- Open Access
Monitoring of gas station attendants exposure to benzene, toluene, xylene (BTX) using three-color chromosome painting
© Santiago et al.; licensee BioMed Central Ltd. 2014
- Received: 28 January 2014
- Accepted: 12 February 2014
- Published: 27 February 2014
Chronic exposure of BTX (benzene, toluene, xylene) may lead to progressive degeneration of bone marrow, aplastic anemia and/or leukemia. In Brazil there is no self-service fuel in gas stations and attendants fill the fuel themselves. Due to this they are chronically exposed to high concentration of BTX. Occupational exposure to benzene has been associated with increased chromosomal aberrations in peripheral blood lymphocytes. Fluorescence in situ hybridization (FISH) using whole chromosome painting (wcp) probes allows the rapid detection of chromosomal aberration. In the present study three-color wcp probes for chromosomes 1, 2 and 4 were used for monitoring 60 gas station attendants.
Blood tests were done and interviews were conducted for each worker. For searching for possible associations between the clinical characteristics and the frequency of chromosomal aberrations the workers were divided into two groups (≤ 10 chromosomal abnormalities per 1,000 metaphases and > 10 chromosomal abnormalities per 1,000 metaphases).The studied workers had a low median age (36 year), albeit long period of BTX exposure (median was 16 years). Low prevalence of smoking and moderate consumption of alcoholic beverages were found in this population. The cytogenetic analysis showed 16.6% (10/60) of workers with a high frequency of chromosomal abnormalities (>10 chromosomal abnormalities per 1,000 metaphases). Translocations were the most frequently observed chromosome aberration. The statistical analysis revealed highly significant differences in skin color (p = 0.002) and a weak significant differences in gender (p = 0.052) distribution between the two groups.
16.6% of the studied population showed elevated frequencies of chromosomal abnormalities, which is highly likely to be correlated with their exposure to BTX during their work. Therefore, further studies are needed for better characterize the work associated damage of the genome in gas station workers. It is necessary to better understand the risks that these workers are exposed, so that we can be effective in preventing diseases and maintaining the health of these workers and possibly the offspring.
In contrast to many developed countries, there are no self service fuel in gas stations in Brazil. Filling in fuel depends on attendants who are chronically exposed to BTX (benzene, toluene and xylene) during the work time. It is well known that benzene induces myelotoxicity in humans; the role of xylene and toluene is still unclear, thus, here we concentrate on the best studied substance among those three, benzene. It causes a variety of hematological disorders including aplastic anemia, myelodysplastic syndrome, and acute myelogeneous leukemia (AML) [1–4]. Benzene is metabolized in the liver to its primary metabolite phenol by cytochrome P4502E1 (CYP2E1) through the benzene oxide intermediate, and is subsequently metabolized by CYP2E1 to hydroquinone (HQ) [5, 6]. HQ is transported to the bone marrow and oxidized to benzochinones, which eventually releases reactive oxygen species (ROS) damaging hematopoietic cells [5, 7].
Many studies have been carried out to determine the hematological alterations and chromosomal aberrations (CA) in benzene exposed workers [8–11]. Reported genetic damages caused by benzene include sister chromatid exchange, DNA cross-linking, DNA adduct formation, and impairment of DNA repair mechanisms . Other studies reported increased levels of chromosomal anomalies such as aneuploidies including monosomy of chromosomes 5 and 7 and trisomies of chromosomes 8 and 21, in the blood lymphocytes of apparently healthy Chinese workers exposed to high levels of benzene (median: 31 ppm, range: 1.6–328.5 ppm). It is well known that these kinds of alterations are also commonly found in leukemia and myelodysplastic syndrome [7, 10, 13]. Thus, chromosomal aberrations may be a precursor of future leukemia risk and other cancers. Additional studies have further strengthened the association between increased levels of CA in human lymphocytes and future cancer incidence and mortality [13, 14].
