Abstract
Oxidative stress and xenobiotic metabolizing enzymes are suspected to be related to carcinogenesis by different cellular mechanisms. Hence, our study aimed at identifying potential relationships between antioxidant defense parameters measured in blood and glutathione S-transferase (GST) genetic polymorphisms of four GST izoenzymes in lung cancer patients and reference individuals. The case–control study included 404 lung cancer patients and 410 non-cancer subjects as controls, matched by age, gender and place of living (central Poland). In control subjects with GSTM3*A/*A, GSTT1 null, GSTM1 null + GSTT1 null, GSTM3*A/*A + GSTT1 null genotype, glutathione peroxidase activity was significantly higher (P < 0.05) than in controls possessing respective potential protective GST genotypes. Controls with GSTM3*A/*A + GSTP1*B genotype presented significantly higher ceruloplasmin activity (P < 0.05) than GSTM3*B + GSTP1*A/*A carriers. Zinc level was significantly higher (P < 0.05) in controls and cases with GSTP1*B + GSTT1 null genotype and in cases with GSTM1 null + GSTP1*B genotype, when compared with respective potential protective GST genotypes. This case–control study indicates that particular defective GST genotypes may enhance the defense against oxidative stress. The potential relationship between the investigated antioxidative enzymes and microelements, and common functional genetic polymorphism of GST was observed mostly in control subjects.
Keywords: Antioxidant defense, Genetic polymorphism, Glutathione S-transferase, Lung cancer
Introduction
Lung cancer is the most common neoplastic disease in Poland and other European countries. Of the 1.2 million cases registered in 2000, 380,000 incidences occurred in Europe. It is estimated that 520,000 lung cancer cases will be registered in Europe in 2050 [28]. International Agency for Research on Cancer data show differences in the incidence of, and mortality from, cancer among men and women. In Poland (in the year 2000), lung cancer constituted the major cause of the incidence of, and mortality from, cancer in men. Among the women it was ranked fourth, preceded by cancer of breast, genitals and large intestine (http://www-dep.iarc.fr).
Overproduction of reactive oxygen and nitrogen species may cause formation of oxidant DNA damages and may also induce disturbances in cellular transduction pathways (Klaunig and Kamendulis [17]. Therefore, the role of antioxidants in preventing tumor progression may be crucial. Several epidemiological studies revealed significant differences in antioxidant status between healthy persons and cancer patients, which indicated disturbances in antioxidant status in cancer risk [19, 21]. Follow-up prospective studies showed associations between high antioxidants/microelements level and high fruit and vegetable consumption and a lower cancer risk, e.g. selenium (Se) and lung cancer risk in a population of Finns [9, 19]. However, several intervention studies with specific micronutrient supplementation, generally failed to prove a protective role of a higher intake of dietary antioxidants on cancer incidence. The findings of the increased risk after β-carotene supplementation in smoking men have indicated unresolved roles of some antioxidants in the carcinogenic process [25]. The studies carried out by Clark et al. [4], suggested a protective role of Se supplementation in cancer development, however, only in the male population of USA. About 35% of cancer deaths has been estimated to be associated with dietary habits, but cancer risk can also be attributable to congenital factors, including mutations in rare and common genes [20]. Some epidemiological case–control studies have shown minor to moderate changes in cancer risk associated with genetic polymorphisms of xenobiotic metabolizing enzymes (XMEs) and antioxidant enzymes, which indicates an pivotal influence of environmental/dietary factors in cancer development [12, 30].
The increasing evidence for genetic and biological variability in nutrient requirements and the advances in the area of nutrient-dependent regulation of the genome machinery, also show interaction of genes and nutrients in humans. XMEs genetic polymorphism may determine effect of specific nutrient by differences in its biotransformation. Modulatory effect of polymorphic XMEs and diet, including antioxidants on cancer development has been indicated by some epidemiological investigations [32].
Our lung cancer case–control study was conducted to investigate the activities of glutathione peroxidase (GPx), GST, ceruloplasmin (Cp) and superoxide dismutase (SOD) and blood levels of Se, zinc (Zn) and copper (Cu) in relation to genetic polymorphisms of glutathione S-transferase (GST) as a one of the approaches towards possible interactions between inherited polymorphic genes and antioxidant defense markers.
