Association of microsomal epoxide hydrolase gene (fast genotype) with lung functions impairment in wood workers

Introduction

Respiratory health problems were markedly observed in wood industry. During furniture making and construction, wood dust particles proved to contain endotoxin and formaldehyde, exposing wood workers to those pollutants [1], [2]. Recently, it was shown that respirable wood dust proportion varies from 6 to 75% of overall wood aerosol in wood processing [3], [4]. Induction of oxidative stress and reactive oxygen species generation by inflammatory cells activation may help in carcinogenesis process due to inhaled wood dust [5]. A recent study mentioned to an association between wood dust exposure (its bioaerosols) with significant increase in respiratory symptoms prevalence (both acute; moderately reversible, or irreversibly chronic) and reduction in lungs functional capacity [6]. Previous studies revealed an association between respiratory disorders and occupational wood dust exposure as asthma, coughing, bronchitis, and overall reduction in ventilatory functions; inform of fall in forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and peak expiratory flow rate (PEFR). Growing evidence indicates an interaction between susceptibility genes and environmental factors playing an important and crucial role in determining health problems in respiratory system [7], [8]. Macrophages represent an essential component of lung defense mechanism against inhaled particles. They have a main vital role in this process where they can expel foreign particle through the lungs in addition to orchestrating inflammatory response when needed [9]. So, alveolar macrophages had a crucial role in inhaled particulate phagocytosis and clearance, in addition to its main role as mediator for different lungs inflammatory responses to fight against those particles. This could be by releasing various proteolytic enzymes, metabolites of arachidonic acid, cytokines and chemokines [3]. The chemokine macrophage inflammatory protein 2 (MIP-2) can mediate lung neutrophilic inflammatory responses. It is released by many different cells as a result of infection or injury; including monocytes and macrophages [10]. Main source of MIP-2 is macrophages (as a part of their response to inflammatory stimuli). Furthermore, MIP-2 can help in polymorphonuclear neutrophils (PMNs) recruitment to injured or infected sites to modulate both inflammatory and immune responses [11]. It can enhance adhesion molecules integrin expression on neutrophils that augment adhesive interactions between endothelium and neutrophils, besides acting as a potent neutrophil chemoattractant [10]. Microsomal epoxide hydrolase 1 (EPHX1) has crucial role in polyaromatic hydrocarbons (PAHs) activation and detoxification as well as aromatic amines [12], and other pollutants in environment [13]. EPHX1 was shown to be expressed in most tissues, with higher concentration to be found in different organs; such as liver, lungs, gonads, bronchial epithelial cells and kidneys [13]. Different reports mentioned an inter-individual marked variations in EPHX1 that resulted from single nucleotide polymorphisms (SNPs) in the coding and promoter region of this gene [14]. In human, EPHX1 gene is located on chromosome 1q42.1. It is 35.48 kb and consists of eight introns and nine exons [15], [16]. In SNP at exon 3 tyrosine is substituted to histidine at position 113 (Tyr113His), at exon 4 SNP histidine was substituted by arginine at position 139 (His139Arg). In exon 3, 113His allele recorded (slow conjugating allele) with reducing enzyme activity (by at least 50%), while in exon 4, Arg139 allele (fast conjugating allele) showed rising activity at least by 25% [17]. Due to this polymorphisms, an alterations in EPHX1 enzyme activity can lead to variations in inter-individual susceptibility to chemical carcinogen that induce cancers [18], [19], [20].

Aim of the study: Our study aimed to find out associations between exposure to wood dust and the rapid decline in lung functions in exposed workers with EPHX1 genotypes with “slow” and “fast” enzyme activities and MIP-2 release.

Subjects and methods

Before the beginning of the study, an approval from the National Research Centre Ethical Committee was obtained.

Results

No significant difference was recorded in age between workers and controls groups (39.5 ± 10.30 and 36.75 ± 6.68 years respectively, p=0.08). All the included workers were exposed for wood dust for more than five years (18 ± 10.9 years).Highly significant reductions were detected in the predicted values of FVC, FEV1 and PEF of workers compared with the controls. While, levels of MIP-2 showed borderline rise (p=0.05) among the workers than the controls (Table 1). Genotype Frequencies of EPHX in exon 3 demonstrated significant difference in worker and control groups. In the exposed workers, the percent of workers with Tyr–Tyr in exon 3 was much lower and the workers with Hist–Hist in the same exon was much higher compared to the controls. While, no significant difference was detected in the frequencies of EPHX genotypes in exon 4 between the two groups (Table 2). The FVC% and FEV1 in Tyr–Tyr and Tyr–Hist genotypes of EPHX in exon 3 showed significant reduction in the workers relative to controls. Also, there was significant decrease in PEF% in Tyr–Tyr genotype of EPHX in exon 3 in workers compared with controls (Table 3). In Table 4, exposed workers with different genotypes of EPHX exon 4 were found to be suffering of significant reduction in FVC% and FEV1% compared to their controls. Also, significant rise in serum levels of MIP-2 was detected in the workers with Hist–Hist genotype of EPHX exon 4. Significant reduction in ventilatory functions was observed in mutant Arg–Arg genotype of EPHX exon 4. Table 5 showed that in workers with EPHX exon 3, FVC% and FEV1% were significantly correlated with MIP-2 in Tyr–Hist genotype. In workers with EPHX exon 4, concerning the relations of the ventilatory functions and MIP-2 to the duration of exposures, FEV1% demonstrated negative correlation with duration of exposure in Hist–Hist genotype of EPHX exon 4, while, PEF% showed negative correlation with duration of exposure in Hist–Hist genotype of EPHX exon 3 and in Hist–Arg genotype of EPHX exon 4.

