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This is a scope review protocol on Long Covid in Workers. The review question is: "What are the main risk factors, clinical management, and management of workers affected by Long Covid?”.

A gasolina é uma mistura complexa de substâncias, dentre elas estão hidrocarbonetos como o benzeno, tolueno, etilbenzeno e xilenos (BTEX), sendo esses compostos emitidos para a atmosfera, com destaque ao benzeno, por ser comprovadamente carcinogênico. Objetivamos avaliar as concentrações de BTEX no ar de postos de revenda de combustíveis no Município do Rio de Janeiro, Brasil, e calcular o risco de câncer associado a estas exposições. Foram realizados dois tipos de amostragem (fixa e móvel) adaptadas da metodologia nº 1.501 do Instituto Nacional de Segurança e Saúde Ocupacional (Estados Unidos), em seis postos de revenda de combustíveis na Zona Oeste da cidade. A amostragem fixa foi feita próxima às bombas de combustível, enquanto a móvel foi realizada através da coleta do ar na zona respiratória dos trabalhadores (frentistas), conforme estes se deslocavam pelo posto. As amostras foram analisadas por cromatografia gasosa com detecção por ionização em chama. Com os resultados das amostragens foram feitos os cálculos de risco à saúde, usando os indicadores Quociente de perigo (HQ) e Risco de câncer (CR), para avaliar os possíveis efeitos não-carcinogênicos e carcinogênicos, respectivamente, nos trabalhadores dos postos de revenda de combustíveis. As concentrações ambientais para a maioria dos compostos BTEX foram abaixo dos limites preconizados, menos para o benzeno, um composto carcinogênico, que apresentou concentrações muito acima dos limites, levando a altos valores de risco de câncer. Os resultados demonstraram que há riscos à saúde dos trabalhadores de postos de revenda de combustíveis, principalmente o risco de desenvolver câncer, devido à exposição excessiva ao benzeno. Gasoline is a complex mixture of substances, including aromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylenes (BTEX). These compounds are emitted into the air, with the special relevance of benzene since it is provenly carcinogenic. The study aimed to assess BTEX concentrations in filling stations in the city of Rio de Janeiro, Brazil, and to calculate the cancer risk associated with such exposures. Two types of sampling were performed (stationary and mobile), adapted from methodology n. 1,501 (U.S. National Institute for Occupational Safety and Health) for aromatic hydrocarbons, in six filling stations in the West Zone of Rio de Janeiro. Stationary sampling was done near the fuel pumps, while mobile sampling was done in the breathing zone of the workers (station attendants) as they moved around the station. The samples were analyzed with gas chromatography flame ionization detector. The sampling results were used to calculate the health risk, using the indicators Hazard quotient (HQ) and Cancer risk (CR) to assess the possible non-carcinogenic and carcinogenic effects, respectively, in filling station workers. Environmental concentrations for the most of the BTEX compounds were below the recommended limits, except for benzene, a carcinogenic compound, which displayed concentrations far above the limits, leading to high cancer risk values. The results showed that there are health risks for filling station attendants, especially the risk of developing cancer from excessive exposure to benzene. La gasolina es una mezcla compleja de sustancias, entre ellas existen hidrocarburos como el benceno, tolueno, etilbenceno y xilenos (BTEX), emitiéndose estos compuestos a la atmósfera, donde se destaca el benceno, al tratarse de un carcinogénico comprobado. Los objetivos fueron evaluar las concentraciones de BTEX en el aire de puestos de reventa de combustibles, en el municipio de Río de Janeiro, Brasil, así como calcular el riesgo de cáncer asociado a estas exposiciones. Se realizaron dos tipos de muestra (fija y movible), adaptadas de la metodología nº 1.501 del Instituto Nacional de Seguridad y Salud Laboral (EE.UU.), en seis puestos de reventa de combustibles de la zona oeste de la ciudad. La muestra fija se realizó cerca de las bombas de combustible, mientras que la móvil se hizo mediante tomas de aire en la zona respiratoria de los trabajadores (de la gasolinera), a medida que estos se desplazaban por el lugar de trabajo. Las muestras se analizaron por cromatografía gaseosa con detección por ionización en llama. Junto a los resultados de las muestras, se realizaron cálculos de riesgo para la salud, usando los indicadores Cociente de peligro (HQ) y Riesgo de cáncer (CR), con el fin de evaluar los posibles efectos no-carcinogénicos y carcinogénicos, respectivamente, en los trabajadores de los puestos de reventa de combustibles. Las concentraciones ambientales para la mayoría de los compuestos BTEX estuvieron por debajo de los limites preconizados, menos en el caso del benceno, un compuesto carcinogénico, que presentó concentraciones muy por encima de los límites, llevando a altos valores de riesgo de cáncer. Los resultados demostraron que existen riesgos para la salud de los trabajadores de puestos de reventa de combustibles, principalmente, riesgo de desarrollar cáncer, debido a la exposición excesiva al benceno.

