The role of polymorphisms in glutathione-related genes in asbestos-related diseases

Asbestos exposure, which still represents an important health problem worldwide, is known to be associated with the development of asbestos-related diseases, including benign pleural diseases (e.g. pleural plaques), asbestosis, lung cancer, malignant mesothelioma (MM) and other types of cancer.1, 2 The pathogenesis of asbestos-related diseases is complicated and not entirely elucidated. Nevertheless, numerous studies have suggested that in addition to a direct mechanical injury, asbestos may stimulate the production of reactive oxygen and nitric species (ROS and RNS) that were shown to have an important role in the pathogenesis of these diseases. ROS and RNS may cause asbestos-related lung injury, DNA strand breaks in mesothelial cells and may increase the risk for developing malignancy.3, 4, 5

To detoxify ROS and consequently prevent the adverse effects of oxidative stress, the human organism possesses antioxidant defence systems. Glutathione (GSH), a tripeptide composed from glutamic acid, cysteine and glycine, is an abundant cellular antioxidant which has a major role in the protection against oxidative injury in cells. It serves as a substrate of many antioxidative enzymes.6, 7 The antioxidant capacity of the glutathione system depends on enzymes involved in its biosynthesis, such as glutamate cysteine ligase (GCL), also known as gamma glutamylcysteine synthetase, as well as on detoxification enzymes, such as glutathione S-transferases (GSTs).6,8, 9, 10

GCL is the rate limiting enzyme of the GSH synthesis and it is suggested to be the major factor that determines GSH level in healthy subjects. The enzyme consists of two subunits: a heavy catalytic subunit (GCLC) and a light modifier subunit (GCLM).6,10 High GSH concentration levels found in many tumors have been associated with the increased GCL activity.11, 12

GSTs are phase II detoxifying enzymes involved in the inactivation of the electrophiles produced by ROS and RNS by catalyzing the conjugation of electrophilic compounds with reduced glutathione.8, 9 In mammals, seven classes of cytosolic GST isoenzymes have been recognized: Alpha, Mu, Pi, Sigma, Theta, Omega and Zeta.13 The crucial GST enzyme in the human lung, which belongs to the Pi class, is GSTP1.14, 15 Two other important polymorphic GSTs are GSTM1 (Mu class) and GSTT1 (Theta class).15, 16

Genes coding for GSH related enzymes are polymorphic. Among the most commonly investigated promoter polymorphisms of the GCLC and GCLM genes are GCLC rs17883901 (c.-129C>T) and GCLM rs41303970 (c.-590 C>T).17, 18, 19, 20 Some studies indicated that polymorphisms in GCLC and GCLM genes are associated with low levels of reduced GSH in vitro, which may explain susceptibility to certain diseases related to oxidative stress.17, 18 The GCLC rs17883901 polymorphism has been suggested to suppress the GCLC gene induction response to oxidants and it has been implicated in coronary endothelial dysfunction and myocardial infarction.17GCLC rs17883901 has also been proposed to modulate the renal disease risk in type 1 diabetes patients.21 The presence of GCLC rs17883901 T allele and GCLM rs41303970 T allele has also been associated with an increased risk of ischemic heart disease.19 However, according to the available literature the association between GCLC and GCLM polymorphisms and asbestos-related diseases has not been studied so far.

Regarding GSTM1 and GSTT1 genes, the most common polymorphism is due to homozygous deletion of these genes (null genotype), which results in the lack of the GSTM1 and GSTT1 enzyme activity.22, 23 In the GSTP1 gene, two common single nucleotide polymorphisms (SNPs) have been described that lead to amino acid substitution and consequently reduced enzyme conjugating activity: GSTP1 rs1138272 (p.Ala114Val) and GSTP1 rs1138272 (p.Ala114Val).22 Hirvonen et al. reported an increased risk for developing MM for individuals with GSTM1 null genotype.24 Similarly, Landi et al. found an increased risk for MM in subject with GSTM1 null allele, while no effect was observed for GSTP1 and GSTT1 polymorphisms.25 In the study of Kukkonen et al., GSTT1 null genotype increased the risk for asbestos-related severe fibrotic changes and GSTM1 null genotype was associated with the greatest thickness of the pleural plaques.26 Our former study showed that asbestosis was associated with GSTT1 null genotype, but not with GSTM1 null genotype.27 Furthermore, we have reported the influence of GSTP1 rs1695 on the asbestosis risk, while no association was found between GSTP1 rs1138272 and asbestosis risk.28

The present study aimed to investigate the influence of GCLC, GCLM, GSTM1, GSTT1 and GSTP1 polymorphisms on the risk for developing pleural plaques, asbestosis and MM. In addition, we also investigated the influence of gene-gene interactions and interactions between glutathione-related polymorphisms and asbestos exposure on the risk for developing these diseases.

