Abstract
In this study, we aimed to investigate disulfide/thiol homeostasis in trichloroethylene (TCE) exposure. The study was carried out in 30 nonsmoker TCE-exposed workers with a variety of occupations. Additionally, 30 healthy nonsmoker volunteers were recruited as the control group. TCE exposure was determined by measuring urinary trichloroacetic acid (TCA) concentration. Median urinary TCA levels of exposed workers (20.5 mg/L) were significantly higher than control subjects (5 mg/L). Thiol and disulfide concentrations were determined using a novel automated method. Disulfide/thiol ratio was significantly higher in the exposed group (p < 0.001). Thiol/disulfide homeostasis was found to be disturbed in TCE-exposed workers. We predict that in TCE-exposed workers this disturbance can be a therapeutic target, and the efficiency of the treatment can easily be monitored by the novel method we used.
Introduction
Trichloroethylene (TCE) is a colorless, volatile, chlorinated organic solvent and is also a widespread environmental contaminant. It has been considered as a human carcinogen (group 1) by the International Agency for Research on Cancer (IARC). 1 In many countries, TCE is commercially produced since 1920 by chlorination of ethylene or acetylene. It has been extensively used in industrial applications such as degreasing and computer chip manufacturing. 2,3 Its use in dry cleaning began in the 1930s but was later replaced by tetrachloroethylene in 1950s. 4 Today, TCE may exist in many products such as printing inks, paints, glues, spot removers, varnishes, and lacquers. Thus, TCE contamination of surface water and groundwater is a major source of pollution. 5
Although TCE and other organic solvents are ubiquitous and have been broadly used in human activities, they comprise a number of potential adverse health effects including immunologic, respiratory, carcinogenic, neurologic, reproductive, cardiovascular, and hematologic effects. 6 TCE has two major urinary metabolites, namely trichloroethanol (TCEH) and trichloroacetic acid (TCA), which are also used as biomarkers of exposure. These metabolites are thought to have important roles in TCE-induced toxicity in human tissues. 7 Oxidative stress has been proved to be one of the main mechanisms of toxic effects of organic solvents by several researchers. 8,9 TCE induces oxidative DNA damage and lipid peroxidation by its metabolites, and this has been implicated either in its toxicity or in carcinogenecity. 10
Oxidative stress is a situation that can be described as an imbalance between oxidant and antioxidant systems favoring the former. 11 This imbalance exists either by the overproduction of reactive oxygen species (ROS), such as superoxide radical (O2−) and hydroxyl radical (OH−), or by the impairment in the elimination of ROS by antioxidant mechanisms. 12 The term “thiol” is used for compounds containing sulfur, and they are important members of the antioxidant cascade as they can destroy free radicals by either enzymatic or nonenzymatic ways. 13 Glutathione (GSH) is the most abundant form of thiol in human cells and maintains an optimal intracellular redox environment for cell function. During oxidative stress, the thiol groups of GSH are oxidized and two GSH molecules form glutathione disulfide. 14 Formed disulfide bonds again could be reduced to thiol groups and so “thiol–disulfide homeostasis” is maintained. Although thiol–disulfide ratio as an index of antioxidant/oxidant status may play an important role in detoxification, signal transduction, regulation of enzymatic activity, and apoptosis, an abnormality in this homeostasis may result with a variety of disorders. 15
In this study, we aimed to investigate the association between biomarker of occupational TCE exposure and thiol–disulfide exchanges. To the best of our knowledge, this study is the first report on the correlation between TCA, a biomarker for occupational TCE exposure, and thiol–disulfide ratio.
Materials and methods
Study population
Thirty male workers who applied for medical examination to Ankara Occupational Disease Hospital and who had high levels of TCA according to the American Conference of Governmental Industrial Hygienists (ACGIH) threshold value (>15 mg/L) were included in the study as the exposed group. Among these 30 employees, 8 were painters, 4 were electric technicians, 8 were welders, and 10 were laboratory technicians. Their mean duration of employment were 16.26 ± 8.02 years. Thirty healthy volunteers, working as office staff, were recruited as the control group. The exclusion criteria for both groups were existence of recent acute or chronic disease and drug usage. Additionally, considering the effect of smoking on thiol levels, both study and control groups were chosen from nonsmokers. Written informed consent was obtained from all the participants, and this study was approved by the Ethical Committee of Yıldırım Beyazıt University.