Materials and methods
Population
The study was conducted in the population of individuals living in the city of Lodz (central Poland, Caucasian ethnicity). There were a total of 404 lung cancer patients of clinical and pathological departments (the study group) and a total of 410 non-malignant patients treated in the Lodz hospitals (the control group). Enrolled individuals had to be residents of the study area for at least 1 year. Controls were matched by gender and age (±3 years). Blood sample was collected, and each participant was interviewed within 3 months following the initial diagnosis or the eligibility for the study. Initial diagnosis of lung cancer was also confirmed histologically within this period. The histological diagnosis of lung cancer comprised squamous cell carcinoma (SqCC), small cell carcinoma (SCC), non-small cell carcinoma (NSCC), and adenocarcinoma (AC). Smoking index (S.I.) was calculated by multiplying daily cigarette consumption and duration of smoking. Primary results of the plasma microelements concentration and GST genetic polymorphism in the investigated groups were presented previously [31]. Characteristics of the study population including GST genotypes frequency are summarized in Table 1.
Table 1.
Characteristics of study population
| Cases | Controls | |
|---|---|---|
| N = 404 | N = 410 | |
| Gendera | ||
| Males | 305 (75.5%) | 300 (73.2%) |
| Females | 99 (24.5%) | 110 (26.8%) |
| Ageb | 58.8 ± 9.1 (30–78) | 59.0 ± 9.3 (32–74) |
| Smoking habitsc | ||
| Non-smokers | 24 (6.0%) | 98 (24.4%) |
| Smokersd | 375 (94.0%) | 304 (75.6%) |
| Smoking index (S.I.)e | 750.3 (40–2,115) | 531.6 (1–2,370) |
| Genotype | ||
| GSTM1 (+) | 207 (55.9)f | 192 (56.8) |
| GSTM1 null | 163 (44.1) | 146 (43.2) |
| GSTM3*A/*B + *B/*B | 31 (26.1) | 44 (31.9) |
| GSTM3*A/*A | 88 (73.9) | 94 (68.1) |
| GSTP1 Ile/Ile | 108 (49.8) | 120 (47.8) |
| GSTP1 Ile/Val + Val/Val | 106 (50.2) | 131 (52.2) |
| GSTT1 (+)g | 99 (83.2) | 99 (71.7) |
| GSTT1 null | 20 (16.8) | 39 (28.3) |
aNumber of individuals (frequency); Chi-square test, NS (no significant)
bMean ± SD (range); Student’s t test, NS
cNumber of individuals (frequency); Chi-square test, P < 0.05
dIncluding individuals who declared to quit smoking >5 years earlier (31 persons in the study and 95 in the control groups)
eDaily cigarette consumption multiplied by duration of smoking (years)
fNumber of individuals (frequency)
gChi-square test, GSST1 (+) versus GSTT1 null, P < 0.03
The Regional Ethics Committee for Scientific Research approved the study protocol, and a written consent was obtained from each participant. All volunteers were informed about the purpose of the investigation and a possible withdrawal from the study.
Blood samples from patients during fasting were collected into Venoject heparinized (lithium heparin) test tubes free from trace elements. Separated plasma and erythrocyte samples were stored at −20°C until determination of antioxidant enzyme activity and microelement concentrations, and 200 μl of whole blood samples was stored at −80°C before genomic DNA isolation. Analyses were conducted within 1 month following sample collection.
Antioxidant defense parameters
The Se concentration in plasma was assayed by graphite furnace atomic absorption spectrometry (AAS) according to Neve et al. method [23] using Unicam Solar 989 QZ apparatus. Zinc and copper in plasma were determined by means of flame AAS [1] with Pye Unicam SP9 800 apparatus. Methods used to determine plasma microelements were constantly tested with parallel detection in the reference material (Nycomed, Seronorm™, Norway). Erythrocyte and plasma GPx activities were assayed according to the method of Paglia and Valentine [26] as modified by Hopkins and Tudhope [13]. Red blood cells (RBC) GST and SOD activities were determined by means of Habig et al. [11] and Beauchamp and Fridovich [3] methods, respectively. Activity of Cp in plasma was assayed according to Sunderman and Nomoto [37].
