Abstracts

Introduction

Benzene requires significant attention due to its toxicity and ubiquitous presence in several areas at low concentrations. It is an organic compound present and/or used as a raw material in several products, such as gasoline and plastic, and is classified by the International Agency for Research on Cancer (IARC) as a group 1 compound, a proven carcinogen to humans ¹1. Cottica D, Grignani E. Evolution of technology and occupational exposures in petrochemical industry and in petroleum refining. G Ital Med Lav Ergon 2013; 35:236-43.^,22. Sahmel J, Devlin K, Burns A, Ferracini T, Ground M, Paustenbach D. An analysis of workplace exposures to benzene over four decades at a petrochemical processing and manufacturing facility (1962-1999). J Toxicol Environ Health A 2013; 76:723-46.^,33. Panko JM, Gaffney SH, Burns AM, Unice KM, Kreider ML, Booher LE, et al. Occupational exposure to benzene at the ExxonMobil refinery at Baton Rouge, Louisiana (1977-2005). J Occup Environ Hyg 2009; 6:517-29.. It is currently one of the ten priority chemicals for study and regulation according to the World Health Organization (WHO) ⁴4. American Conference of Governmental Industrial Hygienists. Benzene CAS number: 71-43-2. Cincinnati: American Conference of Governmental Industrial Hygienists; 2001..

In terms of public health, the most significant benzene contamination route is through respiration. Most inhaled benzene is eliminated by expiration, while the retained portion accumulates, mainly, in fatty tissues ⁵5. Rasera IM. Toxicidade do benzeno, fontes de contaminação e analyses laboratoriais ENT#091;MonographyENT#093;. Novo Hamburgo: Instituto de Ciências da Saúde, Centro Universitário Feevale; 2009.. After absorption, the biotransformation of benzene occurs, primarily, in the liver, with its secondary metabolism occurring in the bone marrow ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.. Several enzymes are involved in this metabolism, including Cytochrome P450E1 (CYP2E1), NADPH:quinone oxidoreductase 1 (NQO1) and Glutathione S-transferase (GST) ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.. The kidneys are the main responsible organs for the excretion process of the generated metabolites ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17..

Effects resulting from acute exposure include headaches, fatigue, dizziness, mucosal irritation, convulsions, excitement, depression and, eventually, death due to respiratory failure ⁷7. Tunsaringkarn T, Soogarun S, Palasuwan A. Occupational exposure to benzene and changes in hematological parameters and urinary trans, trans-muconic acid. Int J Occup Med Environ Health 2013; 4:45-9.^,88. American Conference of Governmental Industrial Hygienists. Threshold limit values for chemicals substances and physical agents and biological exposure indices. Cincinnati: American Conference of Governmental Industrial Hygienists; 2003., while the bone marrow is the main target organ for benzene toxicity ⁹9. Agency for Toxic Substances and Disease Registry. Toxicological profile for benzene. Atlanta: Agency for Toxic Substances and Disease Registry; 2007.^,1010. Sorensen M, Skov H, Autrup H, Hertel O, Loft S. Urban benzene exposure and oxidative DNA damage: influence of genetic polymorphisms in metabolism genes. Sci Total Environ 2003; 309:69-80..

Chronic exposure to low benzene concentrations is associated with certain diseases, such as aplastic anemia and leukemia ²2. Sahmel J, Devlin K, Burns A, Ferracini T, Ground M, Paustenbach D. An analysis of workplace exposures to benzene over four decades at a petrochemical processing and manufacturing facility (1962-1999). J Toxicol Environ Health A 2013; 76:723-46.^,1111. Infante PF. The IARC October 2009 evaluation of benzene carcinogenicity was incomplete and needs to be reconsidered. Am J Ind Med 2011; 54:157-64.^,1212. Pyatt DW, Aylward LL, Hays SM. Is age an independent risk factor for chemically induced acute myelogenous leukemia in children? J Toxicol Environ Health B 2007; 10:379-400.^,1313. Schnatter AR, Rosamilia K, Wojcik NC. Review of the literature on benzene exposure and leukemia subtypes. Chem Biol Interact 2005; 153/154:9-21.. Some studies also suggest that exposure at different concentrations may increase the risk of developing non-Hodgkin’s lymphoma ¹¹11. Infante PF. The IARC October 2009 evaluation of benzene carcinogenicity was incomplete and needs to be reconsidered. Am J Ind Med 2011; 54:157-64.^,1414. Steinmaus C, Smith AH, Jones RM, Smith MT. Meta-analysis of benzene exposure and non-Hodgkin lymphoma: biases could mask an important association. Occup Environ Med 2008; 65:371-8.^,1515. Galbraith D, Gross SA, Paustenbach D. Benzene and human health: a historical review and appraisal of associations with various diseases. Crit Rev Toxicol 2010; 40:1-46.^,1616. Smith MT, Jones RM, Smith AH. Benzene exposure and risk of non-Hodgkin lymphoma. Cancer Epidemiol Biomarkers Prev 2007; 16:385-91.^,1717. Savitz DA, Andrews KW. Review of epidemiologic evidence on benzene and lymphatic and hematopoietic cancers. Am J Ind Med 1997; 31:287-95., multiple myeloma ¹¹11. Infante PF. The IARC October 2009 evaluation of benzene carcinogenicity was incomplete and needs to be reconsidered. Am J Ind Med 2011; 54:157-64.^,1515. Galbraith D, Gross SA, Paustenbach D. Benzene and human health: a historical review and appraisal of associations with various diseases. Crit Rev Toxicol 2010; 40:1-46.^,1818. Infante PF. Benzene exposure and multiple myeloma: a detailed metaanalysis of benzene cohort studies. Ann N Y Acad Sci 2006; 1076:90-109. and various other hematopoietic disorders ²2. Sahmel J, Devlin K, Burns A, Ferracini T, Ground M, Paustenbach D. An analysis of workplace exposures to benzene over four decades at a petrochemical processing and manufacturing facility (1962-1999). J Toxicol Environ Health A 2013; 76:723-46.^,1111. Infante PF. The IARC October 2009 evaluation of benzene carcinogenicity was incomplete and needs to be reconsidered. Am J Ind Med 2011; 54:157-64.^,1212. Pyatt DW, Aylward LL, Hays SM. Is age an independent risk factor for chemically induced acute myelogenous leukemia in children? J Toxicol Environ Health B 2007; 10:379-400.^,1313. Schnatter AR, Rosamilia K, Wojcik NC. Review of the literature on benzene exposure and leukemia subtypes. Chem Biol Interact 2005; 153/154:9-21.^,1919. Ross D. The role of metabolism and specific metabolites in benzene-induced toxicity: evidence and issues. J Toxicol Environ Health A 2000; 61:357-72..

