Published ahead of print on April 17, 2008, doi:10.1164/rccm.200711-1749OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200711-1749OC
Lung Function Loss, Smoking, Vitamin C Intake, and Polymorphisms of the Glutamate-Cysteine Ligase Genes1 Department of Epidemiology and 2 Department of Pulmonology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; and 3 Centre for Prevention and Health Services Research, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Correspondence and requests for reprints should be addressed to H. M. Boezen, Ph.D., Department of Epidemiology, University Medical Center Groningen, E3.29, P.O. Box 196, 9700 AD Groningen, The Netherlands. E-mail: h.m.boezen{at}epi.umcg.nl
Rationale: Smoking-induced oxidative stress contributes to chronic obstructive pulmonary disease, a lung disease characterized by low lung function and increasing mortality worldwide. The counterbalance for this effect may be provided by, for example, increased intake of the antioxidant vitamin C or endogenously acting antioxidant enzymes like glutamate-cysteine ligase (GCL), which is responsible for glutathione biosynthesis. Objectives: To investigate associations of functional polymorphisms in GCL subunits (GCLM and GCLC) with lung function level and its longitudinal course, with vitamin C and smoking habits as potential interactive factors. Methods: Two independent general population samples (Doetinchem, n = 1,152, and Vlagtwedde-Vlaardingen, n = 1,390) with multiple lung function (FEV1, VC) measurements were genotyped for three polymorphisms (C[–129]T, C[–588]T, and a trinucleotide GAG repeat [TNR]) in the subunits of GCL. Genetic effects on lung function level and decline were estimated using linear regression and linear mixed effect models adjusted for confounders. Findings were further investigated for interactions with vitamin C intake in the Doetinchem cohort. Measurements and Main Results: GCLC polymorphisms were significantly associated with lower lung function levels in interaction with pack-years smoked in both cohorts. TNR variants in GCLC were associated with accelerated FEV1 decline in both cohorts in interaction with pack-years. All significant effects were specifically present in subjects within the lowest tertile of vitamin C intake. Conclusions: GCLC is a novel susceptibility gene for low level of lung function in two independent populations. We provide suggestive evidence that this occurs due to an interaction between GCLC polymorphisms, smoking, and low vitamin C intake, which all contribute to the oxidative burden.
Key Words: GCLC • GCLM • polymorphism • chronic obstructive pulmonary disease • oxidative stress
Lung function loss leads to the development of chronic obstructive pulmonary disease (COPD), which is accompanied by increasing morbidity and mortality worldwide (1). The disease has a large personal, societal, and economic impact. COPD is characterized by a slow, progressive airflow limitation that is mainly caused by cigarette smoking and it is predicted to predominantly account for tobacco-caused deaths worldwide (2). The fact that some smokers are resistant to cigarette smoke and thus do not develop clinically relevant COPD suggests that other environmental factors and genetic variations can predispose to COPD development (3). Cigarette smoke contains a large amount of free radicals (4), which disturb the redox balance in the lungs, leading to the elevation of oxidative burden (5). Such an oxidant overdose can injure lung tissue directly by oxidation of cellular components (5) or indirectly by promoting inflammation and tissue degradation (6). When the lungs are chronically exposed to cigarette smoke, a more efficient protection against this oxidative stress is needed. The latter can take place via antioxidants consumed with food (5, 7–9) and via endogenous factors like antioxidant enzymes that are capable of neutralizing certain toxic cigarette smoke compounds (5, 10, 11). Vitamin C, which is abundantly present in the lung epithelial lining fluid, has been linked to a beneficial effect on lung function in several epidemiologic studies (12). Of all endogenous antioxidants, glutathione (GSH) has been shown to be present in the lung in uniquely high concentrations, which are even further increased in smokers (13). GSH plays a crucial role in systems such as multidrug resistance proteins and the GSH transferases (GSTs) that neutralize a variety of xenobiotics (11). De novo GSH synthesis is a two-step reaction, with the first step being a rate-limiting one (i.e., this step is the bottleneck of the whole process) (14). Within this step, two amino acids are conjugated by the glutamate-cysteine ligase (GCL). GCL is a heterodimeric enzyme containing catalytic and modifier subunits, coded by the GCLC and GCLM genes, respectively. Three polymorphisms in the GCLM and GCLC genes have been identified to have a functional role (15–19) and thus are of interest to study in the context of smoking-related lung function loss. We investigated whether previously identified and functional polymorphisms in GCLC and GCLM contribute to the level of lung function and its change over time in a prospective Dutch cohort study, the Doetinchem cohort. In addition, we had the opportunity to assess interactions between genotypes and environmental factors (i.e., vitamin C intake and smoking habits) with respect to lung function parameters in this cohort. A replication study in another population-based cohort, the Vlagtwedde-Vlaardingen cohort, was performed to verify associations found in the Doetinchem cohort regarding genotypes and genotype–smoking interactions.
