Chromosomal Damage Risk Assessment to Benzene Exposure

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  Journal of Environment and Earth ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online)Vol 1, No.2, 2011 37 | Page  Chromosomal Damage Risk Assessment to Benzene Exposureamong Gasoline Station Workers inBangkok Metropolitan, Thailand Tanasorn Tunsaringkarn 1* Panthira Ketkaew 2 Jamsai Suwansaksri 3 Wattasit Siriwong 1  Anusorn Rungsiyothin 1 Kalaya Zapuang 1 Mark Gregory Robson 4,5,6 1.   College of Public Health Sciences, Chulalongkorn University, Institute Building 2-3, SoiChulalongkorn 62 Phyathai Rd, Bangkok 10330, Thailand2.   Department of chemistry, Faculty of Science, King Mongkut’s University of TechnologyThonburi, Bangkok 10140, Thailand3.   Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University,Bangkok 10330, Thailand4.   School of Environmental and Biological Sciences, Rutgers University, 59 Dudley Rd, Rm 204AForan Hall New Brunswick, New Jersey 08854, USA5.   UMDNJ-School of Public Health, Piscataway,   65 Bergen St, Suite 701 Newark, New Jersey08854, USA6.   Thai Fogarty ITREOH Center, Chulalongkorn University, Bangkok 10330, Thailand* E-mail of the corresponding  Abstract  This study was a cross-sectional survey study to assess relative risk (RR) of chromosomal damage throughbenzene exposure in 45 gasoline stations workers compared to 30 controls in central area of Bangkok.Sister chromatid exchange (SCE) is as genotoxic biomarker, performed in white blood cells, and bloodbenzene level (BBL) is a biological marker of benzene exposure was performed by gas chromatography-flame ionization detector (GC-FID) using modified headspace solid-phase micro-extraction (HS-SPME)technique. The results showed that the average blood benzene level of these workers was significantlyhigher than in the controls (  p < 0.001) as well as the frequency of sister chromatid exchange. The sisterchromatid exchange was strongly and positively associated with blood benzene level of gasoline workers (  p  < 0.001) with the chromosomal damage relative risk at 2.50 (  p < 0.001). Keywords : gasoline worker, benzene, sister chromatid exchange, chromosomal damage 1. Introduction  Air pollution has become to be a serious health problem in Bangkok, Thailand, especially amongoccupational workers such as gasoline workers. Exposure to gasoline vapors is classified by theInternational Agency for Research on Cancer as possible cancer risk in humans, mainly on the basis of theestablished carcinogenicity of some chemical components such as benzene (IARC   1989). The mechanismof benzene toxicity, particularly its leukemogenic effects, is far from being fully understood. Contaminationof the environment with volatile organic compounds (VOCs) has become an important issue, since many of these compounds are toxic and may pose health risks of various concerns . To assess the biological riskscaused by gasoline vapor, by biological monitoring using biological marker of oxidative chromosomaldamage and repairing capacity (Lambert et al . 1982; Carrano et al. 1983), as frequency of sister chromatidexchange (SCE) in gasoline workers, may provide useful information about the genotoxic risk associatedwith exposure to this carcinogenic agent that is benzene. In major cities in Asia, the levels of ambient airbenzene are relatively high compared with those in Europe or in the United States (Leong & Laortanakul2003) and there is high prevalence of cancer and leukemia related to petrol station density (Yimrungruang et al. 2008 ; Chang et al. 2009; Weng et al. 2009) . The purpose of this study   was to evaluate the relative risk   Journal of Environment and Earth ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online)Vol 1, No.2, 2011 38 | Page  (RR) of chromosomal damage cause by benzene exposure in gasoline station workers in central area of Bangkok, Pathumwan district, comparing to controls.  