Benzene exposure in humans is monitored through biomarkers that significantly correlate to exposure intensity and/or biological effect caused by the substance ²⁰20. Amorim LCA. O uso dos biomarcadores na avaliação da exposição ocupacional a substâncias químicas. Rev Bras Med Trab 2003; 1:124-32., such as S-phenylmercapturic acid (S-PMA) and metabolic polymorphisms (CYP2E1; NQO1).

S-PMA is an aliphatic metabolite excreted in urine, with only 0.11% of absorbed benzene being biotransformed into this product. Its elimination half-life is of approximately 10 hours, which makes this metabolite a strong candidate for biomarker exposure, due to its high residence time. However, smoking acts as a confounding factor in S-PMA analyses, while urinary S-PMA levels are not subject to dietary interference. Urinary S-PMA has a biological exposure limit value of 25μg.g^-1 creatinine ²¹21. American Conference of Governmental Industrial Hygienists. Threshold limit values (TLVs) for chemical substances and physical agents biological exposure indices for 2013. Cincinnati: American Conference of Governmental Industrial Hygienists; 2013.. However, since benzene is a recognized carcinogenic substance, there is no safe exposure limit established in Brazilian law; therefore, any S-PMA value found in the biological sample is a potential health risk.

Genes coding for various enzymes belonging to the benzene metabolizing routes present polymorphic variations, which may alter enzymatic efficiency, with consequent increases or decreases in the concentrations of toxicologically relevant metabolites. Studies have indicated that genetic polymorphisms of the CYP2E1 and NQO1 enzymes are of great importance in this process ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.^,2222. Kim S, Lan Q, Waidyanatha S, Chanock S, Johnson BA, Vermeulen R, et al. Genetic polymorphisms and benzene metabolism in humans exposed to a wide range of air concentrations. Pharmacogenet Genomics 2007; 17:789-801.^,2323. De Palma G, Mutti A, Spatari G, Andreoli R, Mozzoni P, Carrieri M, et al. Biomarkers of effect and susceptibility to low doses of benzene. G Ital Med Lav 2013; 35:259-62.^,2424. Ross D, Zhou H. Relationships between metabolic and non-metabolic susceptibility factors in benzene toxicity. Chem Biol Interact 2010; 184:222-8.^,2525. Lan Q, Zhang L, Li G, Vermeulen R, Weinberg RS, Dosemeci M, et al. Hematotoxicity in workers exposed to low levels of benzene. Science 2004; 306:1774-6.. Because these are individual characteristics inherent to the exposed organism, these variations should be considered when assessing the potential health impacts of population groups.

Recent studies have led to great interest in the CYP enzymatic system, since this system plays a significant role, mainly in phase I of the metabolism of most endogenous substances and xenobiotics, including organic chemicals such as ethanol, acetone and benzene ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,2727. Hatagima A. Genetic polymorphism and metabolism of endocrine disruptors in cancer susceptibility. Cad Saúde Pública 2002; 18:357-77.^,2828. Coura RS, Marques CFS, Koifman RJ, Koifman S, Cabello PH, Hatagima A. CYP1A1 and CYP2E1 polymorphism frequencies in a large Brazilian population. Genetic and Molecular Biology 2007; 30:1-5.^,2929. Piccoli P, Carrieri M, Padovano L, Di Mare M, Bartolucci GB, Fracasso ME, et al. In vitro CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene. Toxicol Lett 2010; 192:29-33.^,3030. Tang K, Li X, Xing Q, Li W, Feng G, He L, et al. Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in Chinese Han populations from four different geographic areas of mailand China. Genomics 2010; 95:224-9.. In humans, this family of enzymes is present in several organs, mainly in the liver ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47., and is encoded by more than 50 genes, distributed throughout 10 large families, with subfamilies subdivided into isoforms ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3131. Autrup H. Polymorphisms in human xenobiotica metabolizing enzymes as susceptibility factors in toxic response. Mutat Res 2000; 464:65-76..