Subjects Subjects from the Doetinchem cohort study, a prospective part of the larger MORGEN (Monitoring of Risk Factors and Health in The Netherlands) study, were included (7, 9). All subjects were tested for lung function two times with a 5-year interval: measurements of FEV1 and VC (performed with forced expiratory maneuver) were performed with a heated pneumotachometer (Jaeger, Würzburg, Germany). A subsample (n = 1,152) was randomly selected from the total cohort (n = 3,224) with no significant differences in population characteristics (Table E2 in the online supplement). Vitamin C intake at the last survey was calculated using a semiquantitative food frequency questionnaire as described previously (7). The Vlagtwedde-Vlaardingen cohort (n = 1,390) was described previously in detail (20, 21) and was included as a replication sample. This cohort was prospectively followed for 25 years with FEV1 and VC (slow inspiratory maneuver) measurements every 3 years using a water-sealed spirometer (Lode Instruments, Groningen, The Netherlands). There was no overlap in subjects whatsoever between the two cohorts, due to different sampling time frames and sampling from different cities. The study protocol was approved by the University of Groningen Hospital medical ethics committee, and all participants gave their written, informed consent.
Genotyping
Statistical Analysis FEV1 change was calculated as yearly decline (ml/yr) between two surveys in the Doetinchem cohort. The effects of polymorphisms on lung function parameters (FEV1, VC, FEV1/VC at the last survey in both cohorts and annual FEV1 change in the Doetinchem cohort) were tested with linear regression analysis adjusted for age, height, and pack-years smoked, and for sex and initial FEV1 level if appropriate. Linear mixed effect models were used to estimate longitudinal FEV1 course in the Vlagtwedde-Vlaardingen cohort (see the online supplement for details). Genotype–smoking interactions were tested by introduction of interaction terms (i.e., number of pack-years smoked x genotype) into the regression and linear mixed effect models. All interaction terms reported assessed whether the joint effect of pack-years smoked and a certain genotype was larger than the sum of these factors separately. We studied the effect of vitamin C on lung function within the whole Doetinchem cohort (n = 3,224) (see the online supplement for details). To assess whether vitamin C intake affected the relations between genotypes and lung function parameters, we repeated the analyses stratified according to tertiles of vitamin C intake. Statistical analyses were performed using SPSS (version 14.0; SPSS, Inc., Chicago, IL) and S-PLUS (version 7.0; Insightful, Seattle, WA). P values less than 0.05 were considered to be significant.
GCLM and GCLC Genotypes The characteristics of the Doetinchem cohort and Vlagtwedde-Vlaardingen cohort are shown in Table 1. SNPs and the TNR were distributed according to Hardy-Weinberg equilibrium in both cohorts. The C(–129)T SNP and TNR variants in GCLC formed three characteristic groups of genotypes (as shown in Table 2 for the Doetinchem cohort), implying that the rare TNR alleles (i.e., 8 and 9 GAG repeats) are in linkage disequilibrium with the wild-type SNP allele (i.e., C). According to the confirmed functionality (15, 17, 19) and linkage disequilibrium, both GCLC polymorphisms were analyzed by defining the reference genotype combination (bold italic values in Table 2) and simultaneously the two other groups of genotypes (the italic and bold values in Table 2, which were both entered into the statistical model as dummy variables).
The same groups were observed and defined in the Vlagtwedde-Vlaardingen cohort.
Cross-sectional and Longitudinal Analyses on Associations between GCLM and GCLC Genotypes and Lung Function
None of GCLC or GCLM polymorphisms was significantly associated with the change in FEV1 over time in any of the populations investigated (Table 3).
Interaction of Genotypes with Smoking in the Doetinchem and the Vlagtwedde-Vlaardingen Cohorts In the longitudinal analyses, the rare TNR alleles in GCLC interacted with pack-years smoked and contributed to accelerated FEV1 decline in the Vlagtwedde-Vlaardingen cohort only (Table 3, genotype carriers compared with the reference).