2. Research Methods 2.1 Population study A cross sectional surveyed at 11 gasoline stations in Pathumwan district, Bangkok, Thailand, and collectedblood samples analysis from 45 gasoline workers, which 33 non-smokers and 12 smokers, compared to 30non-smoker controls from April to June 2009. All subjects have given informed consent before the study.The Ethical Review Committee for Research Involving Human Research Subjects, Health Science Group,Chulalongkorn University, approved the study. All subjects were healthy and had worked more than sixmonths. 2.2 Sample collection The venous blood samples were drawn from subjects during the 6-8 h shift work, 2 x 2 mL, using plasticheparinized vacuum blood tube, stored at 4 °C, one SCE frequency analysis within 6 h and the other onestored at -20 °C before benzene analysis within 1 month. 2.3 Sample analyses The blood examination for SCE frequency was modified in manner suggested by Tucker and Preston(1996). Aliquots of 0.5 mL of heparin zed blood samples, both worker and control groups, added to 5.0 mLof the culture medium containing Roswell Park Memorial Institute medium (RPMI 1640, Hyclone, Utah,USA), supplemented with 15% fetal bovine serum, 2.5% phytohemagglutinine (PHA, Sigma, Germany)and 1% penicillin-streptomycin. Afterwards 100 µL of 1.3 mg/L 5 ′ -bromodeoxyuridine (BrdU, SigmaChemical Co.) was added to the medium and additionally incubated in the dark room temperature for 96hours. Immediately, added colchicine (0.2 µg/L, Sigma Chemical Co.), collected cultured cells and treatedwith 0.075 mol/L   potassium chloride (KCl) at 37 °C for 10 min to be fixed with methanol-acetic acid (3:1).Standard harvest procedure was performed by a drop of harvested cell pellets spread on clean glass slideand stained by Hoechst No.22358 plus Giemsa technique (Koto et al. 1975). Finally, the slides wereexamined by a light microscope (Nikon E200) in regard to SCE frequency/metaphase cell. The total of 15well-spread metaphases was evaluated in worker and control groups. Counting of SCE frequency was doneby using oil immersion.Blood benzene determination was performed by GC-FID using modified HS-SPME technique(Tunsaringkarn et al. 2004). Briefly, to 0.5 mL of blood sample in glass cap bottle was added 0.2 g of sodium chloride, shaking vortex for 15 s, then controlled and absorbed by SPME in water bath at 50 ° Cwith vibrator for 20 min. Injected in GC (Varian CP 3800) at 220 ° C with flame ionization detector at 220 ° C (used column CP-SIL5 CB, split less). Oven temperature started at 50 ° C for 10 min, then increased 5 ° C/min until 90 ° C and finally increased by 30 ° C/min until 250 ° C for 17 min. The quantity of bloodbenzene was analyzed under relative intensity of chromatographic signal for 40 min. The Limit of Detection (LOD) of benzene was 10.00 µ g/L (ppb) and the average coefficient of determination (r2) was0.999657.Statistical analyses were carried out with the SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA).Descriptive statistics were used for BBL and frequency of SCE in gasoline worker and control groupswhich were presented as mean and standard error of the estimate (Mean ± SE). The comparison betweencontrol and worker parameters were analyzed by independent-t test with a value  p < 0.05 limitation. Theassociation between parameters and SCE was the estimated relationship of them by multiple linearregression, which SCE as dependent variable, BBL, age, sex and cigarette smoking as independentvariables. The relative risk (RR) of chromosomal damage was calculated by comparing the proportion of   Journal of Environment and Earth ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online)Vol 1, No.2, 2011 39 | Page  workers with frequency of SCE higher than the mean plus standard error value of control to proportion of workers with frequency of SCE lower than the mean plus standard error value of the control group. Chi-square analysis was used to determined differences between groups in frequency distribution with 95%confidence interval (CI). 