International Research Journal of Environment Sciences_________________________________ ISSN 2319–1414Vol. 1(5), 48-53, December (2012) Int. Res. J. Environment Sci. International Science Congress Association 48 Relative Study on Blood BTEX, Testosterone Hormone, Kidney and Liver Functions in Gasoline Station Workers, Thailand TunsaringkarnTanasorn, Zapaung Kalaya and Rugsiyothin Anusorn College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, THAILAND Available online at: www.isca.in Received 07th November 2012, revised 24 November 2012, accepted 2 December 2012 Abstract Benzene, toluene, ethylbenzene and xylene are volatile organic compounds (VOCs), usually referred to as BTEX. These compounds can affect human health upon its dose and time of exposures. VOCs are usually found in fuels and other solvents which commonly presented in the environment, however, little known about their effects on the endocrine system. This study aimed to evaluate the relationship between blood BTEX and testosterone hormone, and kidney and liver functions of gasoline station workers. The results showed average blood benzene, toluene, ethylbenzene and m-, p-xylene. o-xylene levels were 284.9, 201.3, 178.7, 35.9, 73.3 µg/L respectively. While average testosterone, blood urea nitrogen (BUN), creatinine, serum glutamic pyruvic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase (ALP) levels were 13.4 nmole/L, 11.8 mg%, 1.0 mg%, 26.0 U/L, 30.9 U/L and 71.8 U/L respectively. Blood testosterone level was inversely related to toluene, m-, p-xylene, o-xylene and total BTEX levels (Linear regression analysis, p0.05). In addition, testosterone level had strongly inverse-relationship to kidney function of BUN and creatinine (linear regression analysis, p0.05 and p0.01). In conclusion, this study supported that BTEX exposures were chronically affected the decreasing of testosterone level in reproductive system as well as to kidney function.Keywords: BTEX, testosterone, liver, kidney, gasoline station. Introduction BTEX (benzene, toluene, ethylbenzene, and xylene), is a group of compounds which belonged to the broader category of volatile organic compounds (VOCs). Benzene is a known carcinogen, and has also been shown to cause blood disorders and impact the central nervous and reproductive systems. Toluene may affect the reproductive system (including an increased incidence of spontaneous abortions) and central nervous system (CNS dysfunction depression and narcosis)3-4. Ethylbenzene and xylene may cause some respiratory effects such as throat irritation, chest constriction, irritation of the eyes, and neurological effects such as dizziness. Chronic (long-term) exposure to ethylbenzene by inhalation in humans has shown conflicting results regarding its effects on the blood, liver and kidney5-8. BTEX compounds can be emitted during various oil and gas operations activities, including flaring, venting, engines, produced water storage tanks, and during the dehydration of natural gas. The use of biomass energy has many unique qualities that provide environmental benefits as well as health affectsThe respective agencies in the government should build up the environmental awareness among people as other country10Testosterone is one of six major androgen hormones made in the interstitial cells which stimulate secondary sex characteristics in males. it helps stimulate spermatogenesis in the testes (with FSH) and associates with sex drive11-12. Women's ovaries and adrenal glands share the responsibility for testosterone production for maintain muscle, ovarian function, bone strength, and contributes to sex drive or libido. Testosterone level of women is one-seventh the amount of testosterone each day that men do13. There were some evidences of solvent exposures on reproductive endocrine effects of lower preovulatory LH levels among women14, lower prolactin and cortisol in cleaning task workers15 and that may disrupt endocrine function in natural animal populations and humans16. Although endocrine disrupting chemicals (EDCs) have been know for many years, but this study has only recently attracted worldwide interest due to growing concerns about the relation of BTEX exposures and one of male sex hormones (testosterone) and its relations to kidney and liver function in gasoline workers. Our object of this study was