The CYP2E1 isoform is the most active in benzene metabolism. CYP2E1 catalyzes the conversion of benzene to benzene oxide (BO), as well as the conversion of hydroquinone (HQ) and catechol to 1,2,4-trihydroxybenzene ²²22. Kim S, Lan Q, Waidyanatha S, Chanock S, Johnson BA, Vermeulen R, et al. Genetic polymorphisms and benzene metabolism in humans exposed to a wide range of air concentrations. Pharmacogenet Genomics 2007; 17:789-801.^,2626. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3232. Rothman N, Smith MT, Hayes RB, Traver RD, Hoener B, Campleman S, et al. Benzene poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C/T mutation and rapid fractional excretion of chlorzoxazone. Cancer Res 1997; 57:2839-42.^,3333. Barry KH, Zhang Y, Lan Q, Zahm SH, Holford TR, Leaderer B, et al. Genetic variation in metabolic genes: occupational solvent exposure, and risk of non-Hodgkin linphoma. Am J Epidemiol 2011; 173:404-13.. The location of the CYP2E1 gene in the human genome is on chromosome 10, in 10q24.3 ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3434. Rossini A, Lima SS, Rapozo DCM, Faria M, Albano RM, Pinto LFR. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro. Braz J Med Biol Res 2006; 39:195-201..

Studies investigating this isoform have reported the presence of polymorphisms in the promoter region at -1293 (restriction endonuclease site RsaI) and at position 7632 of the gene (restriction endonuclease site DraI), whose variants are associated with increased enzyme activity and increased risk for leukemia development, probably due to the high production of toxic intermediates ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.^,2626. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3535. Seaton MJ, Schlosser PM, Bond JA, Medinsky MA. Benzene metabolism by human liver microsomes in relation to cytochrome P4502E1 activity. Carcinogenesis 1994; 15:1799-806.^,3636. Wang L, He X, Bi Y, Ma Q. Stem cell and benzene-induced malignancy and hematotoxicity. Chem Res Toxicol 2012; 25:1303-15..

The enzyme NQO1 acts preferentially in the bone marrow, considered a phase II enzyme in the chemical metabolism. It acts by reducing benzoquinones to dihydroxy-quinones, less toxic, in the benzene metabolism pathway ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.. Thus, the higher the NQO1 activity, the lower the action of intermediary reactive metabolites in the organism ¹⁹19. Ross D. The role of metabolism and specific metabolites in benzene-induced toxicity: evidence and issues. J Toxicol Environ Health A 2000; 61:357-72.^,3737. Snyder R, Witz G, Goldstein BD. The toxicology of benzene. Environ Health Perspect 1993; 100:293-306.^,3838. Nebert DW, Roe AL, Vandale SE, Bingham E, Oakley GG. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet Med 2002; 4:62-70..

In humans, the gene encoding this enzyme is located on chromosome 16 (16q22.1) and exhibits a functional polymorphism originating from a substitution of C→T bases at position 609 of the gene, which determines the exchange of proline by serine at position 187 of the protein ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47..

Studies have shown that the presence of the variant allele is related to decreased enzymatic efficiency and, consequently, protective NQO1 action. Individuals presenting the homozygous genotype may display absence of enzymatic activity.

Currently, there is a need for studies aiming at promoting the joint evaluation of biomarkers, susceptibility and effect in residents living around petrochemical poles exposed to benzene, in order to find possible justifications for the high leukemia hospitalization rates in these areas. In this context, the aim of this study was to evaluate the association of environmental exposure to benzene to several biomarkers in the surrounding Industrial Complex of Campos Elíseos (PICE) population, Municipality of Duque de Caxias, Rio de Janeiro State, Brazil.

Methodology

This study was carried out at the Center of Studies on Worker’s Health and Human Ecology (CESTEH), approved by the Sergio Arouca National School of Public Health (ENSP) Ethics Committee (Opinion 971.927, CAAE: 40514415.0.0000.5240) at the Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil.

The design was of observational descriptive sectional type, applying the description of the resident population surrounding the petrochemical industries, with sampling by convenience, due to the difficult access to this locality.

The geographical study area was the Campos Elíseos district, located in the municipality of Duque de Caxias, near the PICE. The main activity in the area concerns the petrochemical industry. This region is home to several industries, including the Caxias Refinery (Reduc), the largest industrial unit in the Greater Rio de Janeiro area, Braskem, which produces as by-product a pyrolysis gasoline containing about 40% benzene, and the Synthetic Rubberd Factory (Fabor), both displaying significant importance in the development of the study area.

Inclusion criteria

The study group consisted of individuals residing up to 1,000 meters from the petrochemical Campos Elíseos complex, comprising male and female adults (age ≥ 18 years), of different ages and races, living in the area for at least 3 months.