3. Analysis results A total of 75 subjects, 30 controls and 45 gasoline station workers, were included in this study. The averageage of worker and control groups was 31.2 ± 1.4 and 31.2 ± 1.8 years. The gasoline workers were 60%male and 26.7% smoker while the controls were 60% male and all of them were non-smokers. Thefrequency of SCE in workers and controls was 13.62 ± 0.24 and 6.97 ± 0.20 SCE/cell (Table 1 and Figure1). Both, smoking and non-smoking workers, SCE frequencies were significantly higher than those of controls (  p < 0.001). SCE frequency in smokers had higher level than in non-smokers but there was nosignificant difference. The average BBL of workers and controls was 302.84 ± 20.54 and 27.28 ± 15.31 µ g/L, but workers had significantly higher levels than controls (  p < 0.001) as shown in Table 1. But SCEand BBL of male and female were not difference in each group. In addition, the benzene exposure risk ratio(BBL of workers/controls) was 11.83 while the chromosomal damage risk ratio (SCE of workers/controls)was 1.94.The association between BBL and SCE frequency was analyzed by multiple linear regression with SCE asdependent variable and BBL, age, sex and cigarette smoking as independent variables (Table 2). Thefrequency of SCE was positively and significantly associated with BBL (  p = 0.001). Age, sex and cigarettesmoking, were not significantly associated with SCE (  p > 0.05). The frequencies of SCE weredichromatomized into high- and low-frequency groups based on their means plus standard error of estimate(SE) values of control group at 7.17 (6.97 + 0.2). The chromosomal damage relative risk of gasoline stationworkers compared to controls was 2.50 ( χ  2–test, 95% CI = 1.17-5.34,  p < 0.001). 4. Discussion Volatile organic compound in gasoline is a common source of benzene which is classified as carcinogen. Itcan cause serious health effects including genotoxicity. Biomarkers of response indicate biological orbiochemical changes in target tissue or surrogate from chemical action. The biomarkers commonly used arechromosome aberration (CA), micronucleus (MN) and SCE. This study used SCE as genotoxic biomarkerand BBL as exposure biomarker. The results of this study showed significantly higher genotoxic orchromosomal damage in gasoline workers than in controls which supported the previous studies(Vijayalaxmi & Evans 1982; Celik & Akba 2005; Calderón-Ezquerro et al. 2007). The SCE frequency wasnot effective for low smoker workers with less than 10 cigarettes a day (average number of cigarettesmoking 5.2 cigarette/day), but there was a trend of higher chromosomal damage in smokers than in non-smokers. The comparison between smokers and non-smokers should be related to the number of cigarettessmoked a day with a significant difference in moderate and heavy smoker (Vijayalaxmi & Evans 1982;Celik & Akba 2005; Calderón-Ezquerro et al. 2007). The BBL of all workers was significantly higher thanin controls (  p < 0.001), but BBL of smokers trending upwards compared to non-smokers, but notsignificantly different, most non-smokers being women with higher heart rate than men (Ryan et al. 1994;Stein et al. 1997). As the results of women were higher in BBL than in men with same rate of benzeneclearance, it may influence its internal exposure and cancer development. Gender affected more BBL thanbenzene exposure from low cigarette smoking. In addition, BBL of all gasoline workers were higher thanthe biological monitoring of occupationally exposed persons, an exposure equivalent for carcinogenicworking material (EKA-value) of 54 µ g/L (Angerer et al. 1991). It should be considered that BBL inworkers was 5.6 folds of limited level, corresponding to high level of benzene exposure (11.83 folds of exposure risk ratio) and this marker indicating that cells have been exposed to mutagen or carcinogen(Keretetse et al. 2008). The frequency of SCE provided information of cumulative effects of carcinogenswhich was associated with increased risk of cancer (1.9 folds of chromosomal damage risk ratio). Theresults of this study indicated that the frequency of