International Journal of Environment Science and Technology Center for Environment and Energy Research and Studies (CEERS) ISSN: 1735-1472 EISSN: 1735-2630 Vol. 3, Num. 2, 2006, pp. 113-117

International Journal of Enviornmental Science and Technology, Vol. 3, No. 2, Spring 2006, pp. 113-117

Evaluation of benzene exposure in adults and urinary s-phenylmercapturic acid in children living in Adelaide, South Australia

^1*A. R. Bahrami and ²J. W. Edwards

¹Department of Occupational Health, Hamadan University of Medical Science, Hamadan, Iran ²Department of Environmental Health, School of Medicine, Flinders University, Adelaide, Australia

Received 13 January 2006; revised 7 March 2006; accepted 15 March 2006; available online 20 April 2006

*Corresponding Author, E-mail:Bahrami@umsha.ac.ir

Code Number: st06014

ABSTRACT

Benzene Exposure was evaluated in adults and children living in Adelaide, South Australia by measuring benzene and urinary s-phenylmercapturic acid (SPMA). To determine of benzene exposure in each subject the personal passive samplers was used and samples were analyzed by gas chromatography system equipped to flame ionization detector. The level of SPMA was determined by competitive enzymelinked immunosorbent assay (ELISA) in children. The mean concentration of benzene in Summer and Winter were 1.62±1.43 and 1.36±0.87 ppb respectively. There was a significant difference between exposure to benzene for subjects with less and more than 6 hours activity over days of week (p<0.05). The mean urinary concentrations levels of SPMA adjusted to creatinene for children that living less and more than 200 meters distance from main road were 1.56 and 4.67 µmol/mol creatinene, respectively and the significant difference was seen in two groups (p<0.005). Data shows, that SPMA can be utilized as a biomarker for exposure to benzene in children. Exposure to benzene is more for children that living near to main road compare to other children. Adults have more activity in out side of home has more exposure to benzene than other people.

Key words: Benzene, s-phenylmercapturic acid (SPMA), trans, trans-muconic acid (TTMA), Biological monitoring, children

INTRODUCTION

Benzene, a component of petrol, is known carcinogen. A link between benzene exposure and leukemia has been established in some studies (Synder, 2000 and Guenel et al., 2002). The major sources of benzene in ambient air of urban area are motor vehicle exhaust and evaporation loss during handling, distribution and storage of petrol (Leung and Harrison, 1998). Biological monitoring of exposure to benzene includes measurement of benzene or its metabolite in blood, urine and exhale air (Weisel et al., 1996; Ong et al., 1996). Measurement of metabolite in urine prefer to analysis of benzene at blood because collection of urine is painless, easy to obtain, low in cost and easily performed even at subjects are far away from laboratory. An alternative approach involves measurement of the metabolites of benzene in urine including phenol, trans, trans-muconic acid(TTMA) and s-phenylmercapturic acid (SPMA) (ACGIH, 2005; Inoue et al. 2000; Boogaard et al., 1996). TTMA and SPMA were proposed to replace phenol in biological monitoring of benzene (Boogaard et al., 1996;ACGIH, 2005) Benzene in the human system is subsequently oxidizedto benzene oxide and in turn conjugated with glutathione to form SPMA that is excreted in the urine (15). SPMA derives solely from benzene metabolism and is a sensitive and specific biomarker of exposed benzene in low level (Van Sitter et al., 1993, Ghittori et al., 1996). American Conference of Governmental Industrial Hygienists (ACGIH) introduce s-phenylmercapturic acid as biological exposure indices for benzene exposure (ACGIH, 2005). Most of studies have measured benzene in ambient air with passive or active sampler in some stations in streets. These studies have been performed at near retail petroleum outlets and in residential streets but few study was carried out concern to exposure to benzene based on people activity. Some studies also reports cancer in children living near to street and stay at traffic road (Duarte-Davidson, 2001 and Pearson, 2000) but researches concern to measurement of benzene metabolite in urine of children isvery low (Amodio, 2001). The objective of this study is to evaluate atmospheric benzene exposure in adults by personal air sampling and determination of urinary SPMA of children group living near to the main road and comparison of results to children living far from the road. This research was done from 2001 to 2002 at South Australia.