Individuals who agreed to participate in the study signed an informed consent form, as required by the Brazilian Ministry of Health, Resolution n. 466/2012 concerning research involving humans. A total of 194 residents volunteered. Four subjects were excluded from the study, due to insufficient biological material for the laboratory analyses, with 190 remaining.

Information collection

First, participants answered a semi-structured questionnaire for characterizing population exposure and obtaining sociodemographic data and information regarding dietary habits, daily routine, housing conditions and health and illness records, among others. Information was also collected to identify confounding factors in the study. The data were then analyzed in order to investigate possible exposure-outcome associations, and finally, to characterize the risk to which these residents are subjected to in their environment.

Biological material sampling

Urine samples were collected in 20mL polyethylene bottles and sent to the CESTEH Toxicology Laboratory/ENSP/Fiocruz, where they were stored in an ultrafreezer (-80ºC) until the laboratory analyses. Blood samples for molecular analyses and blood cell counts were collected in vacuum tubes containing EDTA K2 as anticoagulant and stored in an ultrafreezer (-80ºC) in the same laboratory.

Organization and characterization of the study groups

To characterize the study groups, a semi-structured questionnaire was applied to obtain data regarding sex, race, age, schooling and professions, and to verify frequency regarding chemical product handling, alcohol consumption and smoking habits in their social circles. The obtained data were used to minimize the influence of the confounding variables, promote the definition of resident profiles and delineate common habits, accurately and qualitatively identify relevant symptoms for assessments concerning possible exposure-outcome associations, and characterize the risk to which these residents are subjected to in their environment.

Analytical procedures

Urinary S-PMA analyses

S-PMA was determined by liquid chromatography coupled to sequential mass spectrometry (HPLC-MS/MS) applying the methodology described by Gomes ³⁹39. Gomes RP. Métodos de extração com partição a baixa temperatura para a determinação de marcadores biológicos de exposição ao benzeno, em urina, por UHPLC-MS/MS ENT#091;Masters ThesisENT#093;. Ouro Preto: Programa de Pós-Graduação em Engenharia Ambiental, Universidade Federal de Ouro Preto; 2016.. Liquid-liquid extraction by low temperature partition (LLE-LTP) was applied, consisting in the addition of a reduced amount of an organic solvent in the sample and subsequent refrigeration of the obtained mixture to -20ºC for at least 3 hours. Under these conditions, the aqueous phase solidifies and the organic solvent is separated, forming a supernatant phase containing the solubilized analyte, which is then removed and analyzed ³⁹39. Gomes RP. Métodos de extração com partição a baixa temperatura para a determinação de marcadores biológicos de exposição ao benzeno, em urina, por UHPLC-MS/MS ENT#091;Masters ThesisENT#093;. Ouro Preto: Programa de Pós-Graduação em Engenharia Ambiental, Universidade Federal de Ouro Preto; 2016..

After extraction, S-PMA concentrations were determined by high performance liquid chromatography (Scientific Surveyor, Thermo Fisher Scientific, Waltham, United States) coupled to a triple quadrupole tandem-MS sequential mass spectrometer with electrospray ionization (ESI) (TSQ Quantum model, Thermo Fisher Scientific, Waltham, United States), using the Xcalibur software (https://www.thermofisher.com/order/catalog/product/OPTON-30487).

Creatinine determination

Creatinine concentrations in urine samples were determined using the Doles Colorimetric Kit (Panamá, Goiás State, Brazil) applying a picric acid reaction in an alkaline medium, after deproteinization, through spectrophotometry ⁴⁰40. Rosa ACS. Avaliação dos níveis basais de metabólitos de agrotóxicos piretroides na população adulta da cidade do Rio de Janeiro: contribuição para a vigilância em saúde no país ENT#091;Doctoral DissertationENT#093;. Rio de Janeiro: Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz; 2017..

CYP2E1 - 1293G>C and NQO1 609C>T polymorphism determinations

Polymorphisms were determined by genotyping using the real-time polymerase chain reaction (PCR) technique. Template DNA samples of each individual were obtained by extraction of genomic DNA by the Salting-Out technique, from 500μL aliquots of whole blood. Quantitation of extracted DNA was performed by fluorescence using the Qubit 2.0 fluorometer (Invitrogen, Life Technologies, Carlsbad, United States). A 50µg.mL^-1 ratio of DNA per optical density was adopted for calculation purposes. Real-time PCR was carried out in a final volume of 8μL, containing 1x4μL of TaqMan Genotyping Master Mix (Thermo Fisher Scientific), 1x0.5μL of the TaqMan SNP Genotyping Assay probes (Thermo Fisher Scientific) specific for each polymorphism, CYP2E1 - 1293G>C (rs3813867) and NQO1 609C>T (rs1800566). Amplification conditions followed the recommendations suggested by the probe manufacturer.