to study the relationship between blood BTEX and testosterone hormone, and between testosterone and kidney and liver functions of gasoline station workers who directly and continuously exposed to these VOCs. Material and Methods Population Study: A cross sectional survey was conducted by collecting 105 gasoline station workers (80 men and 25 women) of 11 gasoline stations located in Pathumwan area (figure-1), central Bangkok, Thailand. All subjects were healthy and had worked at the gasoline stations for more than six weeks. They International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 1(5), 48-53, December (2012)Int. Res. J. Environment Sci. International Science Congress Association 49 were provided with the consent form before the study. Permission to conduct the biological monitoring from human subjects in this study was approved by the Ethical Review Committee for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University. Blood Sample Collection: Blood collection were conducted during their work shift (normally of 6-8 hours shift), using glass heparinize vacuum blood tube and stored at -20 oC before BTEX analysis.Biological Analysis: The kidney function tests [blood urea nitrogen (BUN) and blood creatinine], liver function tests [alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT)] and testosterone hormone were performed at Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University under standard laboratory quality control process. Blood BTEX Analysis: The BTEX analysis was performed using the headspace-solid phase micro-extraction (HS-SPME) technique17. The quantity of blood benzene was analyzed under relative intensity of chromatographic signal for 40 minute. The Limit of Detections (LODs) of benzene, toluene, ethylbenzene and xylene were 10.00, 5.00, 20.00 and 10.00 g/L (ppb) respectively and average coefficient of determination (r) was 0.9988286 for four chemicals (-and - xylene appeared with the same peak). Statistical Analysis: All the statistical analyses were performed using the SPSS 17.0 for Windows Program. Descriptive statistic was used for the characteristics and blood biological levels of gasoline station workers. The relation between testosterone and BTEX levels and between testosterone and biological levels of kidney and liver function tests were analyzed by Multiple Linear Regression. All statistical significant value accepted at 0.05.Figure-1 Study area of Pathumwan area (• gasoline station) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 1(5), 48-53, December (2012)Int. Res. J. Environment Sci. International Science Congress Association 50 Results and Discussion Characteristics of Gasoline Workers: Of the total of 105 gasoline station workers, their mean of age, BMI and work period were 29.9 years, 23.2 kg/m and 5.3 years, respectively (table 1). The general characteristics of age and work period of gasoline station workers were not difference between genders. There were 47.8% smoking and 98.0% drinking in men which were significantly higher than women (Chi-Square Test, p0.001) while the BMI was significantly higher in women than in men (Independent t-test, p0.05). Blood Chemical Levels of Gasoline Workers: Most of blood chemical levels in men were significantly differences from women workers except blood benzene concentration (Independent t-test, table 2). Most of blood BTEX exposure levels were higher in women than in men workers, except benzene. It could be caused by the overall complexity of heart rate dynamics which are higher in women than men18. Blood benzene levels of gasoline station workers found in our study was 5.7 times of the BEI reference19. Testosterone, chemicals of kidney and liver function levels were also higher in men than women workers. Relationship of Testosterone Levels and Characteristics of Gasoline Workers: The relations between testosterone level, BMI, smoking and drinking showedstatistically significances at p0.01, p0.05, p0.05 respectively (table 3). Testosterone level was significantly inverse associated with BMI, which supported previous study of inverse correlation between testosterone and obesity20. There were high correlation of testosterone levels with body composition and its contribution to the balance of lipid metabolism. Moreover, testosterone was positively correlated to smoking and drinking