Fluorescence in situ hybridization (FISH) using whole chromosome painting (wcp) libraries opened new insights in studying the CA in people exposed to mutagens and in delimiting individuals at risk . This approach allows the rapid detection of translocations and other cytogenetic alterations, enabling new possibilities of cytogenetic dosimetry. This kind of test can also be used to address the chromosomal rearrangements detectable in individuals exposed to benzene and delimiting their individual cancer risk [1, 15].
The aim of this study was to describe the cytogenetic changes on chromosomes 1, 2 and 4 in gas station attendants from Rio de Janeiro, Brazil, that had occupational exposition to BTX. The obtained results were used as an indicator for chromosomal damage as a whole, which happened in this population due to their work exposure.
Biometrics data (clinical and demographic) of gas station attendants
36 (± 13.5)
Duration of exposure (years)
Illicit drug consumption
Mean corpuscular volume (fL)
In the medical history of workers vision impairment (22.6%) was the complaint most frequently reported, followed by osteoarticular*a (18.5%), cardiovascular (16.0%) and respiratory tract diseases (12.6%). Hematological diseases (1.8%) were scarce, and no neoplasic diseases were reported.
Chromosomal abnormalities of gas station workers
der(1), t (1;2)
del(1), del(2); -4
A compared to normal controls (see Discussion) elevated frequency of CAs (9.3 per 1,000 metaphases analyzed) was found in the gas station workers population. Among the total CAs the translocations were most frequently found (43.6%), followed by deletions (23.7%); monosomies (10.9%); chromosomal breaks (12.7%); chromosomal fragments (7.3%) and trisomies (1.8%).
Translocations of chromosome 2 were responsible for 20% of total CAs, followed by translocation involving chromosome 1 (18.2%) and gross deletions within chromosome 2 (9.1%). Chromosome 1 showed the highest percentage of CAs (41.8%) that corresponded to: translocations (18.2%), chromosome breakage (7.2%), deletions (7.2%), monosomies (5.4%), and formation of chromosome fragments (3.6%). Chromosome 4 had the lowest percentage of CAs observed (27.3%). Among the CAs of chromosome 4 the most common were deletions (7.2%), followed by translocations (5.4%), monosomies (5.4%), chromosome breaks (3.6%), chromosome fragments (3.6%) and trisomies (1.8%).
Statistical analysis between translocations and others chromosomal abnormalities of chromosomes 1, 2 and 4
Number of chromosomal aberrations
Statistical analysis between translocations and others chromosomal abnormalities of chromosome 1 and 2
Number of chromosomal aberrations
Association between the cytogenetic results and clinical and demographic data
For possible associations between the clinical characteristics and the frequency of CAs the subjects were divided into two groups (≤ 10 CAs per 1,000 metaphases = group 1, and > 10 CAs per 1,000 metaphases = group 2). The statistical analysis revealed highly significant differences in skin color distribution between the groups (p = 0.002). Similarly, we found, when statistical analysis were done, differences only concerning blacks and whites between the two groups (p < 0.01, OR = 9.02 and 95% CI [1.54 to 97.77]). It was observed that blacks have a lower number of CA than whites. Also we observed on the gender analyze that 88% of male workers were in group 1 and 12% in group 2, while it were 60% of female workers in the group 1 and 40% in group 2.The statistical analysis revealed weak significant differences in gender distribution between the two groups (p = 0.052, OR = 4.71 and 95% CI [0.76 to 28.05]).
Associations between the frequency of chromosomal abnormalities and biometrics (clinical and demographic) data
≤ 10 chromosomal abnormalities per 1000 metaphases
>10 chromosomal abnormalities per 1000 metaphases
Time of employment (year)
Typical lymphocytes (%)
Mean corpuscular volume (fL)
Mean corpuscular volume (fL)
In the present study, the frequency of CAs in peripheral blood lymphocytes of Brazilian gas station workers was used as an effect biomarker of BTX exposure. The chromosomes pairs 1, 2 and 4 represent together 21.87% of human genome. Thus they were chosen for the cytogenetic paint analysis as representatives of the entire human genome as previously described by Verdorfer and colleagues .
