GSTT1 and GSTM1 Null Genotypes May Facilitate Hepatitis C Virus Infection Becoming Chronic

Worldwide, >170 million individuals are chronically infected with hepatitis C virus (HCV) [1]. The estimated prevalence in Spain is ∼800,000 cases [2]. Oxidative stress plays an important role in the pathogenesis of HCV-induced damage [3]. Reduced glutathione (GSH) is the main element of the endogenous antioxidant system, which prevents the accumulation of free radicals through the transformation of GSH, catalyzed by glutathione peroxidase, into dimeric oxidized glutathione, which is converted back to GSH by glutathione reductase [3]. Interestingly, in chronic hepatitis C, there is an accelerated turnover of GSH [4].

Glutathione S-transferases (GSTs) are a superfamily of enzymes that catalyze the conjugation of electrophilic chemicals with glutathione. The isoforms most extensively studied in relation to the risk of environment-related human diseases are GSTM1 and GSTT1, because their activity is modulated by genetic polymorphisms. The GST mu 1 (GSTM1) gene and the GST theta 1 (GSTT1) gene frequently have a partial deletion that causes the total absence of enzymatic function (null alleles). Both polymorphic traits are inherited independently. Individuals homozygous for null alleles of either gene are devoid of specific enzymatic activity in all their organs and tissues [5].

GSTM1 and GSTT1 enzymes are expressed in hepatocytes [6] and in Kupffer cells [7]. These have been identified in the cytosol and in mitochondria, where the rate of production of free radicals is highest [8]. Available evidence indicates that GST isozymes, including the M1 and T1 isoforms, are involved in the detoxification of 4-hydroxy-alkenals, among them 9-hydroxynonenal [8]. Moreover, GSTT1 has glutathione-peroxidase activity [9], and it might be directly involved in the detoxification of reactive species produced as a consequence of ongoing HCV infection of the liver.

Because GSH seems to play a relevant role in chronic HCV infection and because polymorphisms of the GSTM1 and GSTT1 enzymes are common, it can be speculated that individuals devoid of enzyme activity would be more likely to develop severe HCV infection. To our knowledge, this topic has not been studied to date. We therefore determined the distribution of GSTM1 and GSTT1 polymorphisms in a group of Spanish patients with chronic hepatitis C and in healthy control subjects, to clarify whether these polymorphisms are involved in the clinical course of HCV infection.

Patients and methods. We studied a cohort of 139 white, unrelated patients of Spanish ancestry and nationality with a diagnosis of chronic HCV infection who were treated at the Department of Gastroenterology of the Hospital Clínico San Carlos (Madrid, Spain) between January 2001 and September 2003. Eighty-two patients were male (mean ± SD age at diagnosis, 43.8 ± 11.3 years), and 57 were female (mean ± SD age at diagnosis, 47.0 ± 12.1 years).

The diagnosis of chronic hepatitis C was based on the results of clinical, biochemical, and viral tests. All patients were positive for anti-HCV antibody and for HCV RNA at the time of inclusion in the study. The HCV genotype was known for 124 patients. Active hepatitis B virus (HBV) infection and HIV infection were excluded by standard analytical procedures. Hepatocellular carcinoma (HCC) was excluded by liver ultrasonography. No patient formerly treated with antiviral therapy was included. All patients were informed and asked to participate in the study before a liver biopsy was performed, and the blood specimen for genotyping procedures was obtained on the same day as the biopsy specimen. Data on tobacco and alcohol use were collected. Heavy drinkers were defined as individuals drinking >50 g of alcohol per day.

The control group was composed of 329 unrelated, healthy, white, Spanish individuals from the same geographical area (198 men with a mean ± SD age of 42.1 ± 11.5 years and 131 women with a mean ± SD age of 44.6 ± 14.5 years). A medical history was obtained from and laboratory tests were done for each individual, to exclude preexisting conditions. The majority were students and staff from the hospitals collaborating in the study. The control group was matched for sex. The study protocol was approved by the local ethics committee, and all subjects gave informed consent before inclusion in the study.

Liver biopsies were performed during the evaluation of patients for chronic hepatitis C antiviral therapy. All liver-tissue specimens were examined by the same pathologist (L.O.), who was not informed of the genotyping results. The hepatic activity index (HAI) [10] was estimated in every case. Quantitative and qualitative detection of HCV RNA was evaluated using Cobas Amplicor Hepatitis C Virus Monitor Assay (Roche Molecular Systems) [11], which was the method available in the study center between 2001 and 2003. The lower limit of detection was 600 IU/mL, and the upper range was 500,000 IU/mL. HCV genotyping was determined using a commercial single polymerase chain reaction (PCR; Cobas Amplicor Hepatitis C Virus; Roche Diagnostics) and a reverse-hybridization system for the amplified product with type-specific probes (the INNO-LiPA line Probe Assay; Innogenetics). Both procedures were performed and interpreted in accordance with the recommendations of the manufacturers.

Genotype analyses were performed on genomic DNA obtained from blood samples. A multiplex PCR assay was used to determine the GSTM1 and GSTT1 genotypes, as described elsewhere [12].

Differences between patients and control subjects in the distribution of GSTM1 and GSTT1 genotypes, which were considered to be dichotomous variables, were analyzed using the Mantel-Haenszel χ² test with Bonferroni's correction or Fisher's exact test, where appropriate. Odds ratios (ORs) with their corresponding 95% confidence intervals (CIs) were simultaneously calculated. Continuous variables were compared using Student's t test and the Mann-Whitney U test, where appropriate. All tests were 2-sided. The calculations were made using the SPSS statistical software package forWindows (version 11.5; SPSS) and Epi-Info 2002 (Centers for Disease Control and Prevention).

