This study included 100 healthy individuals (smokers) and 100 nonsmokers of TABUK-northwestern Saudi Arabia. The subjects were reporting to hospital for routine checkup.

All the Subjects were interviewed using a structured questionnaire regarding epidemiological/demographic data, past history, history of addiction particularly smoking, family history of any significant diseases.

Venous blood was collected in EDTA tubes from all subjects and controls and the high molecular genomic DNA was isolated using DNA extraction QIAamp DNA Blood Mini Kit (51106) and samples were stored at −20˚C until PCR. The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of DNA by Thermo Scientific NanoDrop™. A ratio of ~1.8 to 1.95 was generally obtained.

Briefly, 50 ng of DNA was amplified in a 25 μl multiplex reaction mixture containing 25 pmol of each of the following primers as depicted in Table 1 and 12.5 μl from TGoTaq^® Green Master Mix (cat no M7122) (Promega, USA) which is composed of GoTaq^® DNA Polymerase, 2X Green GoTaq^® Reaction Buffer (pH 8.5), 400 µM dATP, 400 µM dGTP, 400 µM dCTP, 400 µM dTTP and 3 mM MgCl₂. The buffer contains yellow and blue dyes, which function as loading dyes when reaction products are analyzed by agarose gel electrophoresis which prevents the use of toxic Et-Br. The multiplex PCR method was used to detect the presence or absence of the GSTT1 and GSTM1 genes in the genomic DNA samples, simultaneously in the same tube. Each set of reaction included both positive and negative controls. The reaction mixture was subjected to initial denaturation at 94˚C for 1 min, followed by 35 cycles of 94˚C for 58 seconds, 59˚C for 58 seconds, and 72˚C for 58 seconds. The final extension was done at 72˚C for 10 min. The amplification products were separated by electrophoresis through 1% agarose gel stained with ethidium bromide. Length of PCR products from positive for GSTM1 and GSTT1 genotypes yielded bands of 215bp and 480bp respectively while the internal positive control (CYP1A1) PCR product corresponded to 312 bp as depicted in the as depicted in Figure 1. The subjects were classified as either positive (when at least one copy of the gene was present) or null genotypes. Heterozygous and homozygous individuals for GSTM1 (GSTM1 +/0 and GSTM1 +/+) or GSTT1 (GSTT1 +/0 and GSTT1 +/+) have been reported to present similar enzyme expression levels and were considered together for statistical analysis.

The distribution of the GSTM1 and GSTT1 genotypes was evaluated for Hardy-Weinberg equilibrium using chi-squared analysis, as described. Chi Square test or Fisher Exact Probability test was used for analysis of frequency distribution of subjects in various subgroups. Odds ratio and confidence interval were used to estimate risk of contracting disease in presence of various risk factors. Statistical significance was accepted as P < 0.05 (Two tailed). All analyses were done using EPI 6 software (Epi info 6 CDC).

The distribution of the GSTM1 null genotype was more frequent among non smoker individuals (14%) than among smoker individuals (1%) as depicted in Table 1 and Figure 1. Similarly the distribution of the GST T1 null genotype was more frequent among non smoker individuals (14%) than among smoker individuals (1%) as depicted in Table 2 and Figure 2 & Figure 3.

The significant correlation was reported between non smoker individuals and smoker individuals. The difference was statistically significant (P < 0.0003) as depicted in the Table 3.

The significant correlation was reported between non smoker individuals and smoker individuals as depicted in Table 1. The difference was statistically significant (P < 0.0001) as depicted in the Table 4.

The allele frequencies of GSTT1 (normal) and (null) genotypes in according to Chi-sq Hardy-Weinberg equilibrium test calculator for biallelic markers as depicted in Table 5.

The allele frequencies of GSTM1 (normal) and (null) genotypes in according to Chi-sq Hardy-Weinberg equilibrium test calculator for biallelic markers as depicted in Table 6.

The “worst” detoxifier genotype (the combined genotype of GSTM1 null and GSTT1 null) was found in 14 of

GST = glutathione S-transferase; GSTM1 = GST mu; ^*P < 0.05 compared between smokers and non smokers (Fisher exact test).

GST = glutathione S-transferase; GSTT1 = GST theta. ^*P < 0.05 compared between smokers and non smokers (Fisher exact test).

100 (GSTM1) and 28/100 (GSTT1) northwestern Saudi Arabia female populations and 1 of 100 (GSTM1) and 14/100 (GSTT1) northwestern Saudi Arabia male populations. The difference was statistically significant as depicted Table 7. The OR of “worst” detoxifier genotype versus all other genotypes was 2.72 (95% CI = 1.00 - 13.45).

