International Journal of Mevlana Medical Sciences Advanced Technology and Science
ISSN:2147-8236
www.atscience.org/IJMMS
Research Article
Serum Aspartate Transaminase is not a Useful Diagnostic Biomarker in Industrial Toluene Exposure Engin TUTKUN1,*, Sedat ABUSOGLU2, Hinc YILMAZ1, Aysegul BACAKSIZ3, Fatma Meric YILMAZ4, Tulin SOYLEMEZOGLU5, Ali UNLU2 Received 7th July 2013, Accepted 21th July 2013 DOI: 10.1039/b000000x Abstract: Toluene is one of the most widely used volatile organic compounds (VOCs) worldwide in many industrial fields such as industrial paints, adhesives, coatings, inks, and industrial cleaners and used as a starting material for the synthesis of a wide range of chemicals, including benzene. Hippuric acid is excreted in the urine and considered as colorless crystal structure which is often used in biomonitoring of exposure to industrial toluene. 295 non-exposed control subjects and 85 toluene-exposed workers were included to this study. Blood and urine samples were collected for biochemical, haematological and occupational parameters. According to occupational classification, painters were found to have highest urinary hippuric acid levels. Also, there was a weak positive correlation between serum aspartate transaminase (AST) and urinary hippuric acid levels (r=0.129, p=0.012). Serum AST levels may not be considered as a diagnostic tool for toluene-exposed workers. Keywords: Toluene exposure; Aspartate Transaminase; Workers; Urinary Hippuric Acid; Industrial exposure.
1. Introduction Biological monitoring of exposure to hazardous materials in the workplace is mandatory to ascertain the health effects and risks of the workers. In order to maintain the safe health conditions of a workplace, employers are obligated to inform worker injury and illness records, chemical inventories, inspection reports, records of workers’ exposure to hazardous materials and other related scientific and technical investigations in most of the countries. Although the motivation for biological monitoring arises in clinical medicine, forensic toxicology and industrial hygiene, biological monitoring and health surveillance of the worker should serve as a complimentary, but not the main part of occupational health practice. Organizations and governmental regulating agencies play a key role in the integration of science and policy and their mission is to use the available scientific and technical data to set the recommended or regulatory conditions that will protect the workers’ health against hazardous materials in the workplace [1]. NIOSH (National Institute of Occupational Safety and Health) classifies the main monitoring goals as air monitoring, biological monitoring of exposure and biological monitoring of effect and the main goal of the biological monitoring method has been _______________________________________________________________________________________________________________________________________________________________
a.,
Occupational Diseases Hospital, Department of Occupational Toxicology, Ankara, Turkey b. Selcuk University, Faculty of Medicine, Department of Biochemistry, Konya, Turkey c. Hacettepe University, Faculty of Medicine, Department of Nutrition Health, Ankara, Turkey d. Yildirim Beyazit University, Faculty of Medicine, Department of Biochemistry, Ankara, Turkey e. Ankara University, Institute of Forensic Sciences, Forensic Chemistry and Forensic Toxicology, Ankara, Turkey * Corresponding Author: Email:
[email protected]
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emphasized to be consistent with the target of industrial hygiene investigation. A biological monitoring analytical result is a determinant of the exposure of the period in which the sample was taken and it doesn’t represent the whole working period [2]. On the other hand, workers are involved in more than one facility and exposed to multichemicals. During biological monitorization or health surveillance of a worker, an approved exposure to one or more hazardous industrial chemical which is present in the workplace may be accepted as a clue for further investigation of industrial hygiene conditions or preventive measures of that enterprise. In some cases, if exposure with other routes is possible, simultaneous measurement of air concentrations will not clarify the source. Further steps should be carried carefully for detailed evaluation of the occupational safety conditions, including air monitoring. The multidisciplinary approach has provided a significant improvement in both industrial technology and hygienic practices. Organic solvents represent a group of aliphatic and aromatic organic compounds which are usually lipophilic and more or less volatile. The advent of industry