Discussion

Wood dust comprises mixture of complex compounds which varies considerably depending on the different species of tree. It includes several biologically active substances which include carcinogenic and genotoxic agents [22]. Previous studies mentioned that occupational exposure to wood dust is associated with respiratory health problems such asthma, chronic bronchitis, cross-shift decline in FEV1, bronchial hyper-reactivity and lung function impairment [7], [23]. Bacterial endotoxin contained in organic dust can represent an essential causative agent which shared in airway inflammation and airflow obstruction [24]. Exposure to that endotoxin may lead to endothelial cell damage, blood leukocytes influx into the airway, in addition to producing different cytokines, and also excessive oxygen metabolites production like reactive oxygen species (ROS) which could induce oxidative injury to lung [24], [25]. Saad and Hammad [26] referred to the importance of excluding smokers in order to eliminate the potential association between ventilator abnormalities and cytogenetic variations resulting from smoking. Accordingly, smokers were excluded from the present study. The present results revealed significant reductions in predicted percentages FVC and FEV1 among the wood exposed workers compared to their controls, that is in accordance with Sriproed et al. [27] and Saad-Hussein et al. [7]. They found significant reduction in FVC and FEV1 in wood workers compared with their controls. Moreover, Jacobsen et al. [23] recorded a dose–response relationship between decline in FEV1 cross-shift in wood workers and duration of wood dust exposure. In our study, we found clearly significant rise in levels of serum MIP-2 in workers compared to controls. That was in accordance with Long et al. [3] who demonstrated that MIP-2 mRNA expression was induced by pine dust and releasing MIP-2 protein in rat alveolar macrophages. The Current study also revealed a significant difference in the frequencies of EPHX genotypes in exon 3 among exposed workers and controls, while non significant differences in the frequencies of EPHX genotypes in exon 4 among exposed workers and controls. Microsomal epoxide hydrolase (EPHX) represented a critical biotransformation enzyme. It had a special and vital role in potential carcinogens detoxification from endogenous compounds in addition to exogenous chemicals, that eventually transform it to metabolites of low toxicity [20]. Xu et al. [16] had demonstrated that this enzyme was expressed in endoplasmic reticulum, microsome and integral membrane, particularly in the lung, kidney, and liver tissues. Alterations in EPHX expression and activity levels, as a consequence of its polymorphisms might result in variations in the detoxification capability in different individuals. That in turn might increase susceptibility to cancer on exposure to chemicals in certain individuals [16], [20]. The present results revealed significant reduction in levels of predicted FVC and FEV1 in Tyr–Tyr and Tyr–Hist genotypes of EPHX exon 3. While significant decline in PEF% in Tyr–Tyr genotype. In a meta-analysis of different studies on patients suffering from COPD, Wu et al. [28] observed limited variation among heterozygotes and homozygotes in exon 3 and 4 for defining disease risk. Zusterzeel et al. [29] concluded that EPHX, under certain conditions can detoxify highly reactive epoxides in order to prevent modification of essential cellular macromolecules. Otherwise, under certain condition, EPHX may activate other compounds, leading it more toxic compound instead of detoxifying it to less toxic one. The current study revealed that exposed workers with different genotypes of EPHX exon 4 suffered from significant reduction in FVC% and FEV1% compared to their controls. Also, significant reduction in ventilatory functions in mutant Arg–Arg genotype of EPHX exon 4 was detected. Hu et al. [30] indicated that cDNA expression In vitro in different reports mentioned that enzymatic activity of EPHX1 is increased by 25% at least in individuals with fast allele (Arg139). Moreover, our findings are in accordance with other findings which mentioned to an association between that polymorphisms and rapid lung function decline and an association between EPHX1 gene and several lung disorders, as well as lung cancer. So, the findings reflect functional direct effect of EPHX1 gene by regulating of lung EPHX1 levels. The present study illustrated high significant elevation in MIP-2 levels in Hist–Hist genotype of EPHX exon 4. According to Hang et al. [25], genotypes with “slow” enzyme activity can cause generation of ineffective metabolites (resulting from inefficient metabolism of ROS) due to organic dust exposure which finally lead to excessive cytokine and chemokine production. The present work revealed that FVC% and FEV1% were significantly correlated with the levels of MIP-2 in Tyr–Hist genotype of EPHX exon 3. A previous study recorded that macrophages can produce neutrophil chemoattractants, which in turn lead to neutrophils recruitment to the lung. That response is reported in wood dust exposure, where relation between elevated MIP-2 levels and neutrophilia was concluded [9]. So, consequently neutrophils can certainly produce convenient mediators which, when produced excessively could lead to damage of the lung tissues. As example, neutrophil elastase production and reactive oxygen species [31] (which represented key neutrophil responses) were associated with different adverse respiratory effects in the form of change in FVC% and FEV1% [9]. Negative relation was seen between duration of exposure and FEV1% in Hist–Hist genotype of EPHX exon 4, while PEF% showed negative relation with duration of exposure in Hist–Hist genotype of EPHX exon 3 and in Hist–Arg genotype of EPHX exon 4.

Owing to the relatively small number of workers with duration of exposure more than five years in this factory (70 workers), this study could be considered a preliminary work to highlight the association between microsomal epoxide hydrolase gene (fast genotype) and decline in lung functions related to wood dust exposure. Future studies with larger sample size are needed to confirm those findings.

Conclusion

The results could be a basis for workers surveillance in occupational settings. It was concluded that in exon 3, wild type genotype Tyr–Tyr and in exon 4, mutant Arg–Arg genotype (fast genotype) showed significant reduction in ventilatory functions. Excessive effective control in most occupation for health risks is required for reducing incidence of illness at workplace and improving workforce health.