Tobacco smoke is a major cause of many diseases. Although its carcinogenic role via induction of DNA damage and mutation is well established, GSTM1-null was reported to be associated in some studies with increased susceptibility to inflammatory pathologies and increased risk of smoking-related cancers [16] . GST is a family of enzymes that detoxify reactive electrophiles, particularly present in tobacco smoke, reactive oxygen species, and known or suspected carcinogenic compounds Modulation of DNA damage and mutation caused by polymorphisms in detoxification enzymes, including the glutathione S-transferases (GSTs), is a well-established risk factor for tobacco-related carcinogenesis, and a similar change in cellular damage may be involved in the risk of disease associated with tobacco smoking [17] . Of four classes of GST isoenzymes, null genotypes of GSTM1 which belongs to the µ class and of GSTT1 which is in the class are associated with increased risk of smoking- related cancer [18] . The free radical scavenging enzymes, GSTT1 and GSTM1, have an important role in detoxifying systems due to their ability to convert the carcinogenic compounds to excretable metabolites. There is evidence that GSTs are candidate genes on cancer susceptibility. As GST enzymes malfunction in the case of GST homozygous null polymorphism, the risk of cancer may increase in an individual who have GSTs null polymorphisms [19] . GSTT1 gene on chromosome 22q11,2 has two classes denoted as GSTT1-null homozygote for the null allele (GSTT1-0), and GSTT1-1 with at least one functional allele. The GSTT1 enzyme encoded by GSTT1-1 catalyzes the detoxification of monohalomethanes and ethylene oxide, present in cigarette smoke [20] , and deficiency of its activity is associated with high risk of smoking-related cancers [21] .

Tobacco smoking is a strong risk factor in the development of cancers. Tobacco is consumed both in smoking and smokeless forms. Tobacco smoke comprises nearly 60 carcinogenic compounds whereas its unburned form contains 16 identified carcinogens [22] . Among these, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and polycyclic aromatic hydrocarbons (PAH) are considered to be the most important causative agents for the development of lung cancer. PAH require metabolic activation and subsequent binding to DNA (forming bulky “PAH-DNA adducts”) to exert their carcinogenic action [23] . Similar activation of one of the N-nitrosamines, NNK, by the P450 system produces metabolites that form methyl and pyridyloxobutyl DNA adducts. Detoxification of these toxic metabolites occurs via the action of multiple Phase II enzymes, most notably the glutathione-S-transferases. There are many studies in the literature that analyze the effects of GSTT1 and GSTM1

^*OR: Odd ratio ^#RR: Risk ratio ^{^}RD: Risk difference.

polymorphisms on the risk of developing different disease. When the findings of them are examined in detail, it can be seen that different results exist. As the GSTs are important in the detoxification of toxic molecules in tobacco, their variant genotypes should be investigated together in susceptibility to cancer.

The underlying reasons of these differences were the sample size of study, ethnic differences, the smoking habits (heavy/mild/light smoker), the type of endogenous and exogenous genotoxicants and exposure time to them as well as genetic structures of enzymes having role for the detoxification. The genetic structure (in terms of mutation and polymorphisms) of other enzymes having a role for the detoxification, diet and the exposure time to the endogenous and exogenous genotoxicants may affect the age of onset and type of cancer among different populations.

The frequency of the GSTM1 null genotype was more frequent among non-smoker individuals (14%) than the smoker individuals (1%). Similarly the higher frequency of GSTT1 null genotype was reported among nonsmokers (Females) (28%) than the smokers (6%). While the distribution of GSTM1 and GSTT1 null genotypes were described previously for different population as depicted in the Figure 4 and Table 8 but no data has been published from Tabuk Region of northwestern Saudi Arabia.

We investigated the polymorphism at GST loci in the populations of Tabuk Region in northwestern Saudi Arabia. The frequency of the GSTT1 null genotype in the present study was significantly higher than those in the populations of North Indian, South India, England, Turkish-USA, Brazil, Italy, Caucasians and Africa whereas the frequency of null genotype in the present study was significantly lower than those in Japan and China as depicted in the Table 8. The frequency of the GSTM1 null genotype in the present study was significantly lower than those in the populations of rest of the world except Gambia.

Comparing the frequencies of GSTM1 deletion in our study with other studies, we found homozygosity generally comparable with Gambia. Similarly comparing the frequencies of GSTT1 deletion in our study with other studies, we found homozygosity generally comparable with Tunisians (37%) Bahraini (29%) and Lebanese

(37%) and Egypt (29%). Statistically significant differences were detected when we compared smokers and non smokers (P < 0.001), which may be due to gender and ethnic mixture in this population. The homozygosity for the GSTT1 deletion in the four populations Tunisian (37%) Bahraini (29%) and Lebanese (37%) and Egypt (29%) were comparable to those reported for Tabuk population of Saudi Arabia. GSTM1 null genotype are not uniformly distributed among diverse population, but an ethnic and geographic basis of distribution was suggested.

There are wide variations in the frequencies of GSTT1 deletion (GSTT1*0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes.

The polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Nevertheless the activity of these enzymes may differ due to polymorphisms which ultimately results in interindividual susceptibility to cancer development. Our results provided useful information in selecting target SNPs that are likely to have an impact on GST activity and contribute to an individual’s susceptibility to the disease. Furthermore, protective effect of GSTT1 was strongly associated with smokers only. It is suggested that GSTM1 and GSTT1 genotypes do not individually influence susceptibility to develop disease.