Statistical analyses

All statistical analyses were carried out using Excel (https://products.office.com/) and SPSS Statistics v.20 (https://www.ibm.com/) software programs. The frequency of each variable, urinary S-PMA concentrations and metabolic polymorphisms, was first investigated. Subsequently, data distribution was verified by the Kolmogorov-Smirnov test. As a normal distribution for urinary S-PMA was observed, parametric tests were applied. The Mann-Whitney test was used to verify possible differences in the mean concentrations of urinary S-PMA, residence time, smoking and metabolic polymorphisms. Associations between polymorphisms and urinary S-PMA levels were investigated through odds ratio (OR). The representation of the reference values was expressed as means, standard deviations and 95% confidence intervals (95%CI).

Results and discussions

Urinary S-PMA analyses

After optimization of the spectrometric conditions, S-PMA m/z ratios in the negative ionization mode were determined, as well as the multiple reaction monitoring (MRM) transitions, which were used for metabolite quantification, alongside retention time for compound confirmation. The analytical curves were prepared ranging from 10 to 500ng.mL^-1 and were repeated for each sample batch. The recovery percentage of the analyte in the extraction procedure was evaluated using concentrations referring to three points of the analytical curve. For each point, urine was fortified with the predetermined amount of the S-PMA standard prior to extraction, while another extraction was carried out in another aliquot of the same urine, and the same amount of S-PMA was added to the extract, considered as corresponding to 100% recovery. For the extraction method to be considered efficient, the point means should be between 70 and 120% ⁴⁰40. Rosa ACS. Avaliação dos níveis basais de metabólitos de agrotóxicos piretroides na população adulta da cidade do Rio de Janeiro: contribuição para a vigilância em saúde no país ENT#091;Doctoral DissertationENT#093;. Rio de Janeiro: Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz; 2017.^,4141. Directorate-General for Health and Food Safety, European Commission. Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. Madrid: European Commission; 2016. (SANTE/11945/2015)..

All samples were extracted and analyzed over two weeks and the processing order was held after the collection sequence. For each day of analysis, an analytical curve was prepared, comprising three concentration points, and recovery evaluation was carried for one point using a single fresh pool of urine, used as a urine blank, composed of a mixture of five urine samples donated by non-smoking CESTEH workers.

To verify the daily sample processing and quality controls, the angular and linear areas of the curve were observed in the matrix and visually evaluated in the software for equipment analysis and qualitative evaluation module, concerning the fortification areas at the three concentration levels, as described in Rosa ⁴⁰40. Rosa ACS. Avaliação dos níveis basais de metabólitos de agrotóxicos piretroides na população adulta da cidade do Rio de Janeiro: contribuição para a vigilância em saúde no país ENT#091;Doctoral DissertationENT#093;. Rio de Janeiro: Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz; 2017..

The final sample evaluation was performed by assessing the relationships between quantifier and qualifier ions. If this value was within the ion ratio range established as acceptable for qualification on that day of analysis, the area of the 109m/z ion obtained in each sample was then applied to the straight-line equation, to calculate urinary S-PMA concentration values, in μg.g^-1.

From the total of 190 participants, 21 samples displayed quantifiable urinary S-PMA levels by the adopted method, expressed as means, median, maximum and minimum values and 25, 50, 75 and 95 percentiles, displayed in Table 1.

The urinary S-PMA results were tested concerning normality by the Kolmogorov-Smirnov test, and the general data did not present a normal distribution (p < 0.05).

Of the 21 samples presenting quantifiable S-PMA levels, only two were from smokers, determined at 0.88 and 1.56μg.g^-1 creatinine, thus indicating that exposure through smoking did not significantly influence group findings.

The S-PMA levels found herein are similar to findings from other studies investigating this biomarker in non-occupationally exposed populations, and below the biological exposure limit of 25μg.g^-1 creatinine, as defined by the American Conference of Governmental Industrial Hygienits (ACGIH) ²¹21. American Conference of Governmental Industrial Hygienists. Threshold limit values (TLVs) for chemical substances and physical agents biological exposure indices for 2013. Cincinnati: American Conference of Governmental Industrial Hygienists; 2013. regarding occupational exposure.

Gomes ³⁹39. Gomes RP. Métodos de extração com partição a baixa temperatura para a determinação de marcadores biológicos de exposição ao benzeno, em urina, por UHPLC-MS/MS ENT#091;Masters ThesisENT#093;. Ouro Preto: Programa de Pós-Graduação em Engenharia Ambiental, Universidade Federal de Ouro Preto; 2016. applied the same extraction method and separation and detection technique to volunteers who were environmentally exposed to benzene and obtained a S-PMA median of 2.56μg.g^-1 creatinine, similar to that reported in a method and application development study carried out by Fan et al. ⁴²42. Fan R, Wang D, She J. Method development for the simultaneous analysis of trans,trans-muconic acid, 1,2-dihydroxybenzene, S-phenylmercapturic acid and S-benzylmercapturic acid in human urine by liquid chromatography/tandem mass spectrometry. Analytical Methods 2015; 7:573-80., with a median of 3.35μg.g^-1 creatinine found in non-occupationally exposed groups, while the study carried out by Protano et al. ⁴³43. Protano C, Andreoli R, Manini P, Vitali M. Urinary trans,trans-muconic acid and S-phenylmercapturic acid are indicative of exposure to urban benzene pollution during childhood. Sci Total Environ 2012; 435/436:115-23. reported a median of 0.62μg.g^-1 creatinine when evaluating 395 children and adolescents not exposed to direct benzene emission sources. Johnson et al. ⁴⁴44. Johnson ES, Langard S, Lin YS. A critique of benzene exposure in the general population. Sci Total Environ 2007; 374:183-98., in a data review study on exposure of non-occupational populations to benzene (including children) reported mean/median urinary S-PMA values ranging from 1.2 to 16.0μg.g^-1 creatinine among the different groups.