Methods
Morning voiding urine samples were collected in sterile polypropylene bottles from all participants at the end of the shift week. Blood samples were drawn to 16 × 100 mm tubes with red caps without gel (BD Vacutainer, New Jersey, USA). After at least 30 min of incubation, the specimens were centrifuged at 1500g for 10 min. Serum and urine samples were immediately frozen and kept at −80°C until analysis. Urinary TCA levels were detected by gas chromatography (GC)/mass spectrometry head space (Agilent, Tokyo, Japan) using a commercial kit (Eureka, Italy) following manufacturer’s introductions. Briefly TCA, after dilution, is treated with a stabilizing and derivatizing reagent for 1 h at 100°C, and this solution is directly injected into GC. Sensitivity and dynamic range of method are 0.01 mg/L and 0.01–120 mg/L, respectively.
For serum disulfide/thiol homeostasis measurement, we used the method described by Erel and Neselioglu, 16 which is automated using Cobas c501 (Roche Diagnostics, Indianopolis, Indiana, USA). Briefly, 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB; 10 μL, 10 mM in methanol) is used for determining functional thiol groups. A linear standard curve was drawn using 2-mercaptoethanol solutions as calibrators. For samples, two parallel vessels were used. For determining total thiol, 10 μL sample was treated with 10 μL sodium borohydride in 50% methanol–water solution (v/v; R1), which reduces dynamic disulfide bonds to free thiol groups. Excess reductants were eliminated using 110 μL 6.715 mM formaldehyde and 10.0 mM ethylenediaminetetraacetic acid (EDTA) in Tris buffer 100 mM (pH 8.2). For native thiol, 10 μL sample was treated with 10 μL, 10 mM sodium chloride in 50% methanol–water solution (v/v; R1′) and 110 μL 6.715 mM formaldehyde and 10.0 mM EDTA in Tris buffer 100 mM (pH 8.2). Then, the DTNB solution is added. The first absorbance was taken after only adding R1 and R1′ for total and native thiol, respectively, and the second absorbance was taken after the application of formaldehyde and DTNB solutions, and when the reaction trace draws a plateau (assay duration is approximately 10 min). First absorbance was subtracted from the second. The main wavelength is 415 nm, and the secondary wavelength is 700 nm (optionally bichromatic). All the chemicals were purchased from Merck Chemicals (Darmstadt, Germany) and Sigma-Aldrich Chemie (Milwaukee, Wisconsin, USA). Half of the difference between total thiol and native thiol gives the disulfide amount. Disulfide (SS) amounts, disulfide/total thiol percent ratios (SS/SH + SS), disulfide/native thiol percent ratios (SS/SH), and native thiol/total thiol percent ratios (SH/SH + SS) were calculated.
Statistical analyses
Statistical analyses were performed using GraphPad Prism version 6.00 for Windows (trial version, GraphPad Software, La Jolla, California, USA). Coherence to normal distribution was determined using Shapiro–Wilk test. Normal values were presented as mean ± SD, or in the case of non-normally distributed data, as median (minimum–maximum) and interquartile range. For parametric variables, the presence of a statistically significant difference between the groups in terms of continuous variables was examined with Student’s t–test, and Mann–Whitney U test was used for nonparametric variables. Correlation between nonparametric variables were checked using Spearman’s correlation analysis. All results were accepted statistically significant if p < 0.05.
Results
The results of the biochemical measurements and the comparisons between the groups are summarized in Table 1. There was no significant difference between the groups in the name of duration of employment or age. Native thiol, total thiol, and native thiol/total thiol levels of exposed group were significantly lower than control group, while disulfide level in disulfide/native thiol and disulfide/total thiol ratios were significantly higher (Figure 1).
Comparison of parameters between the groups.a
aValues presented as mean ± standard deviation and median (minimum–maximum) [interquartile range].
Mean percent ratios of disulfide to native thiol and total thiol (error lines represent ± standard deviation).
There was a negative correlation between native thiol, total thiol, native thiol/total thiol, and TCA values (r = −0.403, p = 0.001; r = −0.316, p = 0.014; r = −0.336, p = 0.009, respectively). Also a positive correlation was detected between disulfide/native thiol, disulfide/total thiol, and TCA values (r = 0.335, p = 0.009; r = 0.338, p = 0.008). There was no correlation between TCA and disulfide levels (r = 0.206, p = 0.114; Table 2).
Correlation between TCA and other parameters.
TCA: trichloroacetic acid.
