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  399  Address correspondence to Viroj Wiwanitkit, M.D., Departmentof Laboratory Medicine, Faculty of Medicine, ChulalongkornUniversity, Bangkok 10330, Thailand; tel 662 256 4136; fax 662 218 3640; e-mail viroj.w@chula.ac.th. Research Note:  Urine Trans,trans-muconic Acid as a Biomarker for BenzeneExposure in Gas Station Attendants in Bangkok, Thailand  Viroj Wiwanitkit, 1  Jamsai Suwansaksri, 2  and Paweena Nasuan 3 1  Department of Laboratory Medicine, Medical Faculty, Chulalongkorn University, Bangkok  2  Department of Clinical Chemistry, Allied Health Science Faculty, Chulalongkorn University, Bangkok  3  King Rama VII Hospital, Chantaburi Province, Thailand  Abstract. The toxicity of benzene, a chemical used in many industrial processes, involves bone marrow depression and leukemogenesis and is associated with damage to multiple classes of hematopoietic cells andhematopoietic functions. Environmental exposure to benzene causes an increased body burden, which isreflected in several biomarkers, eg, urine trans,trans-muconic acid (ttMA). Associated with theindustrialization of Thailand, a developing country in Southeast Asia, workers in many occupations haveacquired substantial risks of benzene exposure. In this study, benzene exposure was monitored by high-performance liquid chromatography (HPLC) of urine ttMA in 79 persons, including 49 controls and 30gas station attendants. In controls, urine ttMA concentration averaged 0.12 (SD ±  0.03) mg/g creatinine;in gas station attendants, urine ttMA concentration averaged 4.00 (SD ±  12.49) mg/g creatinine (p <0.05). Based on these findings, wider use of urine ttMA determination is recommended as a biomarker foroccupational exposure to benzene. (received 31 July 2001; accepted 5 August 2001) Keywords:  benzene, gas station attendants; occupational and environmental health, biomarker Introduction Benzene is an important chemical agent that is usedin many industrial processes. Its toxicity involvesbone marrow depression and leukemogenesis causedby damage to multiple classes of hematopoietic cellsand various hematopoietic cell functions [1].Potential mechanisms whereby benzene can damagecellular macromolecules include (a) covalent binding of reactive metabolites, and (b) the capacity of benzene metabolites to induce oxidative damage [2].Environmental or occupational exposure tobenzene results in increased body burden, which isreflected in several biomarkers of benzene exposure.These include the urinary levels of benzene and itsmetabolites, as well as adducts of reactive benzenemetabolites with cellular constituents. Assays of suchbiomarkers can be used to identify populations withsignificant exposures to benzene, to estimate benzeneexposure levels, and to delineate differences inbenzene metabolism [3,4].Trans,trans-muconic acid (ttMA) is a minor non-phenolic metabolite of benzene that is excreted inurine. Assay of urine ttMA has been recommendedas a biomarker for benzene exposure [5-7]. The urinemetabolites that might serve as indices of occupa-tional or environmental exposures to benzeneinclude phenol, hydroquinone, ttMA, and S-phenyl-mercapturic acid. Of these, urine ttMA concent-ration is increasingly recognized as a reliable bio-marker that is relatively convenient to measure [8,9].Due to the recent industrialization of Thailand,a developing country in Southeast Asia, workers inmany occupations appear to be at high risk forbenzene exposure. We previously provided the initialevidence that Thai mechanics have high exposureto benzene, based on monitoring their urine ttMA concentrations [10]. Among the other occupations with high risk for benzene exposure are gas stationattendants, who are the focus of this investigation. 0091-7370/01/0400/0399, $0.75; © 2001 by the Association of Clinical Scientists, Inc.  Annals of Clinical & Laboratory Science, vol. 31, no. 4, 2001  400 During their daily work, gas station attendantshave direct contact with petroleum products, sooccupational exposure to benzene cannot be avoided.In Thailand, most gas station attendants come fromrural areas where they receive scanty education aboutpersonal health-care. The present report describes a a pilot study to compare urine ttMA concentrationsin a group of gas station attendants versus those in a comparable group of non-exposed control subjects. Materials and Methods Subjects.  The study group included 30 male gasstation attendants who worked full-time at gasstations in the Ladprao district of Bangkok,Thailand. All of the workers were non-smokers incompliance with the strict “no-smoking” rules of gas stations. The stations were located in the businessarea of Bangkok, where the monitored level of atmospheric benzene is 0.76 - 4.14 ppm [11].The control group included 49 healthy men with low risk for benzene exposure. All werevillagers from rural areas without nearby factories. All were non-smokers. These subjects were selectedas a reference group for our laboratory and they wereincluded in our previous study [10]. All of the 79 subjects provided informedconsent. A random urine sample was collected fromeach subject on a workday between 2 pm and 7 pm,about 4 to 8 hr after the subjects started to work.  Assay for ttMA in urine.  