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Thiol/Disulphide Homeostasis in Asphalt Workers a

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Ömer Hınç Yilmaz MD , Ceylan Bal PhD , Salim Neşelioglu PhD , Murat Büyükşekerci MD , e

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Meşide Gündüzöz MD , Funda Eren PhD , Lutfiye Tutkun PhD & Fatma Meric Yilmaz MD a

Department of Public Health, Yıldırım Beyazıt University, Ankara, Turkey

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Department of Biochemistry, Occupational Diseases Hospital, Ankara, Turkey

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Department of Biochemistry, Atatürk Educational and Research Hospital, Ankara, Turkey

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Department of Pharmacology, Occupational Diseases Hospital, Ankara, Turkey

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Department of Family Medicine, Occupational Diseases Hospital, Ankara, Turkey

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Department of Chemical Engineering And Bioengineering Division, Hacettepe University, Ankara, Turkey

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Yıldırım Beyazıt University Medical Faculty Biochemistry Department, Ankara, Turkey Accepted author version posted online: 31 Jul 2015.

To cite this article: Ömer Hınç Yilmaz MD, Ceylan Bal PhD, Salim Neşelioglu PhD, Murat Büyükşekerci MD, Meşide Gündüzöz MD, Funda Eren PhD, Lutfiye Tutkun PhD & Fatma Meric Yilmaz MD (2015): Thiol/Disulphide Homeostasis in Asphalt Workers, Archives of Environmental & Occupational Health, DOI: 10.1080/19338244.2015.1076760 To link to this article: http://dx.doi.org/10.1080/19338244.2015.1076760

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ACCEPTED MANUSCRIPT THIOL/DISULPHIDE HOMEOSTASIS IN ASPHALT WORKERS Ömer Hınç Yilmaz MDa, Ceylan Bal PhDb, Salim Neşelioglu PhD c, Murat Büyükşekerci MD d, Meşide Gündüzöz MD e, Funda Eren PhD c, Lutfiye Tutkun PhD f, Fatma Meric

Downloaded by [Texas A & M International University] at 03:41 23 August 2015

Yilmaz MD g a

Department of Public Health, Yıldırım Beyazıt University, Ankara, Turkey.

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Department of Biochemistry, Occupational Diseases Hospital, Ankara, Turkey.

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Department of Biochemistry, Atatürk Educational and Research Hospital, Ankara, Turkey.

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Department of Pharmacology, Occupational Diseases Hospital, Ankara, Turkey.

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Department of Family Medicine, Occupational Diseases Hospital, Ankara, Turkey.

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Department of Chemical Engineering And Bioengineering Division, Hacettepe University,

Ankara, Turkey. g

Yıldırım Beyazıt University Medical Faculty Biochemistry Department, Ankara, Turkey

e-mails (respectively) : [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], fatmamericyı[email protected] Corresponding Author: Dr. Ceylan Bal Mobile phone: +90 505 745 84 38

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ACCEPTED MANUSCRIPT Fax: +90 312 580 84 04 e-mail: [email protected]

ABSTRACT. The aim of this study was to investigate thiol/disulphide homeostasis in asphalt


workers who are exposed to polycyclic aromatic hydrocarbons occupationally. The study was carried out in 34 nonsmoker asphalt workers. Additionally 35 healthy nonsmoker volunteers were recruited as control group. Thiol and disulphide concentrations were determined using the novel automated measurement method. Levels of urinary 1-OH-Pyrene were analyzed by liquid chromatography. Disulphide/thiol ratio was significantly higher in exposed group (p = 0.034). Also a positive correlation was detected between disulphide/thiol ratio and 1-OH-Pyrene values (r=0.249; p=0.036). Thiol/disulphide homeostasis was found to be disturbed in asphalt workers. The novel test we used in this study may be useful for evaluating the oxidative status in PAH exposure. KEYWORDS: Polycyclic aromatic hydrocarbons, asphalt

workers, oxidative stress,

thiol/disulphide ratio.

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Polycyclic aromatic hydrocarbons (PAHs) are a group of carbon and hydrogen containing organic compounds which are broadly distributed and relocated in the indoor and outdoor environment.1 PAHs represent chemicals which are formed during the incomplete burning of


wood, coal, gas, oil, garbage and other organic substances like tobacco and charbroiled meat. They may occur naturally or can be synthetic. PAHs are found widely in air, water and soil and there exist more than 100 different chemical compounds whose health effects individually are not exactly alike.2 Human might be exposed to PAHs by several routes; inhalation of polluted air, ingestion, and dermal contact. Also the occupational exposure to PAHs is crucial and the workers who work in aluminum and steel manufacture, in coke plant are under the risk for skin, lung and bladder cancer. Cigarette smoking is another major route of exposure to PAHs since cigarette coke tar generates a large amount of PAHs.3 Asphalt contains PAHs that are present in the fumes emitted when handling hot products containing bitumen during, for example, road paving or roofing. As a complex mixture, emissions from asphalt fume contain hundreds of distinct compounds. Since the asphalt workers generally are not exposed to high levels to asphalt fume it should be a long-term health effect following chronic exposure by inhalation or skin contact.4 It was reported in several epidemiological studies that a possible association existed between respiratory diseases, lung cancer risk and asphalt fume exposure 5, as reported also by IARC. Thiols, either in cystein or in derived molecules of low and high molecular weight, have higher reducing capacities and they are good nucleophiles as they can easily react with one and two