has increased the need for different solvents. Nowadays, they are widely used in different industrial products, including paints, adhesives, glues, lacquers, varnishes and in the production of plastics, textile, printing inks, pharmaceuticals, agricultural products [3]. Millions of workers may also be exposed to mixtures of organic solvents in their daily practise and therefore it is a major occupational health problem [4, 5]. Several epidemiologicial studies indicate that, while some of them display a carcinogenic effect in human, as reported also by IARC (International Agency for Research on Cancer), the others are known to pose toxic effects on physiological system [6, 7]. Toluene is one of the industrial solvents which is used as a chemical substance can cause both acute and chronic adverse IJMMS, 2013, 1(2), 10–14 | 10
effects if workers are not protected during the operation. It is also known as toluol, methylbenzene, phenylmethane, nomethyl benzene. They are non-corrosive, colorless, transparent-looking, volatile, explosive and flammable organic liquid compounds [8]. It is also one of the most widely used VOCs worldwide in many industrial fields such as industrial paints, adhesives, coatings, inks, and industrial cleaners and used as a starting material for the synthesis of a wide range of chemicals, including benzene [9]. It is considered as not classifiable as a human or laboratory animals carcinogen by IARC and WHO (World Health Organization). However, short-term and long-term exposure to toluene (> 100 ppm) can exert toxicological effects for the central nervous system in particular, resulting in confusion, seizure, headache, vertigo, impairment of visuomotor speed, coding speed, shortterm visual memory, although these effects have been studied extensively and well described in humans and animals [10-13]. Hippuric acid (HA) which is also called N-benzoglycin is the end product of toluene. It is excreted in the urine and was accepted as colorless crystal structure which is often used in biomonitoring of exposure to industrial toluene [14]. Several studies have been undertaken to evaluate of HA level for monitoring of exposure to toluene. Even though it is defended that in exposures under 50 ppm, hippuric acid is not a good biomarker and o-cresol should be searched instead; in industrial toluene exposures, generally agreed biomarker is hippuric acid due to our knowledge, toluene intake is transformed into 75% hippuric acid and 25% o-cresol and providing easy measurement in high toluene exposures [15, 16]. In our country, the periodic examination parameters which has been a responsibility of employer does not have a standard guideline. Companies have to employ an occupational physician for surveillance of the health of the workers. The chosen way is mostly to use easy tests such as routine serum chemistries, complete blood count tests. Most of the patients routinely are routinely undiagnosed until the clinical symptoms occur. The aim of this study is to determine the exposure rate of toluenebased on the branches, which is a common ingredient in industrial exposure in our country and to emphasize the importance of selection of ideal biomarkers in industrial toxicology practice. For this reason, the study also investigated the correlations between routine parameters of workers in periodical examinations and hippuric acid levels, which are used as a biomarker for toluene exposure.
2. Materials and Methods 2.1. Study Participants Ankara Occupational Diseases Hospital is one of the three occupational disease hospital and has the largest coverage area by the number of connected city. So, patients from many parts of Turkey and establishments admitted for periodic health examination to hospital which is connected to a total of 67 cities. Therefore, the study plan was designed at Ankara Occupational Diseases Hospital and thus projected that sample group may comprise a large part of the country and approved from the local ethic committee. In this study, we examined 85 toluene-exposed and 295 nonexposed workers on different shifts with occupational exposure
