Oxidative DNA Damage, Oxidative Stress and Genetic Susceptibility-Prognostic Scores in ‘Missing’ COPD Cases

In situ identification of COPD cases at workplace and sample assessment for oxidative stress/ DNA damage as a function of metabolic/antioxidant genotypes (as susceptibility genotypes) offers a novel manner of hazardous workplace-identification for genotoxic/carcinogenic events. In this case-control study, blood/ sera samples of the COPD cases (n=32 identified spirometrically among the stone-crushing workers) and healthy controls (n=19) were assessed for 8-OHdG (DNA damage), glutathione and superoxide dismutase levels (oxidative stress) and genotyped for GSTT1, M1, P1 and MnSOD variants. Significant increase in oxidative damage and lungfunction decline were observed as a function of some genotypes. Predictors of genetic damage included tGSH, SOD and GSTM1. A prognostic index score based on prognostic factors was developed revealing cases were at high(53.12%), intermediate(34.37%) or low(12.50%) risk for progressive DNA damage. These aberrant findings imply workplace exposure. This study provides insight on exposure-effect relationship in workers at stonecrushing sites. Address for correspondence: Gurpreet Kaur Department of Human Genetics, Guru Nanak Dev University, Amritsar143 005, Punjab, India Mobile: 9815963637 E-mail: [email protected] INTRODUCTION Chronic Obstructive Pulmonary Disease (COPD) is an obstructive airway disorder characterized by the slowly progressive and irreversible decrease in forced expiratory volume in one second (FEV1), accompanied by emphysema and chronic bronchitis (Rabe et al. 2007; Cazzola et al. 2015); it develops with decreasing lung function as a function of age in the normal population (Ito and Barnes 2009). Despite the heritability of 40-77 percent, a host of other influences can also exacerbate this condition (Young et al. 2009). Rather the development and progression of COPD involves multiple genes, gene-gene and gene-environment interactions (•idzik et al. 2008; de Jong et al. 2015). Smoking exposure is considered as the most important risk factor for the development of COPD (Chan-Yeung et al. 2007; Kurmi et al.2015) with mild and moderate COPD cases having a three-fold risk of developing lung cancer within ten years, which increases to a ten-fold risk with severe COPD compared to the smokers with normal lung function (ElZein et al. 2012). There also however exists COPDassociated increased mortality from lung cancer in non-ever smokers (Turner et al. 2007; Kiri et al. 2010; Aldrich et al.2015). Despite quarrying and mining activities as important occupational set-ups inducing COPD (Jhoncy et al. 2011; Iftikhar et al. 2009), yet workplace identification of this occupational disease has not come to attention on pursuing literature related to COPD. Also there is uncertainty in prognosis of COPD although a number of validated indices exist (Briggs et al. 2008). As these indices require in-puts from patients and the interpretation by the care-giver/physician, these may not be able to cater to disease-identification in the field. In the present study, the on-site disease-identification using the recommended spirometry evaluation (Briggs et al. 2008; Shiota et al. 2015) to recognize COPD cases at stonecrushing units (dust exposure) was carried out. Such an identification at the workplace gains importance as 50-80 percent of COPD are missedout on the basis of misdiagnosis/co-current diagnosis due to relying on reported symptoms which are not sufficiently sensitive and / or because of failure of persons to report to the health provider (Levy et al. 2009). The effect of various environmental stressors from occupational exposures needs to be assessed for prediction of cancer outcome(s) (Fenech 2002) as 90 percent of cancer is environmental in origin (Hemminki et al. 2006). COPD has also been identified as an independent risk 98 GURSATEJ GANDHI AND GURPREET KAUR factor for lung cancer with inflammation as the pathophysiologic factor for high risk of its progression (Sin et al. 2006; Hillas et al.2015) and smokinginduced COPD associated with lung cancer has also been documented (Koshiol et al. 2009). While mechanisms governing the risk of developing neoplastic disease are not well known (Barreiro 2008), of the various theories, oxidative stress may be playing a pivotal role in its manifestation. In