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Interferon Decreased mtDNA Mutation Rate & Improved Liver
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Journal of Viral Hepatitis
September 2005
Note from Jules Levin: these study data suggest a possible partial explanation for accelerated progression of HCV in HIV+ individuals. Perhaps, HIV & NRTIs cause mtDNA mutations which injure the liver by increasing chronic inflammation, which accelerates progression. Several studies find that after cirrhosis develops in HCv/HIV coinfected person HCV disease can progress 5 times more quickly than in a HCV monoinfected person.
The study authors report: "......The present work, shows that IFN treatment markedly decreases the number of liver mtDNA mutations in individuals with chronic HCV infection, and that this change is correlated with a concomitant decrease in total HAI score..... Mitochondria that undergo replication appear to acquire DNA damage more readily, resulting in an accumulation of mutations, compared with those maintained under resting conditions..... The high frequency of mtDNA mutations in the liver of patients with chronic hepatitis suggests that hepatocytes in such patients continuously undergo malignant transformation during inflammation induced by HCV infection......
.....It is suggested that the treatment of hepatitis patients with interferon (IFN) reduces the incidence of HCC (liver cancer). Normalization of the plasma levels of alanine aminotransferase by IFN treatment reduces the rate of hepatocarcinogenesis, even if the hepatitis virus is not eliminated. (ED NOTE from Jules Levin: these are the principles supporting the notion of Maintenance Therapy: low dose interferon slows HCV disease progression even if viral load is not reduced). Thus, we hypothesized that mtDNA in the liver might be affected by hepatitis virus infection and IFN therapy. The present work, demonstrates that HCV infection increases mtDNA mutation in the liver, so the treatment of HCV-infected patients with IFN significantly decreases the frequency of mtDNA mutation. ......IFN therapy decreased the mtDNA mutation and the decreased number of mtDNA mutations was positively correlated with suppression of the total histological activity index score....."
"Interferon reduces somatic mutation of mitochondrial DNA in liver tissues from chronic viral hepatitis patients"
Authors: M. Nishikawa1, S. Nishiguchi2, K. Kioka3, A. Tamori2 and M. Inoue1
1Biochemistry & Molecular Pathology and 2Hepatology, Osaka City University Medical School and 3Department of Gastroenterology, Osaka City General Hospital, Osaka, Japan
BACKGROUND
Persistent infection with hepatitis C virus (HCV) can ultimately result in the development of hepatocellular carcinoma (HCC). Thus, unlike other types of cancer, HCC is usually preceded by chronic inflammation for 2040 years, although development of HCC can be rapid, particularly in individuals with viral hepatitis and high plasma levels of alanine aminotransferase [1]. Chronic inflammation induced by hepatitis viruses thus plays an important role in hepatocarcinogenesis. The extent of oxidative stress is increased in the liver of individuals infected with hepatitis viruses [2,3], and reactive oxygen species are important inducers of DNA mutations [4].
Mutations accumulate to a greater extent in mitochondrial DNA (mtDNA) than in nuclear DNA, predominantly because mitochondria generate substantial amounts of reactive oxygen species, while they lack histone-like nucleoproteins [5]. Because, expression of the entire mitochondrial genome is necessary for the maintenance of mitochondrial functions, including electron transport, small changes in the mtDNA sequence can result in profound impairment of such functions, thereby enhancing generation of free radicals, which in turn accelerates the rate of DNA mutation.
We have previously reported that the frequency of mtDNA mutations is markedly increased in both noncancerous and cancerous liver specimens from individuals with HCC [6], and showed that the frequency of mtDNA mutations in HCC tissue was greater than that previously described for other types of cancers [79]. Furthermore, the extent of accumulated mtDNA mutations in HCC tissues showed a positive correlation with the degree of malignancy. The genetic instability that results in the high rate of mtDNA mutation in noncancerous liver tissues is consistent with the multicentric hepatocarcinogenesis that is detected clinically.