Assays of ttMA concentrations in the urine samples were performedby HPLC, as previously described [12]. Briefly, 0.5ml of urine was mixed with 2 ml of tris-HCl buffer(pH 10) containing vanillic acid as an internalstandard. This mixture was percolated through a preconditioned ion-exchange column (Dowex I,100-200 mesh, 1 cm diameter, 10 cm height). Afterthe column was rinsed with phosphoric acidsolution, acetate buffer, and deionized water, theanalyte was eluted in 2 ml of a solution comprising equal volumes of 1.5 M sodium chloride solutionand methanol. Of this, 10 µ l was injected onto a HPLC column (C-18 Lichrocart cartridge, 4 mmdiameter, 12 cm length, Merck Co). The mobilephase consisted of 1% (v/v) acetic acid, 10% (v/v)methanol, and 89% (v/v) 5 mM sodium acetate.The flow rate was 1.2 ml/min. The effluent wasmonitored with a UV detector (model LC-3A,Shimadsu Co.) The analyte (ttMA) and internalstandard (vanillic acid) were eluted at 4.2-4.4 and12.6-13.3 min, respectively. The detection limit was0.05 mg/L. Urine ttMA concentration was reportedas mg/g creatinine. Statistics.  Results were expressed as means ±  SD;the p value was calculated by unpaired t-test; p <0.05considered statistically significant. Results  As listed in Table 1, urine ttMA levels in the groupof gas station attendants averaged 4.00 ±  12.49 mg/g creatinine, which was significantly higher than thelevels in the control group (0.116 ±  0.027 mg/g creatinine, p <0.05).The group of gas station attendants includedone subject with an exceptionally high urine ttMA level (64.2 mg/g creatinine). No explanation of thishigh level was evident. However, even after exclusionof this outlier, the urine ttMA levels in the gas stationattendants (1.93 ±  5.66 mg/g creatinine )  wassignificantly elevated in comparison to the controls. Discussion Because of its occurrence in mineral oil and itsformation in many combustion processes, benzene Table 1. Concentrations of trans,trans-muconic acid(ttMA) in urine specimens from gas station attendantsand non-exposed control subjects.GroupNo. ofUrine ttMA level*subjectsmg/g creatinineControl group490.116 ±  0.027(0 - 0.40)Gas station attendants304.00 ±  12.49§(0 - 64.2)* mean ±  SD, with range in parentheses§ p <0.05 vs controls  Annals of Clinical & Laboratory Science   401 is a widespread environmental pollutant. Sincebenzene is hemotoxic and has been classified as a human carcinogen, it is important to monitor andcontrol exposure to benzene vapor in the environ-ment and especially in workplace air [1,2].In developing countries, there is growing recognition of the public health hazards of benzeneexposure, but relatively few of these countries havepolicies, regulations, and programs to combat theproblem. A main purpose of this study was todocument the increased exposure to benzene in a population of low-skilled workers that is widely distributed throughout the developing world.Gas station attendants can be classified as anoccupation of low-skilled workers at high risk of benzene exposure. They are constantly in contact with petroleum products during their daily work.Compared to mechanics, skilled workers who werepreviously investigated [10], gas station attendantsgenerally have a lower socioeconomic status. Likeother migrants from the rural area to Bangkok [13],they have a relatively low educational background.Therefore, they need instruction about self-care andpersonal hygiene in relation to their work withhazardous chemicals. For these workers, protectiveequipment, such as gloves and masks, is necessary and should be provided. Specific policies and regul-ations regarding benzene exposures are needed forgas station attendants as well as for workers in otherforgotten high-risk occupations.References 1.Irons RD, Stillman WS. The effects of benzene andother leukaemogenic agents on haematopoietic stemand progenitor cell differentiation. Eur J Haematol1996;60(Suppl):119-124.2.Snyder R, Hedli CC. An overview of benzenemetabolism. Environ Health Perspect 1996;104:1165-1171.3.Weisel C, Yu R, Roy A, Georgopoulos P. Biomarkersof environmental benzene exposure. Environ HealthPerspect 1996;104:1141-1146.4.Medeiros AM, Bird MG, Witz G. Potentialbiomarkers of benzene exposure. J Toxicol EnvironHealth 1997;51:519-539.5.Bechtold WE, Lucier G, Birnbuam LS, Yin SL, LiGL, Handerson RF. Muconic acid determinationsin urine as a biological exposure index for workersoccupationally exposed to benzene. Am Indust Hyg  Assoc 1991;52:473-478.6.Lee BL, New AL, Kok PW, Ong HY. Urinary trans,trans-muconic acid determined by liquid chrom-atography: application in biological monitoring of benzene exposure.Clin Chem 1993;39:1788-1792.7.Inoue O, Seiji K, Nakasuka H, Watanabe T, YinSN, Li GL, Cai SX, Jin C, Ikeda M. Urinary ttMA:muconic acid as an indicator of exposure to benzene.Brit J Indust Med 1989;46:122-124.8.Ong CH, Kok PW, Ong HY, Shi CY, Lee BL,Phoon WH, Tan KT. Biomarkers of exposure tolow concentration of benzene: a field assessment.Occup Environ Med 1996;53:328-333.9.Boogaard PJ, van Sittert NJ. Biological monitoring of exposure to benzene: a comparison between S-phenylmercapturic acid, trans,trans - muconic acid,and phenol. Occup Environ Med 1995;52:611-620.10.Suwansaksri J, Wiwanitkit V. Urine trans,trans-muconic acid determination for monitoring of benzene exposure in mechanics. Southeast Asian JTrop Med Public Health 2000;31:587-589.11.Muttamara S, Leong ST, Lertvisansak I. Assessmentof benzene and toluene emission from automobileexhaust in Bangkok. Environ Res 1999;81:23-31.12.Wiwanitkit V, Suwansaksri J, Nasuan P. Feasibility of using trans,trans-muconic acid determinationusing high performance liquid chromatography forbiological monitoring of benzene exposure. J Med Assoc Thai 2001;84(Suppl 1):S263-268.13.Pongpaew P, Tungtrongchitr R, Radomyos P,Vudhivai N, Phonrat B, Himmanngan T, et al.Parasitic infection and socio-demographiccharacteristics of urban construction site workers.Southeast Asian J Trop Med Public Health 1993;24:573-576. Urine trans,trans-muconic acid: a biomarker for benzene exposure 
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