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ACCEPTED MANUSCRIPT electron mechanisms. They are prone to reversible or irreversible modifications. Thiols form a number of products as a result of oxidation process hence they can react with disulphides via reversible thiol-disulphide exchange reactions.6 Formed disulphide bonds again could be reduced to thiol groups and so thiol-disulphide homeostasis is maintained. An abnormality in this


homeostasis results with a variety of disorders since thiol/ disulphide ratio play critical roles in antioxidant protection, detoxification, signal transduction, regulation of enzymatic activity, apoptosis and cellular signaling mechanisms.7Cigarette smoking involves some species which reacts with thiols and that inactivate enzymes whose function involves thiol groups. Thus damaging effect of smoking occurs via antioxidant depletion and thiol destruction .8 PAHs have known to disrupt redox balance and generate reactive oxygen species (ROS) as the rising oxidative stress later may cause oxidative damage to biological molecules such as DNA, lipid and protein.9 Benzo(a)pyrene , a representative PAH agent, have been reported to generate ROS during biotransformation stages and later lead to the generation of oxidative DNA adducts.10 The aim of this study was to investigate a novel, relatively cheap, readily available and easily calculated oxidative stress marker, thiol/ disulphide homeostasis, in asphalt workers who are occupationally exposed to PAHs and compare the results with control subjects. Methods Study population: 34 asphalt workers who came for periodic evaluation to Ankara Occupational Diseases hospital between 01.02.2015 and 01.04.2015 were enrolled in the study according to the selection criteria as mentioned below. Considering the effect of smoking on PAH and thiol

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ACCEPTED MANUSCRIPT levels, both study and control group were chosen from non-smokers. Mean working duration of workers was 15.2 ± 6.8 years. All the workers had been working for 8 hours/day and 40 hours/week work shift. Information of lifestyle factors (frequency and amount alcohol consumption), dietary PAH exposures (consumption of grilled, barbequed or smoked meat/


chicken/fish within the 72 h), use of protective equipment (gloves, overalls, masks, protective shoes) and specific work tasks performed during the day was collected by the questionnaires. All of the workers reported working with hot asphalt during the study day and were engaged in mixing and paving. 35 healthy volunteers, working as officers were recruited as control group. The selection criteria for both groups were as follows: a) no existence of recent acute or chronic disease such as diabetes, cardiovascular diseases, cancer, rheumatoid arthritis, chronic kidney disease, neurodegenerative disease b) not having any medication like vitamins, N acetyl cystein, lipoic acid or herbal supplement products c) non smokers d) no exposure of dietary PAH within 72 h hence the mean elimination half life of 1-OH-Pyrene is 18h and is thought to be eliminated totally from the body in four half time period. The study was approved by the Ethical Committee of Yıldırım Beyazıt University and informed consent was obtained from all patients. Collection of biological samples: All samples were taken at the end of the shift week. Urine samples were used for 1-OH-Pyrene (1-OH-PY) and creatinine analyses, serum samples were used for disulphide and thiol measurements. Morning voiding urine samples were collected in sterile polypropylene bottles from all participants and frozen −80◦C until analysis. Blood samples were drawn to 16х100 mm

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ACCEPTED MANUSCRIPT tubes with red caps not containing gel (BD Vacutainer). Serum samples were separated after centrifugation at 1500 g for 10 min and stored at −80 ° C until the analysis time. Biochemical analysis Urinary 1-OH-Pyrene (1-OH-PY) levels detected on high performance liquid chromatography


(HPLC) (Agilent, Tokyo, Japan) using fluorescence detection with a commercial kit (Euraka, Italy). In this method, 1-hydroxypyrene, after enzymatic hydrolysis at pH=5.0 and after purification in a clean up column it is injected into the HPLC System. Urine creatinine levels were studied by enzymatic method with Vitros 5.1 FS device (Ortho-Clinical Diagnostics, Rochester NY). For serum disulphide/thiol homeostasis measurement we used the spectrophotometric method described by Erel and Neşelioğlu 11. Briefly; reducible disulphide bonds were reduced to form free functional thiol groups. Unused reductant sodium borohydride was consumed and removed with formaldehyde, and all thiol groups including reduced and native thiol groups were determined after the reaction with 5,5’-dithiobis-(2-nitrobenzoic) acid (DTNB). Half of the difference between the total thiols and the native thiols was recorded as the dynamic disulphide amount. After the native thiols (SH) and total thiols were determined, disulphide (SS) amounts, disulphide/total thiol percent ratios (SS/SH+SS), disulphide/native thiol percent ratios (SS/SH), and native thiol/total thiol percent ratios (SH/SH+SS) were calculated. Statistical Analyses Statistical analysis of data was made by using SPSS (Version 15.0) (SPSS Inc, Chicago, IL, USA) package program. Coherence to normal distribution analysis was made by using

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ACCEPTED MANUSCRIPT Kolmogorov-Smirnov test. Values were presented as mean±SD or in the case of non-normally distributed data, as median (minimum-maximum). The presence of a statistically significant difference between the groups in terms of continuous variables was examined with Student’s t test for parametric variables and Mann–Whitney U test for non-parametric variables. Spearman’s


correlation analysis was also performed. All results were accepted statistically significant for p