Because Egypt has the highest prevalence of hepatitis C virus HCV worldwide, the burden of HCC has been increasing with a doubled incidence rate in the past 10 years [11].
Continuous oxidative stress has been associated with hepatocarcinogenesis, suggesting that antioxidant treatment may provide some sort of protection against cancer [12].
Since the main action of the oxido-reductive enzymes (superoxide dismutase, catalase, and glutathione peroxidase) that constitute the most important scavenger systems for free radicals is to provide a steady supply of GSH, conjugate GSH with various environmental risk factors [13], as well as to control the action of specific transporters to remove GSH conjugate from the cell [6], a dramatic downregulation of such enzymes has been considered to be a characteristic pathological feature of HCC [14].
Hence, GSTP1 polymorphisms could decrease detoxification when individuals are exposed to HCC risk factors [15] so we hypothesized that GSTP1 polymorphism could be a probable risk factor for the development of HCC.
Also investigating the oxidative stress status of HCC patients by measuring the antioxidant glutathione (GSH) levels and assessing its relation with GSTP1 polymorphism were important objectives of this study.
In this study, 30% of the normal control and 54.4% of the HCC groups had mutant GSTP1 genotype, while 70% of the normal control and 45.5% of the HCC groups had the wild genotype (p = 0.0005).
Consistently, El-Shafie et al. [16] reported a significant difference between the HCC and the control groups regarding GSTP1 genotyping.
Also, Munaka et al. [17] detected the expression of mutant GSTP1 genotype in 33.3% and 23.1% and the wild genotype in 66.7% and 76.9% in the normal control and HCC groups respectively among the Japanese patients.
Mutant type of GSTP1, older age, male gender, and high serum alanine aminotransferase (ALT) were found to be significant independent prognostic factors for HCC risk in this study (p < 0.047, < 0.001, < 0.001 and < 0.001), respectively, which is contradictory to the results of Chen et al. [10], who found that individuals aged ≤ 57 years with AG or GG alleles of GSTP1 had a (2.18 and 5.64) fold risk of developing HCC compared to individuals with AA alleles (p = 0.02 and 0.04) respectively, but no association was found in the older group aged > 57 years. In the present study, patients and control subjects were selected below the age of 57 as we wanted to compare our patient’s population with the group studied by Chen el al [10].
Lu et al. [18] stated that the younger the age, the more likelihood for the exposure to HCC risk factors thus increasing the susceptibility to develop HCC. Since cirrhosis caused by hepatitis C virus infection is the most important predisposing factor for HCC in Egypt, the development of HCC can occur at a relatively older age as cirrhosis takes relatively long duration to turn into HCC.
On the contrary, Zhao et al. [19], found no association between GSTP1 polymorphism and HCC risk. Subgroup analyses by ethnicity showed no significant association between GSTP1 polymorphism and HCC risk among Asians [19] and Japanese people [17], and decreased risk among Europeans [14], while no association was detected among Chinese people who were exposed to high levels of aflatoxin B1 [20].
On studying the association between GSTP1 polymorphism and the clinical status of the HCC patients the only significant association detected with PVT (p = 0.00783).
Consistently, Chen et al. [10] found no association between the estimated clinico-pathological characteristics in HCC patients and gene polymorphisms of GSTP1.
ALT level was also found to be affected by GSTP1 phenotype (p = 0.049). The highest concentrations were detected among the mutant homozygous, followed by the mutant heterozygous, then the wild phenotype groups (p = 0.049).
Similarly, Mannaa et al. [21] found that increased serum levels of AST and ALT were highly correlated with the increased expression of GSTP1. On the other hand, Chen et al. [10] and Li et al. [22] found non-significant associations between genetic polymorphisms of GSTP1 and both ALT and AST.
ALT can produce pyruvate and l-glutamate by reversibly catalyzing the transfer of an amino group from l-alanine to α-ketoglutarate. l-glutamic acid, glycine, and l-cysteine are the amino acids needed for glutathione synthesis in the body. That is how ALT activity can affect the glutathione concentration inside the body [23].
During inflammation, GSH released from hepatocytes can detoxify reactive oxygen species (ROS) generated in the vascular space of the liver. However, diffusion of the ROS into hepatocytes can result from excessive ROS formation. This can lead to intracellular oxidant stress that can activate a series of transcription factors which can induce 500 genes’ expression; some of which could contribute to carcinogenesis [24] and can cause cell injury through mitochondrial dysfunction [25].
A higher level of GSH has got two contradictory actions: it is important for normal cellular functions, signal transduction, and protection against certain carcinogens, but at the same time can slow down any cancer therapy that works by increasing intracellular reactive oxygen species [25].
On the other hand, although an increase in the ROS in cancer cells is part of the carcinogenesis process, such excessive levels of the ROS can also be toxic to the cancer cells. Therefore, controlling the levels of intrinsic ROS by GSH modulation can be an effective way to selectively kill cancer cells without adversely affecting normal cells [26].
Considering the important role of GSTP-1 in the antioxidant defense mechanism, we further measured the levels of glutathione being an important antioxidant. Although the plasma levels of glutathione were lower in the HCC compared to the normal control group in this study, the comparison did not reach statistical significance (p = 0.156). We also could not find any significant relation between GSTP1 polymorphism and the plasma level of GSH (p value 0.569).
Consistently, Tsaiel al [27]. reported that the levels of GSH were significantly lower in patients with hepatitis B virus (HBV)-associated HCC than in the control group. Also, Czeczot el al [28]. found that GSH level was lower in malignant tissues compared to adjacent normal tissues.
Similarly, Li et al. [22] found that plasma GSH and GST levels were statistically lower in HCC patients than in chronic hepatitis C (CHB) patients. These results indicated that HCC patients are under more severe imbalance of oxidants and antioxidants than patients with benign liver conditions [24].
On the contrary, Huang et al. [4] demonstrated that GSH levels were doubled in the HCC compared to the normal group and Li et al. [22] found that GSTP1 protein expression level was significantly correlated with GSH concentration (p < 0.01).
We found that GSH concentration was highest in patients with Child score A, and patients with no ascites (p = 0.043) each. This might be due to the severe imbalance of oxidative stress and antioxidant defense among the patients with advanced disease status.
We could not find any significant relation between GSTP1 polymorphism and alpha-fetoprotein level in serum (p = 0.812). Similarly, Chen et al. [10] found no association between the gene polymorphism of GSTP1 and AFP.
Up to our knowledge, the clinical utility of the plasma GSH level in HCC was not previously studied. So, we tried to test for the validity of plasma GSH to be used as a diagnostic marker for HCC.
GSH and AFP showed sensitivity and specificity of (38%, 53%) and (79%, 94%) respectively. So, plasma levels of glutathione cannot be considered a probable candidate marker for the diagnosis of HCC; however, further studies with larger groups of population are recommended to validate such results.
Yusof et al. [29] estimated the sensitivity of AFP in HCC as 36.7% compared to 53.3% for GSTP1, thus making GSTP1 a more sensitive marker for detection of HCC.