history to organic solvents attended to Ankara Occupational Diseases Hospital clinic service. Hepatitis B and C seropositive participants or having any type of chronical disease, taking any medication and former-current drinkers were excluded from the study. Control group was selected from workers which are not exposed to toluene and other industrial solvents. Urinary hippuric acid levels, which are used as a marker for toluene exposure, liver function tests which include alaninetransaminase (ALT) and AST, creatinine values for renal function tests were recorded. We performed a detailed questionnaire of health status such as cancer history, other chronic diseases, lifestyle, nutrition, smoking habits, occupational exposure. 2.2. Analysis Methods 2.2.1. Hippuric acid in urine Urine samples were refrigerated immediately, transferred to the analytical laboratory and kept frozen until analyzed. Hippuric acid rates were analyzed with “Chromosystems, Agilent 1200 series equipped with an UV detector HPLC with reagent kit for the HPLC-Analysis. This Chromsystems reagent kit provides the rapid and easy determination of the parameters such as hippuric acid. Initially, pipette 1000 µl Internal Standard and add 10 µl urine into reaction vial, vortex briefly. Samples were mixed centrifuged 5 min at 9000 g, then inject 20 µl supernatant into the HPLC system. The UV detector was set at 254nm. The HPLC column was an ZORBAX SB C18, 5 μm, 4.6 mm × 150 mm (Agilent Tecnhnologies, Palo Alto, CA, USA). Chromatography was isocratic in a mobil phase consisting of water-acetonitrile (1:1) at a flow rate of 1 mL/minute. 2.2.2. Biochemical Measurements Whole blood samples were analyzed on the Coulter Gen-S hematology analyzer; blood chemistry such as urea nitrogen (BUN), creatinine (Cr), ALT and AST analyzes were made by Cobas Integra 800 biochemical autoanalyser (Roche Diagnostics, Basel, Switzerland) by using commercial kits supplied by Roche Diagnostic (Mannheim, Germany). 2.3. Statistical Analysis Results are expressed as mean ± SD. “The Statistical Package for Social Sciences for Windows” (SPSS v18) software was used for the statistical analysis. While the mean differences between two groups were compared by using the Student t-test, the Mann Whitney U test was applied for the comparison of median values. The Kruskal-Wallis analysis of variance was utilized for the comparison of more than two groups in terms of metric variables. Apart from all significant tests, Pearson correlation was computed for all parameters. A two-tailed p value of less than 0.05 was considered to indicate statistical significance.
3. Results and Discussion In this study, 380 employees from manufacturing factories in Turkey were included. The ages of the subjects ranged from 32 to 43 years, with a mean ± standard deviation of 10± 2 years. Table 1 represents the distribution of occupational groups that were included in the study.
Table 1. The distribution of occupational groups. Occupational groups
Hippuric Acid
reproductive, neurologic, and cardiovascular effects.
n
%
Painter Engine (Engine-Maintenance-Repair) Carpenter Petroleum- or natural-gas-refining-plant worker Electrical mechanic (Repairs & Manufacturing) Printing Plant Worker Welder Laboratory Workers
146 39 19 9 28 49 43 10
38.4 10.3 5.0 2.4 7.4 12.9 11.3 2.6
Indirect exposure 1600 µg/g.crea.
37 295 85
9.7 77.6 22.3
To examine the dose-dependent effect of exposure to toluene, hippuric acid levels which are < 1600 µg/g.crea. and < 1600 µg/g.crea. were statistical compared with routine blood parameters. There was no statistically significant difference between the levels of ALT, AST, creatinine, White blood cells, Red blood cells, Hemoglobin, Mean corpuscular volume, Hematocrit, Platelet, lymphocyte, monocytes, neutrophils ratio parameters (p>0,05; Table 2).
Figure 1. Urinary hippuric acid levels of different occupational groups.
Table 2. Comparision of biochemical and haematological parameters for subgroups (urinary hippuric acid < 1600 μg/g creatinine and >1600 μg/g creatinine).
Hippuric acid 1600 N=85
P
ALT (U/L)
28.76±23.23
26.91±13.55
0.340
AST (U/L)
30.73±17.75
35.32±25.87
0.113
Creatinine (mg/dL)
0.92±0.77
0.97±1.06
0.586
White Blood Cells (109/L)
7.76±1.64
7.59±1.98
0.374
Red Blood Cells (1012/L)
5.1±0.40
5.05±0.36
0.271
Hemoglobin (g/dL)
15.51±1.05
15.35±1.18
0.076
Mean Corpuscular Volume (fL)
86.36±5.09
85.2±9.21
0.136
Hematocrit (%)
43.92±2.79
43.25±4.24
0.07
Platelets (109/L)
222±50
217±49
0.342
Lymphocytes (%)
30.40±7.55
30.88±7.52
0.539
Monocytes (%)
6.75±1.78
6.84±1.43
0.584
Neutrophils (%)
59.16±7.70
58.62±7.76
0.496
Figure 1 represents the comparision of parameters for hippuric acid urine levels which are < 1600 μg/g.crea. and >1600 μg/g.crea. between different occupational groups. Painters were the most occupationally exposed group in these nine sectors, when hippuric acid levels were compared in terms of occupational groups (p=0,0001). There was a positive correlation between serum aspartate transaminase (AST) and urinary hippuric acid levels (r=0.129, p=0.012) (Figure 2). There was no statistically significant difference for smoking habit between sub-groups. Urinary hippuric acid levels of painters, petroleum or natural-gas-refining-plant workers and printing plant workers were statistically higher compared to other occupations (p=0.0001).