GST genetic polymorphism and antioxidant defense parameters
RFLP-PCR technique was used to determine GSTM1, GSTM3 and GSTP1 genetic polymorphisms. DNA samples were extracted from whole venous blood. GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null genotypes were distinguished, as described previously [8]. In this method, restriction endonuclease HaeII was applied to determine GSTM1 positive genotypes, further analysed as GSTM1 (+) genotype. The genetic polymorphism of GSTP1 in exon 5 (GSTP1 Ile/Ile, Ile/Val, Val/Val genotypes) was identified according to Watson et al. [41] and Kihara et al. [16] by means of Alw26I restriction enzyme, with a slight modification of the protocol. Detection of GSTM3 genetic polymorphism (GSTM3*A/*A, *A/*B, *B/*B genotypes) was determined according to Inskip et al. [15] and To-Figueras et al. [39]. Restriction endonuclease MnlI was used to recognize GSTM3*A allele. PCR technique described elsewhere [29] was introduced to indicate GSTT1 positive and GSTT1 null genotypes. PCR and RFLP-PCR products were identified by means of horizontal agarose electrophoresis with ethidium bromide, along with DNA ladder, which was then visualised and analysed. Samples with ambiguous results were re-tested and 10% of all samples were repeated.
The associations between antioxidant defence variables and GST genotypes were analyzed in both investigated groups. Lung cancer patients and controls were divided into subgroups according to positive and at-risk single GST genotypes (Tables 3, 4, 5, 6.) and combined genotypes (Table 7).
Table 3.
Antioxidant defense parameters in controls and cases with GSTM1-specific genotypes
| Antioxidant defense parameters | Controls | Cases | ||||||
|---|---|---|---|---|---|---|---|---|
| GSTM1 (+) | GSTM1 null | GSTM1 (+) | GSTM1 null | |||||
| N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | |
| RBC GPx (U/g Hb) | 191 | 14.21 ± 3.72 | 144 | 14.30 ± 3.85 | 206 | 13.43 ± 4.62 | 163 | 13.70 ± 4.43 |
| Plasma GPx (U/l) | 191 | 154 ± 42 | 144 | 161 ± 40 | 206 | 166 ± 63 | 161 | 160 ± 53 |
| RBC GST (U/g Hb) | 190 | 4.17 ± 1.56 | 143 | 3.87 ± 1.42 | 207 | 4.81 ± 2.10 | 163 | 4.41 ± 2.04 |
| SOD (U/g Hb) | 191 | 9.52 ± 2.04 | 143 | 9.49 ± 1.98 | 207 | 7.80 ± 3.55 | 163 | 8.30 ± 3.55 |
| Cp (U) | 190 | 597 ± 209 | 142 | 593 ± 183 | 205 | 765 ± 218 | 163 | 766 ± 248 |
| Se (μg/l) | 188 | 53.5 ± 14.6 | 143 | 53.7 ± 14.5 | 207 | 49.0 ± 17.8 | 163 | 50.4 ± 16.3 |
| Zn (mg/l) | 185 | 0.93 ± 0.20 | 135 | 0.94 ± 0.28 | 205 | 0.85 ± 0.26) | 160 | 0.87 ± 0.24 |
| Cu (mg/l) | 186 | 1.16 ± 0.26 | 135 | 1.20 ± 0.28 | 205 | 1.52 ± 0.34 | 160 | 1.54 ± 0.36 |
Table 4.
Antioxidant defense parameters in controls and cases with GSTM3-specific genotypes
| Antioxidant defense parameter | Controls | Cases | ||||||
|---|---|---|---|---|---|---|---|---|
| GSTM3*B | GSTM3*A/*A | GSTM3*B | GSTM3*A/*A | |||||
| N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | |
| RBC GPx (U/g Hb) | 44 | 13.0 ± 3.6* | 92 | 14.6 ± 3.3* | 31 | 13.0 ± 4.2 | 88 | 12.9 ± 3.9 |
| Plasma GPx (U/l) | 44 | 145 ± 37* | 92 | 160 ± 41* | 31 | 175 ± 66 | 87 | 156 ± 60 |
| RBC GST (U/g Hb) | 45 | 3.82 ± 1.40* | 92 | 4.45 ± 1.51* | 31 | 4.97 ± 2.24 | 88 | 4.74 ± 2.25 |
| SOD (U/g Hb) | 44 | 9.51 ± 1.59 | 92 | 9.86 ± 2.23 | 31 | 7.39 ± 3.33 | 88 | 7.96 ± 3.40 |
| Cp (U) | 44 | 565 ± 215 | 92 | 619 ± 218 | 31 | 770 ± 229 | 87 | 737 ± 194 |
| Se (μg/l) | 44 | 55.6 ± 14.2 | 91 | 52.5 ± 13.9 | 31 | 48.2 ± 16.9 | 88 | 47.2 ± 18.1 |
| Zn (mg/l) | 44 | 0.92 ± 0.21 | 87 | 0.92 ± 0.20 | 31 | 0.83 ± 0.26 | 88 | 0.83 ± 0.19 |
| Cu (mg/l) | 44 | 1.11 ± 0.28 | 88 | 1.19 ± 0.26 | 31 | 1.57 ± 0.34 | 88 | 1.54 ± 0.30 |
* Significant different values (P < 0.05): GSTM3*B carriers versus GSTM3*A/*A carriers
Table 5.