In this study, urinary S-PMA data were grouped into two large groups: samples presenting quantifiable S-PMA levels (n = 21), termed Positive for S-PMA, and the remaining samples, whose results were below the limit of quantification of the method (n = 169), classified as Negative for S-PMA.

Subsequently, both groups were compared to the results for hematological parameters, through the association measure of the OR. Hematological components widely described as altered during benzene exposure were selected, namely leukocyte values (< 4,500/mm³), mean corpuscular volume (MCV) (above 89 fl) and neutrophils (sum of segmented rods < 3,000/mm³). The chi-square test was applied and the odds ratios were then calculated. The results are described in Table 2.

The results indicate that samples with quantifiable urinary S-PMA levels, which derive from the benzene metabolism, are associated to changes in neutrophil and MCV parameters. Although a non-statistically significant association was observed, a trend was noted regarding the association between MCV and S-PMA.

Associations between the pathologies detected in this study and urinary S-PMA concentrations were investigated by calculating the OR of these variables. Participants presenting detectable S-PMA levels displayed an increased risk of also presenting one of the hematological alterations identified herein (dehydration, eosinophilia, thrombocytopenia associated to anemia, anemia, thrombocytopenia, eosinophilia, leukocytosis, leukocytosis associated to neutrophilia, leukopenia, and leukopenia associated to neutropenia) (Table 3).

The values observed herein are consistent with those reported in the literature and corroborate that the hematological alterations observed in this study may be associated with benzene exposure, even at low levels, as described by other authors ⁹9. Agency for Toxic Substances and Disease Registry. Toxicological profile for benzene. Atlanta: Agency for Toxic Substances and Disease Registry; 2007.^,2525. Lan Q, Zhang L, Li G, Vermeulen R, Weinberg RS, Dosemeci M, et al. Hematotoxicity in workers exposed to low levels of benzene. Science 2004; 306:1774-6.^,4444. Johnson ES, Langard S, Lin YS. A critique of benzene exposure in the general population. Sci Total Environ 2007; 374:183-98.^,4545. Ye L, Zhang G, Huang J, Li Y, Zheng G, Zhang D, et al. Are polymorphisms in metabolism protective or a risk for reduced white blood cell counts in a Chinese population with low occupational benzene exposures? Int J Occup Environ Health 2015; 21:232-40.^,4646. Fustinoni S, Campo L, Satta G, Campagna M, Ibba A, Tocco MG, et al. Environmental and lifestyle factors affect benzene uptake biomonitoring of residents near a petrochemical plant. Environ Int 2012; 39:2-7..

The OR for other variables related to levels of urinary S-PMA were also calculated, allowing the qualitative identification of possible benzene exposure sources, such as handling of gasoline and other solvents, which increases the risk of presenting quantifiable urinary S-PMA levels in 12% and 41%, respectively. These results were expected, as gasoline contains benzene and the types of solvents used by the population were not classified as to their nature.

Molecular analyses

Genotype and allele frequencies of CYP2E1 - 1293G>C and NQO1 609C>T polymorphisms in the study population

The genotype and allele frequencies of the study population are presented in Table 4.