It is suggested that the treatment of hepatitis patients with interferon (IFN) reduces the incidence of HCC [10,11]. Normalization of the plasma levels of alanine aminotransferase by IFN treatment reduces the rate of hepatocarcinogenesis, even if the hepatitis virus is not eliminated [12]. Thus, we hypothesized that mtDNA in the liver might be affected by hepatitis virus infection and IFN therapy. The present work, demonstrates that HCV infection increases mtDNA mutation in the liver, so the treatment of HCV-infected patients with IFN significantly decreases the frequency of mtDNA mutation.
Summary.
We recently reported that the genetic instability resulting in the high rate of mitochondrial DNA (mtDNA) mutation in noncancerous liver tissue is consistent with the multicentric hepatocarcinogenesis detected clinically. Interferon (IFN) has been reported to reduce hepatocarcinogenesis in individuals with hepatitis virus infection. Liver biopsy specimens were obtained from 26 patients with chronic hepatitis C virus (HCV) infection before and after IFN therapy (total dose: 252 million units). The mean (±SD) age of the study population was 45 ± 9 years and 13 (50%) were male [mode of acquisition: blood transfusion (27%), unknown (73%); viral load: 5.2 ± 1.1 k copies/mL; duration of infection: 17 ± 9 years (65%), unknown (35%); genotype: I (4%), II (80%), III (8%), IV (8%); alcohol intake: positive (31%), negative (69%)]. DNA samples were extracted from the specimens and subjected to direct sequencing. The mtDNA mutation frequency in the D-loop was increased in liver specimens from individuals with HCV infection compared with 21 controls (2.5 vs 0.6, P < 0.001).
IFN therapy decreased the mtDNA mutation (mean difference = 0.7, P < 0.001) and the decreased number of mtDNA mutations was positively correlated with suppression of the total histological activity index score (mean difference = 1.3, P < 0.01).
These results clearly indicate that the mutational rate of mtDNA is strongly associated with IFN therapy. Thus, analysis of mtDNA could provide a new criterion for the therapeutic evaluation of the effect of IFN, and may be useful for the prediction of risk of carcinogenesis.
Tissue specimens
We analysed liver mtDNA in 26 patients with chronic HCV infection who had received IFN-b (a total dose of 252 million units/patient) for 2 months. The mean (±SD) age of the study population was 45 ± 9 years and 13 (50%) were male [mode of acquisition: blood transfusion (27%), unknown (73%); viral load: 5.2 ± 1.1 k copies/mL; duration of infection: 18 ± 9 years (65%), unknown (35%); genotype: I (4%), II (80%), III (8%), IV (8%); alcohol intake: positive (31%), negative (69%)]. The liver specimens were obtained from these patients by needle biopsy before and after IFN therapy. The histological activity index (HAI) score of the liver specimens was evaluated by Knodell's scoring system. Twenty-one control liver samples were obtained from individuals (age: 57 ± 12 years; male/female: 60/40%) without viral infection, but with liver disease other than HCC (including hepatolithiasis and colorectal carcinoma with liver metastasis).
RESULTS
The entire mitochondrial genomes of liver specimens obtained from an individual with chronic hepatitis type C before, just after, and 3 years after IFN treatment, and of one control liver specimen were amplified by PCR and sequenced manually. Compared with a mtDNA sequence deposited in GenBank (accession no.J01415), the mtDNA sequence obtained from the liver specimen of the control subject contained three single-base variants, all of which were located in the D-loop. The mtDNA sequences obtained from the chronic hepatitis type C specimen before IFN therapy contained 24 mutations, only half of which were detected in the mtDNA from specimens obtained just after and 3 years after IFN treatment. Consistent with previous observations [6-9], most of the mutations detected in this present study were homoplasmic.