GCLM. To check for confounding effects, we tested the association between pack-years smoked and GCLC and GCLM genotypes. No significant differences in pack-years smoked were found between the three GCLC genotypes in both cohorts. Carriers of the mutant allele of GCLM SNP smoked significantly more (median of 7.6 pack-years smoked) than the wild-type (median of 4.2 pack-years smoked) in the Doetinchem cohort exclusively.
GCLM–GCLC Interaction in the Doetinchem and the Vlagtwedde-Vlaardingen Cohorts
Effects of Smoking, Vitamin C Intake, GCLC and GCLM genotypes, and Their Interactions on Lung Function in the Doetinchem Cohort
None of the polymorphisms was associated with FEV1, VC, or the rate of FEV1 decline in any tertile of vitamin C intake (see Table E3). The GCLM polymorphism was negatively associated with a lower FEV1/VC within the lowest tertile of vitamin C intake (  FEV1/VC = –1.93%, SE = 0.07, P = 0.01; see Table E3). The analyzed genotype–pack-year interaction terms in the Doetinchem cohort remained significant predominantly in the lowest tertile of the vitamin C intake for all lung function parameters (Table E4). An interaction between rare TNR variants and pack-years smoked additionally contributed to the accelerated FEV1 decline in this tertile exclusively (Table E4). These results implicate that there exists a three-factor interaction between pack-years smoked, vitamin C intake, and any of the GCLC polymorphisms, and this interaction negatively affects FEV1 and VC levels. In the group with the lowest vitamin C intake and the highest number of pack-years smoked, there was particularly a significant impact of the GCLC variations on FEV1 and VC levels in the Doetinchem cohort (Figures 3 and 4).
To check for confounding effects, we tested the association between vitamin C intake and GCLC and GCLM genotypes in the Doetinchem cohort, but no significant differences were observed.
We describe a novel candidate gene for lung function loss, a risk factor for COPD. We show that genetic variations in GCLC, a gene that is important in the reduction of oxidative burden, enhance the harmful effects of smoking on lung function in two independent cohorts in the general Dutch population. This negative effect was mainly observed in individuals with a low vitamin C intake (i.e., a poor exogenous antioxidant protection). The genetics of the GSH metabolism pathway also has been intensively studied in relation to interaction with smoking. The majority of studies have focused on GST polymorphisms (22–24). GST and GCL functions are likely correlated because the latter provides the essential substrate glutathione for GST activity. GCLM and GCLC are predominantly expressed in lung epithelium (25–29), where they belong to the first line of antioxidant defense and may be responsible for the uniquely high GSH content in the epithelial lining fluid (13). Many antioxidant genes, including GCLC and GCLM, display a characteristic expression pattern in airway epithelial cells—the lowest expression in never-smokers, with higher expression in healthy smokers (28, 29) and an even further increase in COPD stage 0 and I as defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) (3, 28). GCLC is less expressed in more severe COPD (GOLD stage II–IV) than in stage I disease (28). The latter indicates inadequate expression of GCLC and consequently lower GSH production, which may contribute to the development of more severe COPD. However, other studies have shown the same level of GCLC and GCLM proteins in bronchial epithelium of smokers with COPD and healthy subjects (25) or even induction of GCLC mRNA in patients with COPD (26). The GCLC C(–129)T SNP has been associated with lower expression of GCLC in endothelial cells in vitro (15). So far, no studies have focused on the in vivo impact of this SNP on GSH metabolism, but our data suggest that this SNP impairs the antioxidant protection in conditions like oxidative stress. We have observed in two independent cohorts that this impaired protection is reflected by a lower VC level, which is further pronounced by smoking. The VC measurement may reflect a slightly different lung capacity in both cohorts given a difference in measurement methodology; nevertheless the effects on VC were present in two populations. GCLC TNR has been studied in vitro in a panel of tumor cell lines (17). Walsh and colleagues found the wild-type (7 GAG repeats) allele to be responsible for the lowest intracellular GSH level. Interestingly, the allele with 8 GAG repeats was responsible for higher cell sensitivity to several chemotherapeutics (17). This contrasts with results in skin fibroblasts associating the 7-GAG-repeat allele with the highest cellular GSH content (19). Moreover, the mutant alleles (8 and 9 GAG repeats) negatively affected GCL activity and GCLC expression for 7/8, 8/8, 8/9, and 9/9 genotypes when compared with 7/7 and 7/9 (19). Likewise, we