The characteristics of the groups of subjects with pleural plaques, asbestosis, MM and subjects without asbestos-related disease are presented in Table 1. A statistically significant difference between the groups was observed for the age (p < 0.001), pack-years of smoking (p = 0.024) and asbestos exposure (p < 0.001). The mean age was the highest for subjects with MM (65 ± 10.7 years), followed by subjects with asbestosis (58.7 ± 9.1 years).

The mean values of pack-years of smoking were the highest in subjects with asbestosis (24.4 ± 18.6) and in subjects with MM (23.2 ± 17.2). Regarding asbestos exposure, the percent of subjects with low asbestos exposure was the highest for the group of subject with no asbestos-related disease (77.5%), followed by the group of subjects with pleural plaques (72.3%) (Table 1).

The genotype frequencies for all studied genetic polymorphisms are shown in Table 2. Genotype frequencies for all investigated SNPs were concordant with HWE.

In univariate logistic regression analysis, no association was found between GCLC rs17883901 and GCLM rs41303970 genetic polymorphisms and asbestos-related diseases.

GSTT1 null genotype was associated with the decreased risk for asbestos-related diseases when analysed together (OR = 0.63; 95% CI = 0.42–0.95; p = 0.026). When analysing the risk for each disease separately, GSTT1 null genotype was associated with the decreased risk for pleural plaques (OR = 0.63; 95% CI = 0.40–0.98; p = 0.026) and asbestosis (OR = 0.51; 95% CI = 0.28–0.93; p = 0.028), but not for MM. No association was found between GSTM1 null genotype and asbestos-related diseases. Regarding GSTP1 polymorphisms, a positive association was found between GSTP1 rs1695 AG + GG vs. AA genotypes for MM only when compared to pleural plaques (OR = 1.39; 95% CI = 1.00–1.94; p = 0.049) (Table 3).

Regarding age, no association was found between age and pleural plaques (OR = 0.98; 95% CI = 0.96–1.00; p = 0.032). A slight association was observed between age and MM (OR = 1.07; 95% CI = 1.05–1.10; p < 0.001), as well as between age and MM when compared to pleural plaques (OR = 1.10; 95% CI = 1.08–1.12; p < 0.001).

The analysis of association between asbestos exposure and asbestos-related diseases revealed a positive association between high and medium vs. low asbestos exposure and all asbestos-related diseases (OR = 1.93; 95 % CI = 1.31–2.85; p = 0.001), between high and medium vs. low asbestos exposure and asbestosis (OR = 3.22; 95% CI = 1.99–5.20; p < 0.001), and between high and medium vs. low asbestos exposure and MM (OR = 4.06; 95% CI = 2.28–7.23; p < 0.001). When analysing the association between high and medium vs. low asbestos exposure and MM compared to pleural plaques, the OR was 3.07 (95 % CI = 1.85–5.12; p < 0.001).

In multivariate logistic regression analysis, the risk of GSTT1 null genotype for all asbestos-related diseases together (OR = 0.62; 95% CI = 0.41–0.94; p = 0.025) and separately for asbestosis (OR = 0.51; 95% CI = 0.27–0.95; p = 0.033) did not change considerably after adjustment for asbestos exposure. Similarly, the risk of GSTT1 null genotype for pleural plaques remained practically unchanged after adjustment for age (OR = 0.63; 95% CI = 0.40–0.99; p = 0.046). On the contrary, the risk of GSTP1 rs1695 AG + GG vs. AA genotypes for MM compared to pleural plaques increased slightly (OR = 1.97; 95% CI = 1.14–3.39; p = 0.015) after adjustment for asbestos exposure and age (Table 4).

In further logistic regression analysis, the interactions between polymorphisms showed no significant influence on the risk for developing asbestos-related diseases (data not shown).

Testing the influence of interactions between asbestos high and medium vs. low exposure and genetic polymorphisms on the risk of asbestos-related diseases, the interaction between asbestos exposure and GSTT1 null polymorphism decreased the risk for developing MM (OR = 0.17; 95% CI = 0.03-0.85; p = 0.031). Similarly, the interaction between asbestos exposure and GSTT1 null polymorphism (OR = 0.11; 95% CI = 0.02–0.49; p = 0.004) and the interaction between asbestos exposure and GSTP1 rs1695 polymorphism (OR = 0.14; 95% CI = 0.03–0.65; p = 0.012) decreased the risk of MM when compared to pleural plaques.