MATERIALS AND METHODS

Measurement of benzene exposure

43 adults living in Adelaide were selected to obtain air samples. A questionnaire that has some question about outside and inside activities of each subject over 24 hours in a week was used and completed by each subject. The samples of air were collected in both summer and winter seasons for subjects living in metropolitan Adelaide, using passive sampler tubes based on method T0-15 of United States Environmental Protection Agency (USEPA, 1997). Subjects were aged 22 to 66 years. Samples were collected from each subject daily for one week in each season. Samples tubes were desorbed thermally and transfer to a gas chromatography (Perkin-ElmerATD400) systemwithSGEBP20 capillarycolumn and a flame ionization detector. Benzene concentrations were corrected for exposure period, using the equivalent air flow rate and have been expressed as parts per billion in air (ppb).

Analysis of urinary SPMA in children

Ninety seven urine samples were selected with simple random sampling from a 1300 preschool children in four areas of Metropolitan Adelaide. All children subjects were preschools, aged 4-5 years. There was a questionnaire, about the possible risk factors for benzene exposure. This questionnaire was completed by children parents. Sterile urine containers together with an information sheet and consent forms were distributed to parents via the kindergartens and childcare centers in each of the selected area. All children had no specific exposure to benzene other than that present in the general environment. The analysis of urine samples in this study was performed in January and February 2003. Urinary samples were acidified as soon as possible after collection and stored at -20 °C until dispatch for analysis. A method that recently developed based on competitive enzyme linked immunosorbent assay (ELISA) was used. This method specific for PMA and validated by correlation with GC/MS data (Aston et al., 2002). For analysis of samples, the frozen urine samples were kept at ambient temperature for 30 min and then centrifuged at 1500 rpm for 5 min. Ten micro liter of standards urine, and samples were added to a microtitre plate which had been coated with a PMA-boving serum albumin conjugate. After addition 100 µLofan anti-PMA polyelonal antiserum, the plates were incubated for 1 hour at room temperature. The micro plate was washed with a solution of phosphate buffer salin containing Tween-20 (0.05%), the bound PMA fraction was incubated for a further hour with an alkaline phosphatelinked anti-sheep antibody (100 µL).After another wash cycle and the addition of enzyme substrate solution (pni-trophenyl phosphate), the absorbance of the product was monitored at 405 nm using a microplate reader. The absorbance ofstandard solutions which inverselyrelated to PMA concentration, were plotted as a function of the logarithm of PMA dose. The urinary creatinine was measured by Jaffe kinetic method without deproteinization on a Boehringer Mannheim Hitachi 917 automatic analyser and were reported following adjustment for creatinine concentration (measured as mmol/mol creatinine to correct for urine dilution effects). Data analysis was performed with SPSS statistical software for windows. Comparison between the SPMA mean values (creatinine adjusted) was carried out with Mannt-whitnegtes test and for benzene concentration was obtained by the student’s t-test. Linear regression analysis was used to describe the correlation between distance from road and SPMA in urine.

RESULTS

The mean value of benzene concentration for all subjects were 1.5 ± 1.80. The mean concentration of benzene measured in population are shown in Table 1, the results indicate a significant difference for subjects with outside activities less than 6 hours and more than 6 hours activities (p<0.005). The mean concentration of benzene for people that have more than 6 hours activity in winter is more than summer. There was not significant different between benzene concentration in summer compare to winter. There was a significant difference between benzene concentrations for subjects their home was less than 200 m to road compare to subjects more than 200 m far from road in winter (p<0.05) but no significant difference was found between two groups in summer. Table 2 shows the levels of the urinary PMA measuredin this studywith and without adjustment for creatinine. There was a significant difference between PMA level for children living less than 200 m from road compare to children living more than 200 m (p<0.05). The urinary SPMA levels, expressed nmol/ L, were two-half fold higher in children near road compare to children far from road and when adjusted for creatinine, still there is significant difference between two groups. No significant difference was seen between the Urinary PMA for children their parents smoker with non-smokers.