The greatest frequency of mtDNA mutations was apparent in the D-loop, especially in the region between nucleotides 100 and 600. All mtDNA samples contained a GAEA transition at nucleotide 263, a TAEC transition at nucleotide 489, and a C insertion between nucleotides 311 and 312. We then compared the number of mutations in the D-loop region (between nucleotides 100 and 600), excluding the three mutations (variants) common to all samples, among control liver specimens and tissues from individuals with chronic hepatitis type C obtained before and after IFN treatment. The individuals with hepatitis type C virus had much more mtDNA mutations in D-loop than control (2.8 vs 0.5, P < 0.01). The mean difference in the number of mtDNA mutations was 0.7 for specimens obtained before and just after IFN therapy (P < 0.001).
The decreased number of mtDNA mutations in D-loop (delta mtDNA mutation) was positively correlated with the decrease (mean difference = 1.3, P < 0.01) in the total HAI score (?Activity).
The variation of the mtDNA mutations were a C deletion at nucleotide 164, a G insertion between 184 and 185, a G A transition at 207, a C insertion between 303 and 304, a C T transition at 317, 320 and 530, and a deletion of GA at 514515.
DISCUSSION
The present work shows that IFN treatment markedly decreases the number of liver mtDNA mutations in individuals with chronic HCV infection, and that this change is correlated with a concomitant decrease in total HAI score.
Although each hepatocyte contains hundreds of mitochondria and each mitochondrion contains one to 10 genomes [14], most of the mutations identified in mtDNA were homoplasmic. Tumour cell mitochondria have been reported to proliferate preferentially when tumour cells are fused with normal cells [7,15], suggesting the presence of a mechanism that stimulates the replication of tumour-associated mutant mtDNA molecules. The D-loop region of mtDNA is important in the replication and expression of mitochondrial genome, because it contains both a leading-strand replication origin and transcriptional promoter regions [16]. Thus, mutations within the D-loop might affect the rate of DNA replication by modulating its interaction with trans-acting factors.
Mitochondria that undergo replication appear to acquire DNA damage more readily, resulting in an accumulation of mutations, compared with those maintained under resting conditions. Our data suggest that mtDNA mutations accumulate during the neoplastic transformation of hepatocytes. In mtDNA harbouring certain mutations might generate abnormal RNAs or proteins, the latter of which may promote leakage of electrons from the mitochondrial electron transport chain. The amounts of endogenously produced reactive oxygen species, such as superoxide and related free radicals, might thus be increased in cells with mutant mtDNA, and the resulting oxidative modification of DNA may contribute to the early stages of hepatocarcinogenesis.
Although activated leucocytes infiltrate into inflammatory liver tissue, the number of mitochondria in infiltrated inflammatory cells is significantly smaller than in hepatocytes. Thus, the contribution of contaminating inflammatory cells to the increased mtDNA mutation frequency observed in HCC tissues is likely to be negligible. Furthermore, mtDNA preparations from liver tissues in individuals with chronic HCV infection were compared with those from paired blood samples. No mutations were found in the blood samples, except for the three common mutations in the D-loop.
Despite our attempts to detect deletions in mtDNA with the use of multiple primers for PCR analysis, no deletion was found in liver tissues from individuals with chronic HCV infection, suggesting that hepatocytes with large mtDNA deletions might be eliminated, while cells and/or mitochondria without such deletions preferentially undergo proliferation.
The high frequency of mtDNA mutations in the liver of patients with chronic hepatitis suggests that hepatocytes in such patients continuously undergo malignant transformation during inflammation induced by HCV infection. The observation that most of the mutations detected were homoplasmic in nature indicates that the mutated mtDNA had become dominant in the liver. Given the clonal nature and large number of mtDNA copies, the unusually high rate of mtDNA mutation in the liver of patients with chronic HCV infection indicates genomic instability, which is likely to contribute to hepatocarcinogenesis. Therefore, analysis of mutations in mtDNA could provide a new criterion for the therapeutic evaluation of the effect of IFN, and may be useful for prediction of the risk of carcinogenesis.
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