Antioxidant defense parameters in controls and cases with GSTP1-specific genotypes
| Antioxidant defense parameter | Controls | Cases | ||||||
|---|---|---|---|---|---|---|---|---|
| GSTP1 Ile/Ile | GSTP1 Val | GSTP1 Ile/Ile | GSTP1 Val | |||||
| N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | |
| GPx RBC (U/g Hb) | 119 | 13.84 ± 3.67 | 131 | 14.18 ± 3.78 | 108 | 13.31 ± 4.39 | 109 | 13.81 ± 4.44 |
| GPx plasma (U/l) | 119 | 151 ± 37 | 131 | 153 ± 40 | 108 | 158 ± 52 | 108 | 164 ± 60 |
| RBC GST (U/g Hb) | 118 | 4.21 ± 1.45* | 131 | 3.95 ± 1.53* | 108 | 4.79 ± 2.14 | 108 | 4.39 ± 2.03 |
| SOD (U/g Hb) | 119 | 9.33 ± 1.93 | 131 | 9.36 ± 1.96 | 108 | 8.23 ± 2.87 | 109 | 8.62 ± 3.05 |
| Cp (U) | 118 | 572 ± 179 | 131 | 603 ± 190 | 107 | 785 ± 225 | 109 | 748 ± 205 |
| Se (μg/l) | 116 | 53.5 ± 12.3 | 130 | 54.8 ± 15.1 | 108 | 51.0 ± 18.3 | 109 | 48.1 ± 16.3 |
| Zn (mg/l) | 115 | 0.90 ± 0.20 | 121 | 0.92 ± 0.19 | 107 | 0.87 ± 0.25 | 107 | 0.89 ± 0.30 |
| Cu (mg/l) | 112 | 1.16 ± 0.25 | 122 | 1.15 ± 0.26 | 107 | 1.57 ± 0.35 | 107 | 1.50 ± 0.34 |
* Significant different values (P < 0.05): GSTP1 Ile/Ile carriers versus and GSTP1 Val carriers
Table 6.
Antioxidant defense parameters in controls and cases with GSTT1-specific genotypes
| Antioxidant defense parameter | Controls | Cases | ||||||
|---|---|---|---|---|---|---|---|---|
| GSTT1 (+) | GSTT1 null | GSTT1 (+) | GSTT1 null | |||||
| N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | N | Mean ± SD | |
| RBC GPx (U/g Hb) | 97 | 14.0 ± 3.5 | 39 | 14.4 ± 3.4 | 99 | 12.9 ± 4.1 | 20 | 12.9 ± 3.7 |
| Plasma GPx (U/l) | 97 | 151 ± 38* | 39 | 167 ± 44* | 99 | 160 ± 62 | 19 | 168 ± 63 |
| RBC GST (U/g Hb) | 97 | 4.22 ± 1.50 | 38 | 4.33 ± 1.50 | 99 | 4.83 ± 2.26 | 20 | 4.66 ± 2.21 |
| SOD (U/g Hb) | 97 | 9.78 ± 2.12 | 39 | 9.66 ± 1.88 | 99 | 7.87 ± 3.43 | 20 | 7.50 ± 3.14 |
| Cp (U) | 97 | 603 ± 229 | 39 | 597 ± 188 | 98 | 725 ± 187* | 20 | 845 ± 252* |
| Se (μg/l) | 97 | 53.4 ± 14.0 | 38 | 53.6 ± 14.2 | 99 | 47.6 ± 17.6 | 20 | 46.8 ± 18.6 |
| Zn (mg/l) | 93 | 0.91 ± 0.22 | 38 | 0.94 ± 0.16 | 99 | 0.82 ± 0.20 | 20 | 0.88 ± 0.24 |
| Cu (mg/l) | 94 | 1.14 ± 0.27 | 38 | 1.22 ± 0.28 | 99 | 1.53 ± 0.31 | 20 | 1.60 ± 0.33 |
* Significant different values (P < 0.05): GSTT1 (+) carriers versus GSTT1 null carriers
Table 7.