A questionnaire analysis showed a population with a low median age, albeit with long period of exposure. We did not detect inappropriate health behaviors on life style questions. The self-reported medical history showed that the main health problems were related to acute symptoms; reported sight changes appeared to be mainly associated with direct irritant action of fuel vapor in eyes (no masks are used). Osteoarticular diseases were most likely due to the long period of time in physical labor. Hematological and neoplasic diseases were rarely/not reported by workers, which was most likely due to the quiet character of the natural history of these diseases and the fact that they lead quickly to absence from work. Also they are late manifesting diseases, which might not be present in the on average young population studied.
It should be remembered that fuel volatile fraction contains chemicals other than BTX that may act as aneugens or clastogens. It is likely that the workers in this study were exposed simultaneously to several other complex chemicals. However, considering the different behaviors of environment chemicals once released, the significant relationship observed with respect to BTX concentration, indicates a specific role for BTX, mainly for the benzene, in this association [9, 16].
Several studies examined cytogenetic endpoints in subjects exposed to petroleum fuels, auto exhaust, or other organic solvents [1, 10, 17–23]. The benzene exposure has been strongly associated with increased chromosomal abnormalities in the lymphocytes in individuals without diseases . Verdorfer and colleagues analyzed with same technique different groups of individuals with different types of exposures . In our study the workers had a higher frequency of chromosomal abnormalities when compared with Verdorfer groups (control group, military occupied in nuclear area and radiology workers) .
In the present search were found 10/60 of workers with high number of chromosome abnormalities (all workers with ≥20 abnormalities per 1,000 metaphase). It is worth to note that in a normal control group only 4/60 individuals with high number of chromosome abnormalities could be expected (≥20 abnormalities per 1,000 metaphase) .
CAs have a direct association with malignancy. An induced chromosomal instability could also predispose cells to further mutations and by that to an increased risk of malignant transformation . Several researchers studying acute exposure of workers to fuel or organic solvents reported gaps and chromosomal breaks as the chromosomal abnormalities most often detected . While chromosomal translocations were described as markers of chronic exposure that dating back up several years of benzene exposure, so the number of translocations may be a parameter for long term exposure to benzene or BTX [15, 25, 26]. Thus, the elevated involvement of chromosomal translocations found in this study should be most likely due to the long years of workers exposure. Also, chromosome breaks detected in conventional cytogenetic studies cannot be detected in FISH (own unpublished data).
It is worth remembering that significant decreases of WBC, RBC and platelet counts already been observed in human populations exposed to high levels of benzene [11, 27]. However, we did not find a relationship between the frequency of CAs and the rates of WBC or RBC counts in the studied population. The presence of isolated thrombocytopenia is a change which was previously described in literature . Remarkably, the statistical positivity association of platelet decreased counting with frequency of CAs was closer. Further and longer studies are needed to associate the effects of BTX exposure between frequency of CAs and hematological changes with broad range of exposures.
In our study a weak association between gender and frequency of CAs were found. It is serious concern the possibility of women’s genome be severely more affected by BTX exposure. Several epidemiological studies support the idea that genotoxic and nongenotoxic events following benzene exposure may be initiators of childhood leukemia in utero . Another study on AML have shown that the disease is usually initiated in utero because the leukemic translocations and other genetic changes are present in blood spots collected at birth [29–31]. Thus, mother exposure to benzene could be just as important as childhood exposures in producing childhood AML and acute lymphoblastic leukemia.
The number of workers with high amount (10/60) and the high frequency of CAs (9.2 per 1,000 metaphase) found shows how necessary it would be to expand this study nationwide, since Brazil has great ethnic and cultural diversity. The results obtained are valuable, but were only obtained from 5 gas stations in Rio de Janeiro city, a pilot study. It is necessary to better understand the risks that these workers are exposed, so that we can be effective in preventing diseases and maintaining the health of these workers and possibly the offspring.