SCE was strongly associated to BBL (  p < 0.001). With  Journal of Environment and Earth ISSN 2224-3216 (Paper) ISSN 2225-0948 (Online)Vol 1, No.2, 2011 40 | Page  every increase of one year in age, the average SCE decreased by 0.25 whilst every increasing of BBL level1 µ g/L, the average SCE increased by 0.44. In fact, the average age of control group was not higher thanthe average in the worker group. Age, cigarette smoking and sex were not significantly associated tochromosomal damage. Some studies showed that no correlation between SCE frequency, duration of exposure, smoking habit, and age (Hoet et al. 2009; Ulker et al. 2008). The gasoline workers had anincreased relative risk of chromosomal damage at 2.50 compared to controls ( χ  2–test, 95% CI = 1.17-5.34,  p < 0.001), which benzene exposure and chromosomal damage risk ratios of the workers were 11.93 and1.94, respectively.However, biomonitoring in peripheral lymphocytes served as an early indicator of chromosomal damage.Biomonitoring of benzene exposure by using BBL among the gasoline workers should show the associationof cancer risk development in gasoline workers prompting to plan a screening program and primaryprevention for them to minimize the risk of cancer. Future studies of genotoxicity in smokers and non-smokers comparison should cover a large population and take into consideration confounders such as age,gender, smoking habits, alcohol consumption and family history of cancer. Acknowledgments This study was supported by Thai Fogarty ITREOH Center (D43 TW007849 NIH FIC), College of PublicHealth Sciences, and Chulalongkorn University Centenary Academic Development Project, Bangkok,Thailand. And, many thanks go to the gasoline station owners and their workers for the agreeablecollaboration. Last, but not least, thanks to Dr. Karl J. Neeser for his advice. References Angerer, J., Seherer, G., Sehaller, K.H., & Müller, J. (1991), “The determination of benzene in humanblood as an indicator of environmental exposure to volatile aromatic compounds”, Fresenius Journal of  Analytical Chemistry   339 , 740-742.Calderón-Ezquerro, C., Sánchez-Reyes, A., Sansores, R.H., Villalobos-Pietrini, R., Amador-Muñoz, O.,Guerrero-Guerra, C., Calderón-Segura, M.E., Uribe-Hernández, R., Gómez-Arroyo, S. (2007), “Cellproliferation kinetics and genotoxicity in lymphocytes of smokers living in Mexico City”,  Human Experimental Toxicology 26 (9), 715-722.Carrano, A.V., & Natarajan A.T. (1988), “Considerations for population monitoring using cytogenetictechniques”,  Mutation Research   204 , 379-406.Celik, K.A., & Akba S.E. (2005), “Evaluation of sister chromatid exchange and chromosomal aberrationfrequencies in peripheral blood lymphocytes of gasoline station attendants”,  Ecotoxicology and  Environmental Safety   60 (1), 106-112.Chang, C.C., Tsai, S.S., Chiu, H.F., Wu, T.N., & Yang C.Y. (2009), “Traffic air pollution and lung cancer infemales in Taiwan: petrol station density as an indicator of disease development”,  Journal of Toxicologyand Environmental Health, Part A   72 (10), 651-657.Hoet, P. De Smedt, E., Ferrari, M., Imbriani, M., Maestri, L., Negri, S., De Wilde, P., Lison, D., &Haufroid, V. (2009), “Evaluation of urinary biomarkers of exposure to benzene: correlation with bloodbenzene and influence of confounding factors”,  International Archives of Occupational and Environmental Health   82 , 985-995.IARC (International Agency For Research On Cancer). (1989), “Occupational exposures in petroleumrefining; crude oil and major petroleum fuels”, Monographs on the evaluation of carcinogenic risks tohumans. Vol. 45, Lyon, France.Keretetse, G.S., P. J. Laubscher, P.J., Du Plessis, J.L., Pretorius, P.J., Van Der Westhuizen, F.H., VanDeventer, E., Van Dyk, E., Eloff, F.C., Van Aarde, M.N., & Du Plessis, L.H. (2008), “DNA damage andrepair detected by the comet assay in lymphocytes of African petrol attendants: A pilot study”.  Annals of Occupational Hygiene   52 (7), 653-662.
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