Antioxidant defense parameters referred to GST combined genotypes in controls and cases
| Antioxidant defense parameter* | N | Mean ± SD | N | Mean ± SD | N | Mean ± SD |
|---|---|---|---|---|---|---|
| Controls | ||||||
| GSTM1/GSTT1 +/+ | GSTM1/GSTT1 +/− | GSTM1/GSTT1 −/− | ||||
| Plasma GPx (U/l) | 62 | 150 ± 39 | 56 | 156 ± 41 | 18 | 171 ± 43 |
| GSTM3/GSTT1 +/+ | GSTM3/GSTT1 +/− | GSTM3/GSTT1 −/− | ||||
| Plasma GPx (U/l) | 32 | 141 ± 26 | 77 | 156 ± 44 | 27 | 171 ± 36 |
| RBC GPx (U/g Hb) | 32 | 12.5 ± 3.1 | 77 | 14.6 ± 3.7 | 22 | 14.5 ± 2.8 |
| GSTM3/GSTP1 +/+ | GSTM3/GSTP1 +/− | GSTM3/GSTP1 −/− | ||||
| Cp (U) | 45 | 543 ± 143 | 90 | 603 ± 216 | 51 | 620 ± 199 |
| GSTP1/GSTT1 +/+ | GSTP1/GSTT1 +/− | GSTP1/GSTT1 −/− | ||||
| Zn (mg/l) | 68 | 0.90 ± 0.23 | 87 | 0.90 ± 0.18 | 21 | 0.99 ± 0.17 |
| Cases | ||||||
| GSTM1/GSTT1 +/+ | GSTM1/GSTT1 +/− | GSTM1/GSTT1 −/− | ||||
| Zn (mg/l) | 56 | 0.77 ± 0.19 | 56 | 0.87 ± 0.21 | 7 | 0.96 ± 0.22 |
| GSTP1/GSTT1 +/+ | GSTP1/GSTT1 +/− | GSTP1/GSTT1 −/− | ||||
| Zn (mg/l) | 25 | 0.85 ± 0.26 | 80 | 0.81 ± 0.19 | 14 | 0.93 ± 0.23 |
+/+, Both protective GST genotypes; +/−, either protective GST genotypes; −/−, neither protective GST genotypes
* Significantly (P < 0.05) varies between double protective and double defective GST genotype
Statistical analysis
The significance of differences between groups was calculated by means of Student’s t test, the likelihood ratio by Chi-square test [7] and one-way analysis of variance [33, 36]. A value of P < 0.05 was considered to reflect statistical significance. Based on genetic epidemiology data, the GSTM1 null genotype [34], GSTM3*A/*A [39], GSTP1 Val (one and both copies) [41], GSTT1 null [14] were considered as high-risk genotypes.
Results
Study groups and hospital patients did not differ in their age and gender. There was a very small number of non-smoking lung cancer patients (6.0%), as compared with controls (24.4%) (Table 1). There was lack of statistical significance in the distribution of GSTM1, GSTM3 and GSTP1 genotypes between the investigated groups. The frequency of GSTM1 null genotype was similar in lung cancer patients (44.1%) and in controls (43.2%). The prevalence of the most frequent GSTM3*A/*A at-risk genotype was also similar in the study (73.9%) and the control (68.1%) groups; the same applied to wild-type GSTP1 Ile/Ile genotype (49.8 vs. 47.8%). The frequency of GSTT1 homozygous deletion (GSTT1 null genotype) was significantly (P < 0.03) higher in controls than in lung cancer patients (28.3 vs. 16.8%; P < 0.03) (Table 1). There was no difference in combined GST genotypes (analyzed as follows: GST both protective, GST either protective, GST neither protective) between two groups. Moreover, we have not observed differences in GST genotype frequencies in non-smokers and smokers within both investigated groups.