The present study investigated the influence of genetic polymorphisms in GSH related genes, the interactions between these polymorphism, and interactions between polymorphisms and asbestos exposure on the risk of asbestos-related diseases.

The present study revealed a protective effect of GSTT1 null genotype on the risk of all studied asbestos-related diseases together and particularly on the risk of pleural plaques and asbestosis. The explanation of these findings could be that in some instances GSTT1 may catalyse toxification and not detoxification reaction, leading to even more reactive conjugate.15 This observation is in agreement with the results of our previous study, in which GSTT1 null genotype also decreased the asbestosis risk.27 On the other hand, in the present study GSTM1 null genotype showed no effect on the risk of asbestos-related diseases, which is also consistent with the results of our previous study.27 Similar findings were observed by Jakobsson et al., who reported no association between GSTM1 deficiency and parenchymal and pleural abnormalities among the workers exposed to asbestos30, and also by Hirvonen et al., who revealed no increased risk for the asbestos-related pulmonary disorders in subjects with homozygous deletion of GSTM1 gene.16 Contrary to the results of our study, Kukkonen et al. reported that GSTT1 null genotype increased the risk of asbestosis and GSTM1 null genotype was related to the greatest thickness of the pleural plaques.26 Although Landi et al. observed an increased risk for MM in subjects bearing GSTM1 null allele25, in our current study, no association was found between either GSTM1 null genotype or GSTT1 null genotype and MM risk.

The results of our study showed that GSTP1 rs1695 AG + GG vs. AA genotypes increased the MM risk, while GSTP1 rs1138272 polymorphism did not affect the risk of this malignoma. On the contrary, GSTP1 polymorphisms did not influence the MM risk in the study by Landi et al.25

Our study revealed no influence of GCLC rs17883901 and GCLM rs41303970 on the risk for asbestos-related diseases. Our results suggest that these two polymorphisms are not related to the susceptibility to asbestos-related diseases. To our knowledge, no other studies investigated the role of polymorphic genes involved in GSH synthesis in asbestos-related diseases.

Our study confirmed the impact of high and medium vs. low asbestos exposure on the risk for asbestosis and MM, which is consistent with the findings of previous studies.31,32, 33, 34 However, our results also showed that nearly 46% of subjects with MM, 52% of subjects with asbestosis and 72% subjects with pleural plaques had low asbestos exposure. This suggests that asbestos-related diseases can also develop when asbestos exposures are low, which was indicated especially for MM.35,36

In this study, the interactions between investigated GSH related gene polymorphisms did not influence the risk for developing asbestos-related diseases. On the other hand, we observed that the interaction between GSTT1 null polymorphism and asbestos exposure decreased the risk for developing MM, although there was no independent association between GSTT1 null and MM when compared to controls with no asbestos-related disease. In other words, GSTT1 null genotype modified the association between high and medium vs. low asbestos exposure and MM and acted protectively on the risk of this malignant disease.

Another interesting finding of this study showed that the interaction between GSTP1 rs1695 AG + GG vs. AA genotypes and asbestos exposure decreased the risk of MM when compared to pleural plaques, despite the fact that in univariate analysis both GSTP1 polymorphism and asbestos exposure were associated with an increased risk of MM. The relation between benign pleural plaques and the risk of MM has not been clearly proved so far. Although pleural plaques may be the endpoint and the development of pleural plaques may be an entirely independent process from the development of MM37, it is likely that there is a relation between pleural plaques and MM.38 The present study suggests a modifying and protective effect of GSTP1 rs1695 genotypes on the association between asbestos exposure and MM risk when compared to pleural plaques.

Considering the potential limitations of the study, the data on asbestos exposure were not available for all subjects, especially not for patients with MM. Consequently, the analyses of the interactions between genetic polymorphisms and asbestos exposure could be performed only for the subgroup of MM patients.

On the other hand, the study also brings novel findings and has some important strengths. Firstly, according to our knowledge, this is the first study to investigate the association between GCLC rs17883901 and GCLM rs41303970 genetic polymorphisms and asbestos-related diseases. Secondly, it included relatively large numbers of subjects with different asbestos-related diseases from genetically homogenous population and investigated functional genetic polymorphisms in different GSH related genes.

In conclusion, our findings suggest that among genetic polymorphisms in GSH related genes, GSTT1 null polymorphism may be associated with the risk for developing pleural plaques and asbestosis and may also modify the association between asbestos exposure and MM and therefore act protectively on the risk for this malignoma. This study also revealed a modifying and protective effect of GSTP1 rs1695 polymorphism on the association between asbestos exposure and MM risk when pleural plaques were considered as controls.