HAART. Liver mitochondrial damage was assessed through mtDNA and gene expression levels, as well as electron microscopic ultrastructural analyses.
In this prospective cohort study, HIV/HCV coinfected men were either HAART-naive or off HAART more than 6 months (OFF-HAART), or on stable HAART for longer than 6 months (ON-HAART). At the time of liver biopsy, all were HCV therapy naive, free from chronic liver diseases and had no opportunistic infections within the last month. Demographic and clinical characteristics such as age, HCV genotype, CD4 cell count, albumin, bilirubin, aspartate aminotransferase, alanine aminotransferase, platelet count and lactate were comparable between the ON-HAART and OFF-HAART groups.
Two ultrasound-guided liver tissue biopsies were collected between 2003 and 2006 from each subject. The first one was used for pathology using modified Ishak-Knodell scale scoring whereas the second was used for mtDNA and mtRNA quantifications and electron microscopic stereological morphometry. The mtDNA/nuclear DNA (nDNA) ratio was determined as described previously [11,12]. Mitochondrial mRNA was quantified similarly for both a mtDNA-encoded (COX1) and a nDNA-encoded (COX8) gene, and normalized to the housekeeping gene _-actin mRNA level. Tissue for electron microscopic analysis was fixed in 2.5% glutaraldehyde and postfixed in a mixture of 2% osmium tetraoxide and 2% potassium ferrocyanate. The following hepatocyte ultrastructural characteristics were quantitatively and stereologically analyzed: hepatocyte volume using the star volume method [13]; hepatocyte mitochondria, glycogen and lipid volume fractions using the point counting method [14]; and mitochondria cristae surface density, using the line intercept method [15]. To eliminate potential bias, a standardized random sampling protocol was established by a single examiner, and the number of images analyzed for each parameter was determined as that providing a stable and low coefficient of error.
Overall, no statistically significant difference was observed between ON-HAART and OFF-HAART samples, for any of the studied parameters (Table 1). No differences were seen in mtDNA and mtRNA levels, but there was an overall positive correlation between COX1 and COX8 expression levels (R = 0.63, P = 0.006). Volume fractions of hepatocyte metabolic constituents did not significantly differ between ON-HAART and OFF-HAART samples, however, hepatocytes were weakly but significantly larger in the ON-HAART group compared with the OFF-HAART one (P = 0.05), and median cristae surface density was 28% lower ON-HAART (Table 1). All study participants were infected with either HCV genotype 1 or 3a, and the latter was associated with significantly higher intrahepatocyte lipid accumulation [median (interquartile range = 1.3 (0.6-2.3)% vs. 12.0 (8.0-14.1)%, P = 0.002].
HIV/HCV coinfected individuals are more at risk of hepatotoxicity than HIV or HCV monoinfected ones [2,4,6,16]. It has been hypothesized that NRTI-induced liver mitochondrial toxicity may further exacerbate liver damage and contribute to lower tolerance of HCV therapy [5,17]. NRTIs of the dideoxynucleotide type [D-drugs: d4T, ddI and zalcitabine (ddC)] are blamed for mitochondrial toxicity in both clinical and in-vitro studies [11,18]. Decreased liver mtDNA levels in HIV/HCV coinfected patients on D-drugs, but not in those on non-D-drug-containing HAART have been reported [19]. We found no evidence of increased liver mitochondrial damage in association with current HAART. As the majority of ON-HAART subjects were receiving non-D-drug HAART regimens, our results are consistent with the previous finding [19] and suggest low liver mitochondrial toxicity for non-D-drug HAART. Mitochondrial gene expression of both nDNA and a mtDNA-encoded genes did not differ between the two groups, yet showed a significant correlation between them, suggesting the absence of mtDNA-specific alterations in expression patterns.
We rigorously and objectively evaluated hepatocyte cell volume, mitochondria volume fraction, cristae surface density, lipid and glycogen volume fraction by quantitative electron microscopy. No difference in any of the studied parameters was detected between ON-HAART and OFF-HAART liver samples, except for the cell size, which was marginally larger in ON-HAART samples. This appears to be inconsistent with two studies in HIV/HCV coinfected individuals that suggested an association between HAART and increased liver mitochondrial ultrastructural alterations [9,10]. Only two of our participants received d4T and none was on ddI or ddC. Exposure to different NRTIs in these small studies may partially explain this difference. Hepatomegaly has been observed in HIV patients [20] and coinfection with HCV increases the risk of steatosis or steatohepatitis, which may enlarge hepatocytes. However, the relationship between hepatomegaly and HAART remains unclear since mitochondrial dysfunction can also lead to steatosis and steatohepatitis [21,22]. Although our data do not suggest increased mitochondrial damage in ON-HAART individuals, it is possible that enlarged hepatocytes reflect weak hepatotoxicity. As noted by others [23], HCV genotype 3 was statistically significantly associated with higher lipid volume fraction (Table 1), validating the stereological methodology used in this study.
In conclusion, although the sample size was limited, this study provided no evidence that patients coinfected with HIV and HCV show increased liver mitochondrial damage if on concomitant HAART. These results suggest that HAART with predominantly non-D-drug-containing regimen may not be a reason to delay or avoid the start of HCV antiviral therapy in coinfected individuals. This is especially true as liver disease progression and not drug-induced hepatotoxicity is the most common cause of mortality in this population [24].
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