The activity of GPx and SOD in RBC as well as Se and Zn concentrations in plasma were significantly (P < 0.001) lower in lung cancer patients than in controls. In contrast, RBC GST activity, Cp activity and Cu level in plasma were significantly higher (P < 0.001) (Table 2).
Table 2.
Antioxidant defense parameters in controls and lung cancer patients
| Antioxidant defense parameter | Controls | Cases | P< | ||
|---|---|---|---|---|---|
| N | Mean ± SD | N | Mean ± SD | ||
| GPx RBC (U/g Hb) | 395 | 14.7 ± 4.2 | 400 | 13.6 ± 4.6 | 0.001 |
| GPx plasma (U/l) | 398 | 159 ± 41 | 398 | 162 ± 59 | NS |
| GST RBC (U/g Hb) | 394 | 4.02 ± 1.47 | 401 | 4.63 ± 2.18 | 0.001 |
| SOD (U/g Hb) | 383 | 9.51 ± 1.97 | 403 | 7.99 ± 3.54 | 0.001 |
| Cp (U) | 392 | 590 ± 192 | 398 | 760 ± 227 | 0.001 |
| Se (μg/l) | 383 | 54.3 ± 14.3 | 399 | 49.7 ± 17.1 | 0.001 |
| Zn (mg/l) | 372 | 0.93 ± 0.24 | 390 | 0.86 ± 0.25 | 0.001 |
| Cu (mg/l) | 373 | 1.17 ± 0.26 | 390 | 1.53 ± 0.35 | 0.001 |
The possible association between antioxidants and GST genotypes was studied in both groups. Lung cancer patients and controls were divided into subgroups according to protective and at-risk single GST genotypes (Tables 3, 4, 5, 6) and combined GST genotypes as follows: both protective, either protective, neither protective (Table 7).
Significant differences in some antioxidative defense variables between individuals with protective and at-risk GST genotypes were found. Controls with GSTM3*A/*A genotype had significantly higher (P < 0.05) activity of RBC GPx, plasma GPx, and GST RBC than carriers of GSTM3*B single or both alleles (Table 4). Non-cancer subjects possessing GSTP1 Ile/Ile wild-type genotype had significantly elevated (P < 0.05) RBC GST activity (Table 5) in comparison with the subjects with at least one GSTP1 Val allele, while GSTT1 null genotype carriers had significantly higher (P < 0.05) activity of GPx in plasma, as compared with GSTT1 (+) ones (Table 6). In controls with combined defective GST genotypes (GSTM1/GSTT1 and GSTM3/GSTT1), the activity of GPx in plasma was significantly higher (P < 0.05) than in individuals with both respective protective genotypes (Table 7). In subgroup with defective GSTM3/GSTT1 genotype, the activity of RBC GPx was significantly higher (P < 0.05) than in those with GSTM3/GSTT1 protective genotype. Similarly, in GSTM3/GSTP1 defective genotype carriers, Cp activity was significantly higher (P < 0.05), and in both defective GSTP1/GSTT1 subgroup plasma Zn level was significantly higher (P < 0.05) than in individuals with particular relevant protective GST genotype (Table 7).
Lung cancer patients with homozygous deletion of GSTT1 gene presented significantly higher activity of Cp, than individuals with GSTT1 (+) genotype (Table 6). In this group, among individuals with combined at risk GSTP1/GSTT1 and GSTM1/GSTT1 genotype showed significantly higher (P < 0.05) Zn level as compared with relevant protective GST genotype (Table 7).
Discussion
It is well documented that the levels of antioxidants and microelements differ between individuals with certain types of cancer and healthy individuals, including lung cancer [18, 24, 40]. Antioxidative enzymes are essential in protecting against reactive oxygen and nitrogen species, regulating the redox potential of the mammalian cell [17]. The higher activity of RBC GPx and SOD in controls than in lung cancer patients, as observed in this report, may indicate an insufficient defense against oxidative stress among cancer individuals.
Lung cancer patients also showed significantly lower Se and Zn concentration in plasma. These findings are in agreement with a study on children with different types of malignancy that also revealed a significantly lower level of that trace element. Several studies among patients with different malignancies indicated a 5–35% decrease in Se level as compared with healthy controls [2, 43].