Results. The distribution of GST genotypes is shown in table 1. There was a marked excess of GSTT1 null genotypes in case patients, compared with control subjects (OR, 2.76 [95% CI, 1.77–4.30]; P < .001), and a much lower excess of GSTM1 null genotypes (OR, 1.54 [95%CI, 1.02–2.35]; P = .032). There was a significant excess of patients with both GSTT1 and GSTM1 null genotypes, compared with control subjects (OR, 3.65 [95% CI, 1.98–6.75]; P < .001).

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Figure 2.

GSTM1 and GSTT1 genotype distribution and glutathione S-transferase (GST) active genotypes in patients with chronic hepatitis C and healthy control subjects.

Scores of the 4 independent items of the HAI—periportal and bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation, and fibrosis—did not differ between null and active GSTT1 and GSTM1 genotype carriers or among the 3 categories of active genotypes. Similar nonsignificant results were obtained when we divided the necroinflammatory global score (the sum of items 1, 2, and 3 of the HAI) and the fibrosis score (item 4 of the HAI) into 2 categories: no differences in the distribution of the studied genotypes and of their combinations were observed (data not shown).

A total of 72 patients for whom histological results were available received combination therapy with pegylated interferon (IFN) and ribavirin. Thirty-four patients achieved a sustained viral response, and 19 had primary therapeutic failure. The remaining 19 patients had viral relapse or were unable to complete therapy because of intolerance. No significant differences in the distribution of the studied genotypes were found among the different response groups. Age at inclusion, age at diagnosis when known (for illicit drug users and those who received blood products), sex, smoking and drinking habits, liver biochemical results, and viral load were not related to the distribution of the genotypes studied.

Discussion. Our results suggest that the GSTT1 null genotype and, to a lesser degree, the GSTM1 null genotype and the combined double-null haplotype increase the risk of evolution of HCV disease to the chronic phase. The alternative explanation (i.e., that null genotypes increase the risk of acquiring acute HCV infection) seems unlikely and is difficult to investigate because of the low rate of clinically evident acute hepatitis C. GSTs are detoxifying phase II enzymes that are present in the cytosol and mitochondria of several types of liver cells. They catalyze conjugation with glutathione and the elimination of electrophilic toxic metabolites. Because oxidative stress is a very important factor in the pathogenesis of chronic hepatitis C [3], genetically induced differences in antioxidant mechanisms might modulate the natural history of the disease.

Although the control group was not completely matched for age, differences between case patients and control subjects were negligible, considering the marginal influence that age might have on the frequency of presentation of GST genotypes [13]. Moreover, we included in the group of control subjects a subgroup of patients >90 years old [12], and we observed no differences in the GST genotypes studied. This makes it unlikely that the age differences between case patients and control subjects (1.7 years for men and 2.4 years for women) influenced the outcome of the study.

We have found no published data on GST polymorphisms in patients with chronic hepatitis C. It is of interest to note that there was no relationship between the distribution of the genotypes studied and the severity of histological damage as measured by the HAI scoring system. According to our findings, GST polymorphisms might be involved in the early stages of the evolution of HCV infection, but, to date, we have no data on their possible influence at later stages—that is, the development of cirrhosis or even HCC. In a recent report by our group [12], which aimed to study GSTT1 and GSTM1 polymorphisms in Spanish patients with HCC, 63 of 184 patients had underlying HCV-induced liver cirrhosis but were free of chronic, active HBV infection and had not been heavy alcohol drinkers. We have retrospectively analyzed the data from these 63 subjects (all of whom fulfilled clinical and ultrasonographic criteria for advanced liver disease), and we detected an excess of the GSTT1 null genotype (36.6% vs. 23.1% in healthy control subjects; OR, 1.91 [95% CI, 1.04–3.52]; P = .025), which reinforces the influence of this genetic trait on the risk of entering the chronic phase of HCV-induced liver disease, although it does not suggest any relationship later during the course of the disease (in this case, the observed difference should have been greater than that found during earlier stages of the chronic infection, which we have noted in the present study).

A considerable amount of time and effort have been devoted to the investigation of established chronic HCV infection, rather than the identification of factors that help clear the virus during the acute phase of the disease. However, a significant proportion of patients with acute hepatitis C clear the virus spontaneously. The mechanisms underlying this clearance are not well understood, but almost all of these patients display a strong, multispecific, and sustained CD4⁺ and CD8⁺ T cell response; if this response is impaired early, viral clearance fails [14]. The CD4⁺ T cell response is a dominant Th1 response that produces antiviral cytokines (interleukin-2, IFN-γ, and tumor necrosis factor—α) but, when it is ineffective in eradicating infection, persistent hepatic injury may result [15]. We propose that the GSTT1 null genotype might induce a subtle but functionally significant deficit in the hepatic antioxidant and reactive oxygen species (ROS)—inactivating mechanisms. This deficit might be reinforced by the simultaneous lack of GSTM1 enzymatic function. As a consequence, more ROSs and electrophilic compounds generated during the acute phase of HCV infection would be available in individuals devoid of both GST enzymes. We speculate that this effect could be advantageous if it contributes to the Th1 response elicited by HCV and aimed to clear the virus; unfortunately, this occurs only in a minority of patients. In the remaining patients, the deficit of antioxidant mechanisms induced by lower GST enzyme activity might help to amplify and perpetuate the inflammatory reaction that leads to the chronic phase of hepatitis C. Our data, however, should be considered preliminary, and they need to be confirmed by independent studies.

• Received October 27, 2006.
• Accepted December 8, 2006.

• © 2007 by the Infectious Diseases Society of America