Discussion
TCE can be released to the environment in the course of its production, formulation, and use. Besides the occupational exposure in industrial settings, the most important routes of exposure to TCE for general population are ingestion of contaminated drinking water and inhalation of the compound in ambient air. The target for TCE toxicity is central nervous system, as indicated by several available human and animal data. 17 Indeed, with the exception of nonspecific effects on the neurologic system, the most common organ-specific toxicity of TCE, for example, nephrotoxicity and hepatotoxicity, is induced by its metabolites. 18
TCE is metabolized mainly by hepatic oxidation to trichloroacetaldehyde, which can be either reduced to TCEH or further oxidized to TCA. Both metabolites are excreted in urine. 19 According to the ACGIH recommendations, we used urinary TCA levels at the end of the workweek as a metric for assessing occupational TCE exposure. 20 In our study, mean urinary TCA levels of workers who were exposed to TCA (20.5 mg/L) were significantly higher than the control subjects (5 mg/L). The mean urinary TCA levels in this study was lower than that reported by Cheng et al. 21 but was similar to that reported by Singthong et al. 22
The mechanism underlying the toxicity of TCE is diverse and not definitely understood yet. As it was reported by previous studies, exposure to volatile organic solvents induced oxidative stress in animal and cell line studies. 23,24 TCE was shown to exert DNA damage and genotoxic effects, probably, by oxidative stress in a cell line study. 25 8-Hydroxydeoxyguanosine (8-OH–dG) is an important biomarker of DNA oxidation indicating generalized oxidative stress. Abusoglu et al. reported that urinary 8-OH–dG levels in TCE-exposed workers were significantly higher than control subjects. 7 As an add-on to these reports, in this study, we have investigated the disulfide–thiol homeostasis in workers exposed to TCE.
Thiol chemistry is a rapidly growing field in basic and applied bioscience. We used the method developed by Erel and Neselioglu. 16 This method measures dynamic thiol/disulfide homoeostasis in a rapid, automated, inexpensive, and spectrophotometric way. Results are highly reproducible and reliable. In this method, both sides of the homoeostasis (native thiol–disulfide) are determined for the first time in the same assay. 16
Our hypothesis was that disulfide–thiol ratio increases in TCE-exposed workers since previous studies reported a close relation between TCE toxicity and oxidative–antioxidative processes. 10 The role of oxidative stress in the pathophysiology of solvent toxicity also supports our hypothesis. 8,9 We found the serum total and native thiol levels significantly decreased and disulfide levels increased in TCE-exposed workers compared with the control group (Table 1). There was a negative correlation between native thiol, total thiol, and TCA values (r = −0.403, p = 0.001 and r = −0.316, p = 0.014, respectively). As a member of antioxidant cascade, thiol group destroys ROS so total thiol level measurement can be used to evaluate the oxidative status. It is possible to suggest that TCE exposure induced an oxidative stress in the tissues hence the thiol levels decreased. We also found that disulfide–total thiol and disulfide–native thiol ratios in TCE-exposed group were higher than the control group. Also a positive correlation was detected between disulfide/native thiol, disulfide/total thiol, and TCA values (r = 0.335, p = 0.009; r = 0.338, p = 0.008).
The thiol–disulfide equilibrium allows rapid and dynamic regulation, composes redox signaling, and occupies a central place as a target of oxidative stress. This property makes the disulfide–thiol ratio in blood serum useful as a clinical measure of oxidative stress. 16 Recently, Kundi et al. showed that disulfide/thiol ratio increased significantly in coronary atherosclerosis, a disorder that is strongly associated with oxidative stress. 15 Altiparmak et al. reported that plasma disulfide/thiol ratio levels increased in patients with cardiac X syndrome, a disorder characterized by microvascular dysfunction, which is also associated with oxidative stress. 26 The decrease in thiol levels was shown to be related with increased oxidative stress in these patients.
To our best knowledge, this is the first study to show an association between the TCE exposure and disulfide thiol homeostasis in TCE-exposed workers. The novel test we used in this study shows an increase in disulfide bonds that can still be reduced. The restoration of disulfide thiol homeostasis can be a therapeutic target in TCE exposure. There are certain drugs that contain native thiol groups like N-acetylcysteine and α-lipoic acid. N-Acetylcysteine is a drug that contains cysteine amino acid and an acetyl group and can be used to restore thiol levels. 27
In conclusion, thiol/disulfide homeostasis was found to be disturbed in TCE-exposed workers. Theoretically restoring thiol/disulfide hemostasis can be a therapeutic target in TCE exposure, and the novel test we used in this study may provide accurate and precise data for determining and monitoring the in vivo efficiency.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.