Over the last decade, when genetic and biological variability in nutrients requirements was recognized, it has been essential to understand the role of nutrients, including trace elements and other diet-related factors, in inhibiting or promoting the carcinogenic process and first of all in preventing cancer development [6]. Therefore, gene–nutrient interactions should be widely examined through possible interactions of various genes with specific nutrients. One of the approaches is to analyze individual genotype with a specific focus on common genetic polymorphisms of XMEs. It is very important that the process of activation by phase I enzymes and detoxification by phase II ones includes not only environmental and dietary xenobiotics, but also protective components of diet [35]. Furthermore, various dietary compounds can influence the modulation of biotransformation enzymes, through different mechanisms, including ARE and/or XRE-mediated regulation [38].
This study indicated an interaction between some GST genotypes with respect to particular antioxidants and microelements with the functions in the antioxidant defense. The values of blood antioxidant defense parameters were significantly higher in control subjects with particular single or combined GST defective genotypes than in controls possessing potential protective wild-type GST genotypes. Interestingly, the number of such associations was lower in the lung cancer group. Controls with suspected to be at-risk GSTM3*A/*A genotype showed significantly higher activity of GPx and GST in red blood cells and GPx in plasma compared to those with at least one GSTM3*B allele. It is supposed that the wild-type GSTM3*A allele of polymorphic GSTM3 izoenzyme, mainly expressed in lung tissue, may be associated with lung cancer risk [42]. Moreover, a higher activity of plasma GPx was also found in controls lacking the GSTT1 gene, than in those possessing it. The higher activity of GST RBC in controls with wild-type GSTP1 Ile/Ile genotype may rather reflect genotype–phenotype interactions, as it was found higher GST activity in lung tissue samples with GSTP1 Ile/Ile genotype than with GSTP1 Ile/Val and Val/Val genotypes [41]. A protective role of some defective combined GST genotypes, associated with the higher activity of GPx, GST and Cp was also seen in non-cancer individuals, as was the case with significantly higher Zn level in individuals with double defective GSTP1/GSTT1 genotype.
Modulation of antioxidant levels by genetic polymorphism of XMEs was previously found in several studies. In middle-aged men, smokers and non-smokers with GSTM1 null genotype, significantly higher glutathione and vitamin C levels were observed, compared with those possessing protective GSTM1 genotype. However, protective GSTT1 genotype was associated with higher vitamin C level and glucose-6-phosphate dehydrogenase activity, than those observed in GSTT1 null genotype. GST activity measured in lymphocytes was significantly higher in carriers of GSTP1 codon 105 than in wild-type genotypes [5].
The European Prospective Investigation on Cancer and Nutrition (EPIC) study also showed that defective GST genotype may enhance defense against oxidative stress due to modulation of some nutrient and enzymatic antioxidants. Moreover, nutrients modulated by genetic polymorphisms may affect PAH-DNA adduct formation. In Italian participants of the EPIC study strong negative associations between DNA adducts and α- and β-carotene levels was found, however, only in participants having GSTM1 null genotype, but not GSTM1 + genotype. A borderline negative association was also observed in the case of α- and γ-tocopherol levels in plasma of homozygotes lacking the GSTM1 gene. Yet the study did not show any association between GSTM1 genotypes and the levels of other plasma micronutrients: β-cryptoxanthin, lutein, lycopene, zeaxanthin, retinal, and total carotenoids [27]. Similarly, some authors also reported inverse associations between plasma antioxidant levels and DNA adducts in individuals with specific GST polymorphisms [10, 22].
In the present study, some association between antioxidative defense markers and GST genetic polymorphisms also occurs in the lung cancer patients, however, it is less pronounced than in controls. Among lung cancer individuals with some defective genotypes, GSTM1 null/GSTT1 null and GSTM3*A/*A/GSTT1 null a significantly elevated Zn level was observed as compared with patients with protective combined GST genotypes. This may indicate that protective activity of some metabolic genes against oxidative stress occurs mainly in healthy, non-cancerous persons.
In conclusion, our findings may be an important contribution towards the identification of the role played by enzymatic antioxidants, microelements and genetic polymorphism of glutathione S-transferase interactions for cancer-risk. It is thought that these common genetic polymorphisms may modify the bioavailability, metabolism, affinity and activity of several micronutrients and antioxidants, and thus influence oxidative stress. We have proposed one of the approaches towards the interaction between nutrients and genes, which may have a potential to identify susceptible populations and/or individuals and to apply genotype-related dietary recommendations.
Footnotes
Part of the study was presented at the Fourth International Symposium on Trace Elements in Humans, 2003, Athens, Greece.
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