This study was approved by the local ethics committee (Instituto Nacional de Câncer – INCA, Brazil). All subjects were informed for each individual about the nature of the study, the potential benefits, and the risks. Participation was voluntary and written informed consent was obtained from each subject before study participation.
The study included 50 male and 10 female workers recruited on 6 gas stations, in Rio de Janeiro city. A trained interviewer questioned the members of the study population regarding their age, sex, race, life-style (smoking habits, alcohol and illicit drugs consumption, etc.) and medical and work histories.
Peripheral blood samples were collected for complete hemogram, biochemistry tests and cytogenetics. The cytogenetic analyses were made for delimiting workers at risk and for allowing associations between the frequency of CA and clinical characteristics.
Blood samples, 2 ml of heparinized whole blood, were collected by venipuncture. For each sample two cultures were performed according standard technique of lymphocyte cultures. Chromosomes were prepared according to standard procedures after 48 hours of cultivation .
FISH was done as previously reported  using home made wcp probes for chromosomes 1, 2 and 4 . Per gas station worker 100 metaphases should be analyzed; this was possible in 47 cases. In the remainder 13 cases 34 to 97 metaphases were available.
Hematological and biochemistry analysis
The hematological analysis consisted of complete hemogram measuring of hemoglobin, hematocrit, mean corpuscular volume and white blood cell counts. The biochemistry analysis consisted of measuring gamma glutamyl transpeptidase (Gamma GT), aspartate transaminase (AST), glutamic pyruvic transaminase (TGP), lactate dehydrogenase (LDH), bilirubin, creatinine and c-reactive protein.
All blood tests were analyzed in the central laboratory of INCA, according to standard haematological methods.
Subjects were divided into two groups (≤ 10 chromosomal abnormalities per 1,000 metaphases and > 10 chromosomal abnormalities per 1000 metaphases) and compared to clinical characteristics (age, time of employment, race, hemoglobin, leukocytes, etc.) by either Mann–Whitney test or chi-square test (if either quantitative or dichotomic variable). The Mann–Whitney test was performed because the variables quantitative did not have a Gaussian distribution in most of situation. For cytogenetic analyses the Chi-Square Goodness-of-Fit test was performed to evaluate statistical differences of chromosomal aberrations distribution among chromosomes 1, 2 and 4. For all statistical tests p < 0.05 was considered significant. All analyses were carried out using the PASW software (Version 18, Inc., Chicago, IL, USA).
a*Osteoarticular is defined as disease relating to, involving, or affecting bones and joints.
We thank the subjects who volunteered in the study and the workers of the central laboratory of INCA that provided the hemogram.
Programa de Oncobiologia, Rio de Janeiro, Brazil, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Brazil (APQ1 E-26/111.422/2013).