found the rarest genotypes (i.e., 8/8, 8/9, and 9/9 genotypes) to constitute a risk factor for smoking-induced lower FEV1 and FEV1/VC levels in both cohorts. In addition, rare TNR variants were associated with excess lung function decline while interacting with smoking in the Vlagtwedde-Vlaardingen cohort and in individuals with the lowest vitamin C intake in the Doetinchem cohort. In contrast, the mutant TNR alleles had a protective effect on FEV1 as found in patients with mild cystic fibrosis (30). Because previous functional studies focused on specific cell lines (i.e., tumor cells from different tissues and skin fibroblasts) or subjects with cystic fibrosis, further research is needed to determine the role of this TNR in normal, lung-derived cells that may differently respond to oxidant stimuli. It is furthermore important to study this in the context of GSH excretion, which is especially important in the lung (10, 13). We speculate that rare TNR alleles (i.e., 8 and 9 GAG repeats) may increase the sensitivity of pulmonary cells to cigarette smoke by the impairment of GCL function and ultimately lower GSH levels in lung epithelial fluid. This negative effect might be then further enhanced by insufficient vitamin C intake. The GCLM C(–588)T SNP has been associated with COPD in a Chinese population (18). This likely can be explained by the observation that GCLM induction in response to oxidative stress in vitro is lower in mutant T-allele carriers than wild-type carriers (16), which may result in lower GSH levels in vivo (18). Our results indicate that the same allele in the GCLM C(–588)T SNP interacts with smoking and thus contributes to excess lung function decline in the Doetinchem cohort only. We did not observe a similar association in the Vlagtwedde-Vlaardingen cohort. We also observed opposite effects of the interaction between this SNP and pack-years on FEV1 and FEV1/VC ratio level in the two cohorts, although they were mostly not significant. This clearly suggests that the observed contradictory associations for this SNP are spurious rather than caused by the "cohort effect" that occurs when different characteristics of two cohorts alter the genetic effect. This potentially spurious finding may also be due to a confounding effect of pack-years smoked, which was observed for this SNP in the Doetinchem cohort. A remarkable and new finding is that the significant interaction of the GCLC genotype with pack-years smoked, as observed in both cohorts, specifically existed in individuals with the lowest vitamin C intake in the Doetinchem cohort. A logical rationale for these effects might be an in vivo interaction between vitamin C and GSH (31), which are both crucial components of the tissue antioxidant defense. The latter compound serves as a reducing agent for the oxidized form of vitamin C in a spontaneous reaction or in enzymatic reactions catalyzed by the glutaredoxins or omega GSTs (31). Moreover, both GSH and vitamin C are present in a high concentration in the lung (10), which additionally implicates their special role in this tissue. In summary, our studies in two independent Dutch populations show that functional polymorphisms in the GCLC gene, which is involved in GSH-mediated lung antioxidant protection, are importantly contributing to a reduced lung function level and accelerated lung function decline, particularly in heavy smokers and in individuals with poor intake of vitamin C. Our study provides possible strategies for neutralizing harmful effects of smoking, because particularly individuals with a low vitamin C intake experience the harmful effects of smoking in the presence of GCLC polymorphisms. Further molecular and epidemiologic studies are needed to determine the role of vitamin C and genetic factors like GCLC in lung function loss and consequently in the pathogenesis and progression of COPD.
The authors thank the staff of the genotyping unit of the Centre for Medical Biomics, particularly Dr. Gerrit van der Steege, Elvira Oosterom, Marcel Bruinenberg, and Mathieu Plateel, for their help with genotyping all polymorphisms described in this article.
Supported by GUIDE (Graduate School for Drugs Exploration), University Medical Center Groningen, University of Groningen, The Netherlands; and the Netherlands Asthma Foundation (NAF3.2.02.51). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200711-1749OC on April 17, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 27, 2007; accepted in final form April 4, 2008
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ABSTRACT
AT A GLANCE COMMENTARY
12.5; 
P < 0.05 when compared with any of the other smoking groups; 
P < 0.05 in the linear regression for FEV1 adjusted for height, sex, and age when compared with any of the other eight groups, represented by bars (except for heavy smokers with medium vitamin C intake where P = 0.06). C.I. = confidence interval; py = pack-years smoked.