The allele and genotype distribution obtained in this study is similar to that reported in other studies evaluating the same polymorphisms in populations subjected to varying occupational and environmental exposure benzene concentrations ²⁶26. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3434. Rossini A, Lima SS, Rapozo DCM, Faria M, Albano RM, Pinto LFR. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro. Braz J Med Biol Res 2006; 39:195-201.^,3838. Nebert DW, Roe AL, Vandale SE, Bingham E, Oakley GG. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet Med 2002; 4:62-70.^,4747. Wan J, Shi J, Hui L, Wu D, Jin X, Zhao N, et al. Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning. Environ Health Perspect 2002; 110:1213-8.^,4848. Gattás GJ, Soares-Vieira JA. Cytochrome P450-2E1 and glutathione S-transferase mu polymorphisms among Caucasians and mulattoes from Brazil. Occup Med (Lond) 2000; 50:508-11.^,4949. Rossini A, Rapozo DC, Amorim LM, Macedo JM, Medina R, Neto JF, et al. Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population. Genet Mol Res 2002; 3:233-40.^,5050. Canalle R, Burim RV, Tone LG, Takahashi CS. Genetic polymorphisms and susceptibility to childhood acute lymphoblastic leukemia. Environ Mol Mutagen 2004; 4:100-9.^,5151. Silva J, Moraes CR, Heuser VD, Andrade VM, Silva FR, Kvitko K, et al. Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes. Mutagenesis 2008; 23:415-22.^,5252. Maciel ME, Oliveira FK, Propst GB, Bicalho MG, Cavalli IJ, Ribeiro EMSF. Population analysis of xenobiotic metabolizing genes in south Brazilian Euro and Afro-descendants. Genetics and Molecular Biology 2009; 32:723-8.^,5353. Silveira VS, Canalle R, Scrideli CA, Queiroz RGP, Toni LG. Role of the CYP2D6, EPHX1, MPO, and NQO1 genes in the susceptibility to acute lymphoblastic leukemia in Brazilian children. Environ Mol Mutagen 2010; 51:48-56.^,5454. Pinheiro DS, Rocha Filho CR, Mundim CA, Júnior PM, Ulhoa CJ, Reis AAS, et al. Evaluation of glutathione S-transferase GSTM1 and GSTT1 deletion polymorphisms on type-2 diabetes mellitus risk. PLoS One 2013; 8:e76262.. The genotype distribution displayed a Hardy-Weinberg shift for the NQO1 and CYP2E1 genes. Several studies concerning genes encoding metabolic enzymes that are candidates for susceptibility to toxic substances and related diseases have been carried out ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.^,2626. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3535. Seaton MJ, Schlosser PM, Bond JA, Medinsky MA. Benzene metabolism by human liver microsomes in relation to cytochrome P4502E1 activity. Carcinogenesis 1994; 15:1799-806.^,3636. Wang L, He X, Bi Y, Ma Q. Stem cell and benzene-induced malignancy and hematotoxicity. Chem Res Toxicol 2012; 25:1303-15.^,3838. Nebert DW, Roe AL, Vandale SE, Bingham E, Oakley GG. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet Med 2002; 4:62-70.^,5555. Ross D. Function and distribution of NQO1 in human bone marrow: potential clues to benzene toxicity. Chem Biol Interact 2005; 30:137-46.. The greater or lesser susceptibility to the development of certain pathologies due to differences in metabolism determined by genetic variability has been increasingly studied.

To evaluate the relationship between the data and the hematological parameters obtained in this study, an association measure (OR) was carried out. Hematological alterations related to benzenism were selected ⁵⁶56. Departamento de Ações Programáticas Estratégicas, Secretaria de Atenção à Saúde, Ministério da Saúde. Risco químico: atenção à saúde dos trabalhadores expostos ao benzeno. Saúde do trabalhador, Protocolos de Complexidades Diferenciadas, Série A Normas e Manuais Técnicos. Brasília: Ministério da Saúde; 2006., namely leukocytes (< 4,500/mm³), MCV (above 89 fl) and, mainly, neutrophils (< 3,000/mm³). The chi-square test was applied and the OR calculated. Results are displayed in Table 5.

The results indicate that being a carrier of the variant allele may be a risk factor for changes in leukocyte and MCV values, although this was not statistically significant, probably due to the small sample size. This association between indicators can be explained by the presence of the variant allele that, in the case of CYP2E1, is associated with increased enzyme activity and increased risk, probably due to the high production of toxic intermediates ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.^,2626. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3535. Seaton MJ, Schlosser PM, Bond JA, Medinsky MA. Benzene metabolism by human liver microsomes in relation to cytochrome P4502E1 activity. Carcinogenesis 1994; 15:1799-806.^,3636. Wang L, He X, Bi Y, Ma Q. Stem cell and benzene-induced malignancy and hematotoxicity. Chem Res Toxicol 2012; 25:1303-15., while in NQO1 a decreased enzymatic activity and accumulation of toxic intermediates increased the risk for leukemia development ¹⁹19. Ross D. The role of metabolism and specific metabolites in benzene-induced toxicity: evidence and issues. J Toxicol Environ Health A 2000; 61:357-72.^,3838. Nebert DW, Roe AL, Vandale SE, Bingham E, Oakley GG. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet Med 2002; 4:62-70.. The values obtained in this study are similar to those reported in other studies, which also indicate increased risk for the same hematological alterations in the presence of the variant allele ²²22. Kim S, Lan Q, Waidyanatha S, Chanock S, Johnson BA, Vermeulen R, et al. Genetic polymorphisms and benzene metabolism in humans exposed to a wide range of air concentrations. Pharmacogenet Genomics 2007; 17:789-801.^,4747. Wan J, Shi J, Hui L, Wu D, Jin X, Zhao N, et al. Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning. Environ Health Perspect 2002; 110:1213-8.^,5757. Zhang GH, Ye LL, Wang JW, Ren JC, Xu XW, Feng NN, et al. Effect of polymorphic metabolizing genes on micronucleus frequencies among benzene-exposed shoe workers in China. Int J Hyg Environ Health 2014; 217:726-32..

When evaluating the association between the genotypes of both investigated polymorphisms and the different hematological changes as a group - (1) eosinophilia; (2) plateletopenia + anemia; (3) anemia; (4) plateletopenia; (5) plateletopenia + eosinophilia; (6) leukocytosis; (7) leukocytosis and neutrophilia; (8) leukopenia; and (9) leukopenia and neutropenia -, the presence of the NQO1 609C>T variant allele was suggested as a risk factor, despite the lack of statistical significance, probably due to the small sample size. This was not observed for the CYP2E1 - 1293G>C polymorphism. Although these results show no association in the literature, it is possible to observe that benzene exposure is present, due to the indicators assessed in this study.