- Smith MT: Advances in understanding benzene health effects and susceptibility. Ann Rev Pub Health 2010, 31: 133–148. 10.1146/annurev.publhealth.012809.103646View ArticleGoogle Scholar
- Glass DC, Gray CN, Jolley DJ, Gibbons C, Sim MR, Fritschi L, Manuell R: Leukemia risk associated with low-level benzene exposure. Epidemiol 2003,14(5):569. 10.1097/01.ede.0000082001.05563.e0View ArticleGoogle Scholar
- Beelte S, Haas R, Germing U, Jansing PJ: Paradigm change in the assessment of myeloid and lymphoid neoplasms associated with occupational benzene exposure. Med Klin 1983,104(3):197.View ArticleGoogle Scholar
- Holecková B, Piesova E, Sivikova K, Dianovskỳ J: Chromosomal aberrations in humans induced by benzene. Ann Agricult Env Med 2004,11(2):175.Google Scholar
- Kim SY, Choi JK, Cho YH, Chung EJ, Paek D, Chung HW: Chromosomal aberrations in workers exposed to low levels of benzene: association with genetic polymorphisms. Pharmacogenet Genomics 2004,14(7):453–463.View ArticleGoogle Scholar
- Ross D: The role of metabolism and specific metabolites in benzene-induced toxicity: evidence and issues. J Toxicol Environment Health 2000,61(5–6):357–372.View ArticleGoogle Scholar
- Smith MT: The mechanism of benzene-induced leukemia: a hypothesis and speculations on the causes of leukemia. Environment Health Perspect 1996,104(Suppl 6):1219. 10.1289/ehp.961041219View ArticleGoogle Scholar
- Swaen GM, Van Amelsvoort L, Twisk JJ, Verstraeten E, Slootweg R, Collins JJ, Burns CJ: Low level occupational benzene exposure and hematological parameters. Chem Biol Interact 2010,184(1):94–100.View ArticlePubMedGoogle Scholar
- Maffei F, Hrelia P, Angelini S, Carbone F, Barbieri A, Sanguinetti G, Violante FS: Effects of environmental benzene: micronucleus frequencies and haematological values in traffic police working in an urban area. Mutat Research 2005,583(1):1–11. 10.1016/j.mrgentox.2005.01.011View ArticleGoogle Scholar
- Ji Z, Weldon RH, Marchetti F, Chen H, Li G, Xing C, Eskenazi B: Comparison of aneuploidies of chromosomes 21, X, and Y in the blood lymphocytes and sperm of workers exposed to benzene. Environment Molec Mutagen 2012,53(3):218–226. 10.1002/em.21683View ArticleGoogle Scholar
- Qu Q, Shore R, Li G, Jin X, Chi Chen L, Cohen B, Li K: Hematological changes among Chinese workers with a broad range of benzene exposures. Am J Indust Med 2002,42(4):275–285. 10.1002/ajim.10121View ArticleGoogle Scholar
- Joo WA, Kang MJ, Son WK, Lee DY, Lee E, Kim CW: Monitoring protein expression by proteomics: human plasma exposed to benzene. Proteomics 2003,3(12):2402–2411. 10.1002/pmic.200300616View ArticlePubMedGoogle Scholar
- Zhang L, Rothman N, Wang Y, Hayes RB, Yin S, Titenko-Holland N, Dosemeci M, Wang Y, Kolachana P, Xi L, Li G, Smith MT: Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase cells. Environ Mol Mutagen 1999,34(4):260–268. 10.1002/(SICI)1098-2280(1999)34:4<260::AID-EM6>3.0.CO;2-PView ArticlePubMedGoogle Scholar
- Liou SH, Lung JC, Chen YH, Yang T, Hsieh LL, Chen CJ, Wu TN: Increased chromosometype chromosome aberration frequencies as biomarkers of cancer risk in a blackfoot endemic area. Cancer Res 1999,59(1):1481–1484.PubMedGoogle Scholar
- Verdorfer I, Neubauer S, Letzel S, Angerer J, Arutyunyan R, Martus P, Gebhart E: Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals. Mutation Res 2001,491(1):97–109.View ArticlePubMedGoogle Scholar
- Gebhart E, Neubauer S, Schmitt G, Birkenhake S, Dunst J: Use of a three-color chromosome in situ suppression technique for the detection of past radiation exposure. Radiation Res 1996,145(1):47–52. 10.2307/3579194View ArticlePubMedGoogle Scholar