DISCUSSION AND CONCLUSION

The results of this study showed that, activities undertaken by each subject over of 24 h are importance factor in exposure of people to benzene. This study measured mean of benzene concentration in 24 h over weekly exposure, but most of studies in other countries have measured benzene in ambient air with active or passive samplers in special sites in street. The mean concentration of benzene in London, Hong Kong, Manila, Bangkok, Mexico city, Rome, Milan and Tehran were ranged from 4.00 to 39.90 ppb (Duarte-Davidson et al., 2001; Lee et al., 2002; Geeand Sollar, 1998; Brocco et al., 1997; Bahrami 2001; Fernando et al., 1999). If the results are compared with the above-mentioned results, the mean concentration of benzene in Adelaide (1.50 ppb ± 1.80) was less than other studied locations that may concern to extension of the city in great area with range of 40 to 25 km and length of 90 to 50 km (UBD, 2003). Motor vehicles can be considered to the main sources of air pollution in the Adelaide metropolitan, in which, the total number of motor vehicles registered in South Australian have increased steadily since 1979 at an overall annual growth rate of 2.1 percent per year. In south Australia at 1995, there were 653 motor vehicles per 1000 persons, an increase of 2.4 percent from the 1993 motor vehicle census. (Environment Protection Authority, 1998). The present study focuses on the accumulating of urinary SPMA in children using anewlydeveloped, technically simple, based on competitive enzyme linked immunosorbent assay (ELISA) specific for SPMA. The comparison of quantitative results generated by ELISA and GC/MS was done by Astone et al., and indicate a correlation (R=0.92) (Aston et al., 2002). Data in this study demonstrated there are significant difference in urinary SPMA levels in children depending on distance of location from road. It may concern to pollution in road emitted by vehicle. The individual variable such as potential home benzene exposure to passive smocking, cooking and heating were not significant confounding factors in subjects living in south Australia. There was not any significant difference between measurement of urinary SPMA in winter and summer and also among four areas in Adelaide. It may concern to concentration of benzene in seasons and extension of Adelaide in a great area. No significant difference between children their parents were smoker with no-smoker. It may concern to strong interference of outside sources of pollution. This is the first study, which examined urinary SPMA levels of children in South Australia. Studies of urinary PMA levels in other countries is also rare. At the present time the measurement of urinary SPMA is the method most frequently used for the biological monitoring of workers who are exposed to benzene. Biological monitoring for benzene and metabolite for environmental exposure has not been used extensively. In studybyChioet al., (2000) measuring PMAin people living near petrochemical industrial areasin Korea the geometricmean and themaximum amount ofdata were 7.89 and 109.68 µg/g creatinine respectively. In another studyat Koria (Fang et al., 2000) correlation coefficient for urinaryPMA in children near to thepetrochemical with benzenein ambient airwas0.90. TheurinarySPMA at the present study was less than study by Yoonho and otherscarried out in industrialarea. It mayconcern to benzene concentration in air. PMA a more reliable biomarker than TTMA for monitoring benzene exposure(Boogaard, 1995). Somestudies reported that TTMA is not a high sensitive biomarkers for exposure to benzene in low concentration because sorbic acid (common food preservative) converted to TTMA and is a positiveinterference (Giovanni et al., 2001; Barbiers et al., 2002). PMA also was recognized to be an appropriate biomarkers for monitoring of benzene because it is unique metabolite derived from benzene and has a longer half-life. (Boogaard and Sitter, 1995; Van Sittert et al., 1993). It isconcluded that SPMAcan be utilized as a biomarker for exposure to benzene in children. The children and adults that living near to main road have more exposure to benzene. Adults that more activity in outside of home has more exposure to benzene in ambient air compare topeople stayat home. It is recommended that authorities decrease amount of benzene in petrol and develop an emission database transport and monitor benzene in main road.

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