Ye et al. ⁴⁵45. Ye L, Zhang G, Huang J, Li Y, Zheng G, Zhang D, et al. Are polymorphisms in metabolism protective or a risk for reduced white blood cell counts in a Chinese population with low occupational benzene exposures? Int J Occup Environ Health 2015; 21:232-40. also observed that the presence of the variant allele confers a greater susceptibility to hematological changes related to benzene. Thus, participants who presented the CC genotype displayed a reduction in white cell counts. This corroborates the study carried out by Wan et al. ⁴⁷47. Wan J, Shi J, Hui L, Wu D, Jin X, Zhao N, et al. Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning. Environ Health Perspect 2002; 110:1213-8., who described that the CC genotype confers greater susceptibility to benzene-benzene intoxication for CYP2E1 - 1293. In addition, the same study noted increased risk for the development of acute lymphoblastic leukemia in heterozygous individuals.

The association of the variant allele of the two analyzed polymorphisms with the risk for benzene hematoxicity development after exposure has also been reported in other studies ⁵⁸58. Yang Y, Tian Y, Jin X, Yan C, Jiang F, Zhang Y, et al. A case-only study of interactions between metabolic enzyme polymorphisms and industrial pollution in childhood acute leukemia. Environ Toxicol Pharmacol 2009; 28:161-6.^,5959. Li G, Yin S. Progress of epidemiological and molecular epidemiological studies on benzene in China. Ann N Y Acad Sci 2006; 1076:800-9.. Zhang et al. ⁵⁷57. Zhang GH, Ye LL, Wang JW, Ren JC, Xu XW, Feng NN, et al. Effect of polymorphic metabolizing genes on micronucleus frequencies among benzene-exposed shoe workers in China. Int J Hyg Environ Health 2014; 217:726-32. evaluated the influence of genetic polymorphisms on the frequency of micronucleus formation in workers in the benzene-exposed footwear industry and found a small increase in the risk of micronucleus formation in individuals with the variant allele.

Studies described in the literature that aimed to study the CYP2E1 as promoter of region polymorphism at -1293 (RsaI restriction site) justify that the variant form is associated with increased enzyme activity and increased risk for the development of leukemia, probably due to the higher production of toxic intermediates ⁶6. Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E. NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure: a literature review. Toxicol Lett 2008; 182:7-17.^,2626. Mitri S, Fonseca AS, Otero UB, Tabalipa MM, Moreira JC, Sarcinelli PN. Metabolic polymorphisms and clinical findings related to benzenepoisoning detected in exposed Brazilian gas-station workers. Int J Environ Res Public Health 2015; 12:8434-47.^,3535. Seaton MJ, Schlosser PM, Bond JA, Medinsky MA. Benzene metabolism by human liver microsomes in relation to cytochrome P4502E1 activity. Carcinogenesis 1994; 15:1799-806.^,3636. Wang L, He X, Bi Y, Ma Q. Stem cell and benzene-induced malignancy and hematotoxicity. Chem Res Toxicol 2012; 25:1303-15.. However, the presence of the NQO1 variant allele decreases the protective action of the enzyme, due to lower enzymatic activity and consequent toxic metabolite accumulation ³⁸38. Nebert DW, Roe AL, Vandale SE, Bingham E, Oakley GG. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet Med 2002; 4:62-70.^,5555. Ross D. Function and distribution of NQO1 in human bone marrow: potential clues to benzene toxicity. Chem Biol Interact 2005; 30:137-46..

Conclusions

This study carried out a biological characterization associating the evaluation of the internal dose biomarker (S-PMA) with susceptibility biomarkers (metabolic polymorphisms) and hematological alterations suggestive of benzene exposure in Campos Elíseos residents, evidencing the current precarious situation of resident individuals.

Urinary S-PMA concentrations presented results similar to those reported in other studies evaluating non-exposed populations. Thus, according to the results, there is no evidence to suggest that Campos Elíseos exposure is high when compared to other regions. However, when associations between S-PMA levels and other variables were investigated, increased risk with increased urinary S-PMA levels was observed, i.e. the greater the presence of benzene, the higher the occurrence of hematological alterations and associated pathologies. However, further studies are required in the Campos Elíseos region, including the determination of environmental indicators.

The allele and genotype frequencies of metabolic polymorphisms CYP2E1 - 1293 and NQO1 609C>T were determined in the studied population, and the presence of the variant alleles was associated to certain blood cell count alterations, possibly related to benzene exposure.

The need to carry out new studies in this region and in other regions, particularly near petrochemical industries, was demonstrated herein, and further investigations concerning relationships between the concentrations or the presence of biomarkers assessed herein, as well as others, with environmental benzene levels and potential pathologies are required.

Acknowledgments

This research was funded by Brazilian National Research Council (CNPq), Brazilian Graduate Studies Coordinating Board (Capes), and Oswaldo Cruz Foundation (Fiocruz).

References

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