- Zhang L, Eastmond DA, Smith MT: The nature of chromosomal aberrations detected in humans exposed to benzene. Crit Rev Toxicol 2002,32(1):1–42. 10.1080/20024091064165View ArticlePubMedGoogle Scholar
- Carere A, Antoccia A, Cimini D, Crebelli R, Degrassi F, Leopardi P, Marcon F, Sgura A, Tanzarella C, Zijno A: Genetic effects of petroleum fuels. II. Analysis of chromosome loss and hyperploidy in peripheral lymphocytes of gasoline station attendants. Environ Mol Mutagen 1998,32(2):130–138. 10.1002/(SICI)1098-2280(1998)32:2<130::AID-EM8>3.0.CO;2-#View ArticlePubMedGoogle Scholar
- Santos-Mello R, Cavalcante B: Cytogenetic studies on gas station attendants. Mutat Res 1992,280(4):285–290. 10.1016/0165-1218(92)90059-9View ArticlePubMedGoogle Scholar
- Silva JM, Santos-Mello R: Chromosomal aberrations in lymphocytes from car painters. Mutat Res 1996,368(1):21–25. 10.1016/S0165-1218(96)90036-1View ArticlePubMedGoogle Scholar
- Bukvic N, Bavaro P, Elia G, Cassano F, Fanelli M, Guanti G: Sister chromatid exchange (SCE) and micronucleus (MN) frequencies in lymphocytes of gasoline station attendants. Mutat Res 1998,415(1):25–33.View ArticlePubMedGoogle Scholar
- Zhang L, Lan Q, Guo W, Li G, Yang W, Hubbard AE, Smith MT: Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers. Chem Biol Interact 2005,153(4):117–122.View ArticlePubMedGoogle Scholar
- Wright EG: Inherited and inducible chromosomal instability: a fragile bridge between genome integrity mechanisms and tumourigenesis. J Pathol 1999,187(1):19–27. 10.1002/(SICI)1096-9896(199901)187:1<19::AID-PATH233>3.0.CO;2-1View ArticlePubMedGoogle Scholar
- Zhang L, Lan Q, Guo W, Hubbard AE, Li G, Rappaport SM, Smith MT: Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene. Carcinogenesis 2011,32(4):605–612. 10.1093/carcin/bgq286PubMed CentralView ArticlePubMedGoogle Scholar
- Smerhovsky Z, Landa K, Rössner P, Brabec M, Zudova Z, Hola N, Pokorna Z, Mareckova J, Hurychova D: Risk of cancer in an occupationally exposed cohort with increased level of chromosomal aberrations. Environ Health Perspect 2001,109(?):41–45.PubMed CentralView ArticlePubMedGoogle Scholar
- Pressl S, Stephan G: Chromosome translocations detected by fluorescence in situ hybridisation (FISH)-a useful tool in population monitoring? Toxicol Let 1998, 96: 189–194.View ArticleGoogle Scholar
- Aksoy M, Ozeris S, Sabuncu H, Yanardag R: Exposure to benzene in Turkey between 1983 and 1985:a hematologic study on 231 workers. Br J Ind Med 1987,44(11):785–787.PubMed CentralPubMedGoogle Scholar
- Ruiz M, Vassalo J, Souza CAD: Alterações hematológicas em pacientes expostos cronicamente ao benzeno. Rev Saúd Pública 1993,27(2):145–14130.View ArticleGoogle Scholar
- Greaves MF, Wiemels J: Origins of chromosome translocations in childhood leukaemia. Nat Rev Cancer 2003,3(9):639–649. 10.1038/nrc1164View ArticlePubMedGoogle Scholar
- McHale CM, Wiemels JL, Zhang L, Ma X, Buffler PA: Prenatal origin of childhood acute myeloid leukemias harboring chromosomal rearrangements t(15;17) and inv(16). Blood 2013,101(11):4640–4641.View ArticleGoogle Scholar
- Wiemels JL, Cazzaniga G, Daniotti M, Eden OB, Addison GM: Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 1999,354(9189):1499–1503. 10.1016/S0140-6736(99)09403-9View ArticlePubMedGoogle Scholar
- Liehr T, Claussen U: FISH on chromosome preparations of peripheral blood. In FISH-Technology, Springer-labmanual. Edited by: Liehr T, Rautenstrauss B. Berlin: Springer; 2002:73–81.View ArticleGoogle Scholar
- Liehr T, Claussen U: Current developments in human molecular cytogenetic techniques. Curr Mol Med 2002,2(3):283–297. 10.2174/1566524024605725View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.