COPD, oxidative stress has been observed as ensuing from decreased FEV1 (Kluchová et al. 2007), substantial inflammatory response increasing cytokines as triggered by exogenous dust particles (Yang et al. 2011) and decreased antioxidants because of depletions of glutathione peroxidase, superoxide dismutase (SOD), catalase, ascorbic acid and vitamin E (Borm et al. 2004). The impaired oxidant-antioxidant status can cause cellular damage; DNA damage can result from the action of reactive oxygen species (Jackson and Loeb, 2001) and is the underlying cause of mutations leading to cancer (Bernstein, 2012).Chromosomal damage (increased micronuclei frequency) in peripheral blood lymphocytes has been extensively used for predicting risk of cancer (Fenech et al. 2011) and oxidative DNA damage is also implicated in carcinogenesis, ageing and age-related neurodegenerative diseases (Fortini et al. 2003, Nishigori et al. 2004). The major form of oxidative DNA damage is 8hydroxy-2‘-deoxyguanosine (8-OHdG) resulting from G→T and A→C base substitutions. It may lead to mutagenesis if unrepaired and is directly correlated with lung carcinogenesis (Gackowski et al. 2003). The lesion 8-OHdG is an established biomarker of oxidative stress/oxidative DNA damage and being potentially mutagenic, it is useful as an intermediate marker of a disease end-point like cancer (Cheng et al. 1992). Therefore in the present study, 8-OHdG level was assessed as a pre-lesion of neoplasia in peripheral blood leukocytes (PBL) of COPDidentified cases at stone-crushing units. Although the leukocytes are not the direct target of the exposure at this workplace, they may possibly be affected by the accumulated unmetabolized toxic compound(s) in the lung (Gackowski et al. 2003). This hence prompted the assessment of oxidative DNA damage in the peripheral blood leukocytes and also because of the nonaccessibility of the target (lung) cells. The biomarkers of exposure and effect, and clinical disease (cancer) may further be influenced by susceptibility genotypes and their gene products as pre-dispositional factors (Their et al. 2003). Also as DNA damage and DNA repair have a major role in carcinogenesis and from occupational settings, the susceptible metabolic genotypes (gene products) may inherently be associated in causing genetic damage. Therefore genotyping of the occupational workforce was carried out for glutathione-S-transferase (GST) gene variants (both for disease susceptibility and genetic damage) since GST alleles have been documented to have an association with COPD (Young et al. 2011b). Furthermore, a reduced expression of these alleles has also been observed in the air passage of COPD patients (Imboden et al. 2007; Lakhdar et al. 2011) and hence the expression of glutathiones-transferases was also estimated. Association of the Val/Ala variants of manganese superoxide dismutase (MnSOD) with lung cancer (Wang et al. 2001) further justified the genotyping of this allele and assessing its expression. Variant forms of these susceptible genes are generally common in the population. Due to their specificities for substrates they interact with during environmental exposures, they can increase the risk for disease-causation (Lan et al. 2000). Incidentally, GST and SOD enzymes are also involved in the metabolic and oxidative stress pathways (Borm et al. 2004), and since stone-crushing is an inflammation-triggering occupation (Vallyathan et al. 1995), the assessment of the amounts of these enzymes was thought appropriate. The purpose of the present study was twofold. On one hand to identify COPD cases from workplace exposure (occupation-related disease) and hence assist in identifying ‘missing’ COPD cases using recommended (spirometry) measurements (Briggs et al. 2008; Young et al. 2011a) and COPD categorizations (GOLD 2003). The other (main) purpose was to determine the propensity (Prognostic Index/score) for genetic damage and by extension an increased likelihood for carcinogenesis as ensuing from the combined effects/interactions of prognostic (risk) factors in COPD cases (the workers exposed to industrial-type prevalent conditions) at stonecrushing units. This entailed the evaluation for the presence of oxidative stress (GSH and SOD) and oxidative DNA damage in workers at stonecrushing units genotyped for the GST and MnSOD genes. GENOMIC DAMAGE AND GENETIC SUSCEPTIBILITY IN COPD CASES 99