of workers, as reported by Shiels et al21Relationship of Testosterone Levels and BTEX Exposures: The relations between testosterone level, toluene, -, -xylene, -xylene, total xylene and total BTEX statistically showed significances at p0.05, p0.05, p0.05, p0.01 and p0.05 respectively (Linear regression analysis with adjusted for smoking, drinking, BMI and work period) (table 4). The testosterone levels were significantly inverse related to toluene, xylene and total BTEX. This result supported that toluene and xylene could affect testosterone level while benzene and ethyl benzene may be more specific to be genotoxicity at target organ including their toxicities of hematotoxicity and neurotoxicity22-26. However, high level of ethyl benzene can caused liver and kidney damage and has embryotoxic and teratogenic effects. Table -1 Characteristics of gasoline workers Parameter Men N=80 Women N=25 Mean ±± SD P-value* Age (years) 29.4 ± 9.5 31.3 ± 8.7 29.9 ± 9.3 NS BMI (kg/m 3 ) 22.5 ± 4.4 25.3 5.9 23.2 ± 4.9 0.05 Work period (years) 5.1 ± 6.0 6.0 ± 7.6 5.3 ± 6.4 NS Smoking N(%) 35 (43.8) 1 (4.0) - 0.001 Drinking N(%) 56 (70.0) 7 (28.0) - 0.001 Significant difference between men and women workers Table -2 Blood chemical levels of gasoline station workers Blood chemical levels Men N=80 Women N=25 Mean SD P-value* Benzene (µg/L) 275.8 ± 88.2 314.1 ± 150.9 284.9 ± 106.9 NS Toluene (µg/L) 175.0 ± 100.6 285.4 ± 164.9 201.3 ± 127.3 0.01 Ethylbenzene (µg/L) 144.5 ± 99.1 288.1 ± 262.1 178.7 ± 164.6 0.05 m-,p-Xylene (µg/L) 27.8 ± 26.1 61.9 ± 48.1 35.9 ± 35.6 0.01 o-Xylene (µg/L) 55.7 ± 59.2 128.9 ± 126.6 73.3 ± 85.9 0.01 Total Xylene (µg/L) 83.7 ± 84.3 174.5 ± 114.7 105.5 ± 99.8 0.01 Total BTEX (µg/L) 684.7 ± 320.3 1062.1 ± 555.9 775.4 ± 419.5 0.01 Testosterone (nmole/L) 16.3 ± 6.7 1.0 ± 0.9 13.4 ± 8.5 0.001 BUN (mg%) 12.4 ± 3.1 9.8 ± 2.8 11.8 ± 3.2 0.001 Creatinine (mg%) 1.0 ± 0.2 0.8 ± 0.1 1.0 ± 0.2 0.001 SGOT (U/L) 28.1 ± 20.9 19.5 ± 5.8 26.0 ± 18.8 0.05 SGPT (U/L) 34.0 ± 28.1 21.0 ± 10.7 30.9 ± 25.6 0.05 ALP (U/L) 78.1 ± 40.7 51.6 ± 15.9 71.8 ± 38.0 0.01 Significant difference between men and women workers International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 1(5), 48-53, December (2012)Int. Res. J. Environment Sci. International Science Congress Association 51 Table -3 Relation between testosterone levels and the charactersof gasoline station workers Parameter Median Reference Linear regression analysis Standardized coefficients 95% CI P-value B Standard Error Testosterone (nmole/L) M: 15.0 W: 0.8 M: 9.9-27.8 W: 0.2-2.9 Age (years) 29.0 - -0.119 0.110 -0.339 to 0.100 0.283 BMI (kg/m 3 ) 22.1 - -0.177 0.055 -0.286 to -0.667 0.002 Work period (years) 3.0 - -0.014 0.070 -0.153 to 0.125 0.842 Parameter Median Reference Logistic regression analysis Standardized coefficients 95% CI P-value B Standard Error Smoking N (%) 34.3 - 0.051 0.026 1.000 to 1.107 0.052 Drinking N (%) 60.0 - 0.058 0.026 1.000 to 1.116 0.027 Dependence variable: Testosterone Table -4 Relation between blood BTEX and testosterone levels in gasoline station workers Parameter Median Reference Linear regression analysis a Standardized coefficients 95% CI P-value B Standard Error Testosterone (nmole/L) M: 15.0 W: 0.8 M: 9.9-27.8 W: 0.2-2.86 Benzene (µg/L) 280.8 - -0.010 0.008 -0.025 to 0.006 0.218 Toluene (µg/L) 185.8 - -0.017 0.007 -0.030 to 0.003 0.014 Ethylbenzene (µg/L) 150.8 - -0.010 0.006 -0.021 to 0.002 0.096 m-,p-Xylene (µg/L) 30.9 - -0.059 0.025 -0.109 to -0.010 0.019 o-Xylene (µg/L) 43.3 - -0.026 0.010 -0.047 to -0.006 0.013 Total Xylene (µg/L) 75.2 - -0.025 0.009 -0.042 to -0.008 0.004 Total BTEX (µg/L) 713.5 - -0.005 0.002 -0.009 to 0.000 0.019 Dependence variable: Testosterone Adjusted for smoking, drinking, BMI and work periodTable -5 Relation between testosterone and kidney and liver functions in gasoline station workers Parameter Median Reference Linear regression analysis a Standardized coefficients 95% CI P-value B Standard Error Testosterone (nmole/L) M: 15.0 W: 0.8 M: 9.9-27.8 W: 0.2-2.9 BUN (mg%) 12.0 6.0-20.0 0.646 0.263 0.124 to 1.1680.016 Creatinine (mg%) 0.9 0.5-1.5 12.436 4.128 4.237 to 20.634 0.003 SGOT (U/L) 21.0 40 3.455E-02 0.045 -0.055 to 0.1240.446 SGPT (U/L) 24.0 40 4.126E-02 0.035 -0.028 to 0.1110.240 ALP (U/L) 64.0 26.0-117.0 -1.10E-02 0.024 -0.058 to 0.036 0.643 Dependence variable: Testosterone Adjusted for smoking, drinking, BMI and work period International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 1(5), 48-53, December (2012)Int. Res. J. Environment Sci. International Science Congress Association 52 Relationship of Testosterone Levels and Liver and Kidney Functions: The testosterone levels were positively and strongly related to kidney function of BUN and creatinine at p0.05 and p0.01 but not related to liver function (table 5). In addition, testosterone level was also found to be significantly related to kidney function of BUN and creatinine as well as blood BTEX levels by using linear regression analysis. Similar result was done in male rat by Muraoka27. He found the correlation between the serum concentration of total and free testosterone and each parameter of renal function by using linear regression analysisandtestosterone replacement affected the deterioration of renal function in middle-aged rats. However, several reports suggested that testosterone has a stimulatory effect28-29 such as testosterone increases collagen synthesis by vascular smooth muscle cells in culture28, administration of testosterone increases the accumulation of collagen and elastin in the aorta of normal and cholesterol-fed animals31. Conclusion This study revealed that BTEX exposures were chronically affected the decreasing of testosterone level in reproductive system which depended on dose and duration of exposure. But at present study, the testosterone level was still in normal range. It should be followed up of these workers. Female workers may be more affected than male workers cause of higher blood BTEX levels. In addition, testosterone level had relation to kidney function.Acknowledgements This study was suppoteded by Surveillance Center on Health and Public Health Problem Surveillance Center on Health and Public Health Problem under Centenary Academic Development Project, Chulalongkorn University and the College of Public Health Sciences, Chulalongkorn University. Lastly, the authors should thank to Dr. Kriangkrai Lerdthusnee for his advice and rewriting this paper. Abbreviations BTEX: Benzene, Toluene, Ethylbenzene and Xylene BUN: Blood urea nitrogen Cr: Creatinine SGOT: Serum Glutamic Oxaloacetic TransaminaseSGPT: Serum Glutamic Pyruvic TransaminaseALP: Alkaline Phosphatase CI: Confidence IntervalACGIH: American Conference of Governmental Industrial Hygienists ATSDR: Agency for Toxic Substances and Disease Registry NIOSH: National Institute for Occupational Safety and Health US EPA: US Environmental Protection Agency Conflict of Interests: This study was none conflict of interest References 1.American Conference of Governmental Industrial Hygienists, TLVs® and BEIs® based on the documentation of the threshold limit values for chemical substances and physical agents and biological exposure indices, ACGIH, Cincinnati, OH (2010)2.US EPA, Benzene (noncancer effects) (CAS No. 71-43-2) In Support of Summary Information on the Integrated Risk Information System (IRIS), Environmental Protection Agency Washington, DC (2002)3.Agency for Toxic Substances and Disease Registry, Toxicological Profile for Toluene (Update), US Public Health Service, US Department of Health and Human Services, Atlanta, GA (1994)4.US EPA, Integrated Risk Information System (IRIS) on Toluene, National Center for Environmental Assessment, Office of Research and Development, Washington, DC (1999)5.US EPA, Integrated Risk Information System (IRIS) on Ethylbenzene, National Center for Environmental Assessment, Office of Research and Development, Washington, DC (1999)6.ATSDR, Toxicological Profile for Ethylbenzene (Update), Public Health Service, US Department of Health and Human Services, Atlanta, GA (1999)7.National Toxicology Program, Toxicology and Carcinogenesis Studies of Ethylbenzene (CAS No. 100-41-4) in F344/N Rats and B6C3F1 Mice (Inhalation Studies), TR No. 466. Bethesda, MD: US, Department of Health and Human Services, Public Health Service, National Institutes of Health (1999)8.US EPA, Toxicological Review of Xylenes (CAS No. 1330-20-7). In Support of Summary Information on the Integrated Risk Information System (IRIS). US Environmental Protection Agency Washington, DC. Available from: http:// www.epa.gov/ iris/toxreviews /0270tr.pdf. (2003)9.Ansari A., Biomass: Energy and Environmental