4. Conclusion Volatile organic compounds (VOCs) are toxic pollutants ubiquitous in the general environment. Exposure to them has been known to result in immunologic, respiratory, carcinogenic, IJMMS, 2013, 1(2), 10–14
Figure 2. Correlation of urinary hippuric acid and serum AST levels.
VOCs are widely used as constituents of household chemicals such as paints, inks, aerosol sprays and fuels, and threaten health, especially of workers exposed to high concentration of VOCs. Benzene, toluene, ethylbenzene, and xylene (collectively BTEX) among VOCs are commonly found in occupational or nonoccupational environments [17]. Toluene is used widely, not only in glue and adhesive industry, but also in house-holds where toluene exposure and abuse can occur. Urinary hippuric acid and o-cresol excretion rates are used as markers for toluene exposure [18]. In a study, Tas U et al. reported that toluene inhalation significantly increased serum ALT, AST and tissue malondialdehyde (MDA), and decreased serum albumin, but did not affect serum alkaline phosphatase (ALP), total bilirubin levels and tissue superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity in toluene-exposed rats compared to control rats [19]. Liu J et al. reported personal exposures to benzene, chloroform, ethylbenzene, tetrachloroethene, toluene, trichloroethene, ortoxylene, meta and para-xylene, 1,4-dichlorobenzene, and methyl tert-butyl ether (MTBE) and the relationship with biochemical parameters. The first two canonical correlations were 0.3218 and 0.2575, suggesting a positive correlation mainly between the six VOCs (benzene, ethylbenzene, toluene, o-xylene, m-,p-xylene, and MTBE) and the three biochemical liver tests (albumin, ALP, and gamma glutamyl transferase (GGT)) and a positive This journal is © Advanced Technology & Science 2013
correlation mainly between the two VOCs (1,4-dichlorobenzene and tetrachloroethene) and the two biochemical liver tests (Lactate dehydrogenase (LDH) and Total Bilirubin). Subsequent multiple linear regressions show that exposure to benzene, toluene, or MTBE was associated with serum albumin, while exposure to tetrachloroethene was associated with LDH and total bilirubin. In conclusion, exposure to certain VOCs as a group or individually may influence certain biochemical liver test results in the general population [20]. Although serum AST levels were not determined, the other markers identify the effect of VOCs to liver function. In our study, there was a weak correlation between exposure to toluene and serum AST levels. In a study with 289 printing factory employees, Guzelian P et al found that pericentral fatty liver with mild "reactive hepatitis" is the most likely diagnosis in workers exposed to solvents for whom common causes of mild liver test abnormalities have been excluded. An increased ALT/AST ratio may represent a convenient, previously unrecognized indicator of this condition. Also, in our study, printers were most toluene-exposed group but in contrast to this study we did not find a difference between toluene-exposed and control group. This might be due to minor elevations of liver function tests in our study group [21]. Although the serum AST levels of toluene-exposed group of this study were higher compared to control group, this difference did not represent a statistical significance. In a study with 77 petrochemical industry workers FernándezD'Pool J et al. reported he serum activities of the liver enzymes, the concentration of bile acids and urinary phenols were not influenced by the exposure to the solvents [22]. There are contradictory results about the elevation of liver enzymes and exposure to organic solvents. Although the results are in the reference range, the changes of activities of liver enzymes in the reference ranges may reflect the possible liver injury caused by organic solvent exposure. This may be a limitation of our study. We have no record of date of recruitment liver enzyme values of participants. In addition to concurrent radiological findings for evaluating the exposure, the increases of liver enzymes’ activities may help us to determine the organic solvent exposure independently from general population reference ranges.
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Acknowledgements No funding from any pharmaceutical firm was received for this project, and the authors' time on this project was supported by their respective employers.
Declaration of Interest
[16]
[17]
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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