Concerns in Developing Country,I Res. J. Environment Sci. 1(1), 54-57, (2012)10.Syed Ussain S., Sepuri S. and Buddolla V., Environment and Their Legal Issues in India, I. Res. J. Environment Sci.1(3), 44-51, (2012)11.Kukucka Mark A. and Misra Hara P. HPLC determination of an oxytocin-like peptide produced by isolated guinea pig Leydig cells: stimulation by ascorbate, Arch. Androl., 29(2), 185–90 (1992) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 1(5), 48-53, December (2012)Int. Res. J. Environment Sci. International Science Congress Association 53 12.Khalid A., Structure and Function of Testosterone. Demand Media, Inc, Available from: http://www.ehow.com/about _6869171_ structure-function testosterone.html#ixzz1t8RT2wkA (1999-2012)13.Aphrodite Women’s Health, Testosterone in women, Available from: http://www.aphroditewomenshealth.com /news/20020311214759_health_news.hml (2002)14.Reutman S.R., LeMasters G.K., Knecht E.A., Shukla R., Lockey J.E., Burroughs G.E. and Kesner J.S., Evidence of reproductive endocrine effects in women with occupational fuel and solvent exposures, Environ. Health Perspect.,110, 805-811 (2002)15.Pérez-Cadahía B., Lafuente A., Cabaleiro T., Eduardo Pásaro E., Méndez J. and Laffon B., Initial study on the effects of Prestige oil on human health, Environ. Int., 33(2), 176-185 (2007)16.Porte C., Janer G., Lorusso L. C., Ortiz-Zarragoitia M., Cajaraville M. P., Fossi M. C. and Canesi L., Endocrine disruptors in marine organisms: Approaches and perspectives, Comp Biochem Physiol C Toxicol Pharmacol., 143, 303–315 (2006)17.Tunsaringkarn T., Choochat N. and Theppitaksak B., Headspace – Solid Phase Microextraction for determination of benzene, toluene, ethylbenzene, xylene and mtbe in blood, Thai J Health Res., 18(1), 49-59 (2004)18.Ryan S. M, Goldberger A. L., Pincus S. M., Mietus J. and Lipsitz L. A., Gender- and age-related differences in heart rate dynamics: are women more complex than men?,J Am Coll Cardiol., 24, 1700-1707 (1994)19.ACGIH, Threshold Limit Values and Biological Indices, ed, Cincinnati, OH (2001)20.De Maddalena C., Vodo S., Petroni A. and Aloisi A.M., Impact of testosterone on body fat composition. J Cell Physiol., Wiley Periodicals, Inc. doi: 10.1002/jcp.24096 (2012)21.Shiels M.S., Rohrmann S., Menke A., Selvin E., Crespo C.J., Rifai N., Dobs A., Feinleib M., Guallar E. and Platz E.A., Association of cigarette smoking, alco holconsumption, and physical activity with sex steroid hormone levels in US men, Cancer Causes Control.,20, 877–886 (2009)22.NIOSH, Registry of toxic effects of chemical substances: Ethyl Benzene. Cincinnati, OH: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, Division of Standards Development and Technology Transfer, Technical Information Branch (1991) 23.Snyder R., Witz G. and Golstein B.D., The toxicology of benzene, Environ. Health Perspect., 100, 293-306 (1993)24.Golding B.T. and Watson W.P., Possible mechanisms of carcinogenesis after exposure to benzene, IARC Scientific Publications,150, 75-88 (1999)25.Fabaini R., De Bartolomeo P., Rosigonoli M., Scamosci, Lepore L. and Morozzi G., Influence of culture condition on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells, Environ. Mol. Mutagen., 37, 1-6 (2001)26.Tunsaringkarn T., Suwansaksri J., Soogarun S., Siriwong W., Rungsiyothin A., Zapuang K., Robson M., Genotoxic monitoring and benzene exposure assessment of gasoline station workers in metropolitan Bangkok: sister chromatid exchange (SCE) and urinary trans, trans-muconic acid (t,t-MA), Asian Pac. J. Cancer Prev., 12(1), 223-227 (2011)27.Muraoka K., Effects of Testosterone Replacement on Renal Function and Apoptosis on Mesangial and Renal Tubule Cells in Rats, Yonago Acta medica., 41, 37–44 (2001) 28.Fischer G.M., Bashey R.I., Rosenbaum H. and Lyttle C.R., A possible mechanism in arterial wall for mediation of sex difference in atherosclerosis, Exp. Mol. Pathol., 43, 288–296 (1985)29.Franchimont P. and Bassleer C., Effects of hormones and local growth factors on articular chondrocyte metabolism, J. Rheumatol., 18, 68–70 (1991)30.Leitman D.C., Benson S.C., and Johnson L.K., Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells,J. Cell Biol., 98, 541–549 (1994) 31.Silbiger S. and Neugarten J., The impact of gender on progressive renal functional impairment, Am. J. Kidney Dis., 25, 515–33 (1995)