Gasoline is a complex mixture of substances, including aromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylenes (BTEX). These compounds are emitted into the air, with the special relevance of benzene since it is provenly carcinogenic. The study aimed to assess BTEX concentrations in filling stations in the city of Rio de Janeiro, Brazil, and to calculate the cancer risk associated with such exposures. Two types of sampling were performed (stationary and mobile), adapted from methodology n. 1,501 (U.S. National Institute for Occupational Safety and Health) for aromatic hydrocarbons, in six filling stations in the West Zone of Rio de Janeiro. Stationary sampling was done near the fuel pumps, while mobile sampling was done in the breathing zone of the workers (station attendants) as they moved around the station. The samples were analyzed with gas chromatography flame ionization detector. The sampling results were used to calculate the health risk, using the indicators Hazard quotient (HQ) and Cancer risk (CR) to assess the possible non-carcinogenic and carcinogenic effects, respectively, in filling station workers. Environmental concentrations for the most of the BTEX compounds were below the recommended limits, except for benzene, a carcinogenic compound, which displayed concentrations far above the limits, leading to high cancer risk values. The results showed that there are health risks for filling station attendants, especially the risk of developing cancer from excessive exposure to benzene.A gasolina é uma mistura complexa de substâncias, dentre elas estão hidrocarbonetos como o benzeno, tolueno, etilbenzeno e xilenos (BTEX), sendo esses compostos emitidos para a atmosfera, com destaque ao benzeno, por ser comprovadamente carcinogênico. Objetivamos avaliar as concentrações de BTEX no ar de postos de revenda de combustíveis no Município do Rio de Janeiro, Brasil, e calcular o risco de câncer associado a estas exposições. Foram realizados dois tipos de amostragem (fixa e móvel) adaptadas da metodologia nº 1.501 do Instituto Nacional de Segurança e Saúde Ocupacional (Estados Unidos), em seis postos de revenda de combustíveis na Zona Oeste da cidade. A amostragem fixa foi feita próxima às bombas de combustível, enquanto a móvel foi realizada através da coleta do ar na zona respiratória dos trabalhadores (frentistas), conforme estes se deslocavam pelo posto. As amostras foram analisadas por cromatografia gasosa com detecção por ionização em chama. Com os resultados das amostragens foram feitos os cálculos de risco à saúde, usando os indicadores Quociente de perigo (HQ) e Risco de câncer (CR), para avaliar os possíveis efeitos não-carcinogênicos e carcinogênicos, respectivamente, nos trabalhadores dos postos de revenda de combustíveis. As concentrações ambientais para a maioria dos compostos BTEX foram abaixo dos limites preconizados, menos para o benzeno, um composto carcinogênico, que apresentou concentrações muito acima dos limites, levando a altos valores de risco de câncer. Os resultados demonstraram que há riscos à saúde dos trabalhadores de postos de revenda de combustíveis, principalmente o risco de desenvolver câncer, devido à exposição excessiva ao benzeno.La gasolina es una mezcla compleja de sustancias, entre ellas existen hidrocarburos como el benceno, tolueno, etilbenceno y xilenos (BTEX), emitiéndose estos compuestos a la atmósfera, donde se destaca el benceno, al tratarse de un carcinogénico comprobado. Los objetivos fueron evaluar las concentraciones de BTEX en el aire de puestos de reventa de combustibles, en el municipio de Río de Janeiro, Brasil, así como calcular el riesgo de cáncer asociado a estas exposiciones. Se realizaron dos tipos de muestra (fija y movible), adaptadas de la metodología nº 1.501 del Instituto Nacional de Seguridad y Salud Laboral (EE.UU.), en seis puestos de reventa de combustibles de la zona oeste de la ciudad. La muestra fija se realizó cerca de las bombas de combustible, mientras que la móvil se hizo mediante tomas de aire en la zona respiratoria de los trabajadores (de la gasolinera), a medida que estos se desplazaban por el lugar de trabajo. Las muestras se analizaron por cromatografía gaseosa con detección por ionización en llama. Junto a los resultados de las muestras, se realizaron cálculos de riesgo para la salud, usando los indicadores Cociente de peligro (HQ) y Riesgo de cáncer (CR), con el fin de evaluar los posibles efectos no-carcinogénicos y carcinogénicos, respectivamente, en los trabajadores de los puestos de reventa de combustibles. Las concentraciones ambientales para la mayoría de los compuestos BTEX estuvieron por debajo de los limites preconizados, menos en el caso del benceno, un compuesto carcinogénico, que presentó concentraciones muy por encima de los límites, llevando a altos valores de riesgo de cáncer. Los resultados demostraron que existen riesgos para la salud de los trabajadores de puestos de reventa de combustibles, principalmente, riesgo de desarrollar cáncer, debido a la exposición excesiva al benceno.

Resumo Introdução Este estudo avaliou alterações respiratórias, auditivas e citogenéticas em trabalhadores de um estaleiro em Angra dos Reis, no Estado do Rio de Janeiro, relacionadas à exposição de pintores a solventes, metais e ruído no ambiente de trabalho. Métodos Foram avaliados função pulmonar, perdas auditivas e índice de reconhecimento da fala, e alterações citogenéticas pelo teste de aberrações cromossômicas. Foram avaliados manganês e chumbo em sangue por espectrometria de absorção atômica. Os indicadores de efeito utilizados para chumbo foram ALAD e ALA-U, determinados por espectrofotometria e cromatografia líquida, respectivamente. Resultados Seis dos 9 trabalhadores avaliados apresentaram alteração funcional respiratória. Quase 70% dos 18 trabalhadores avaliados apresentaram audição reduzida, com associação entre PAIR e chumbo em sangue. O percentual médio de recuperação da ALAD foi de 32,9%, com médias de ALA-U de 1,7 mg g-1 creatinina, 4,65 µg dL-1 para Pb-S e 10 µg L -1 para Mn-S entre os trabalhadores. Foram observadas associações entre ALA-D ativada com Mn-S e com a presença de aberrações cromossômicas. As alterações citogenéticas identificadas foram aneuploidias, separação prematura centromérica e aberrações como formação de anéis, quebras e união de cromátides irmãs. Conclusão os trabalhadores do estaleiro apresentam alterações que podem ser associadas à exposição ocupacional.

It is estimated that approximately 0.4% of the total leachate produced in a landfill is destined for treatment plants, while the rest can reach the soil and groundwater. In this context, this study aimed to perform leachate toxicity evaluations through immune system cytotoxic assessments, genotoxic (comet assay) appraisals and antioxidant system (superoxide dismutase - SOD; catalase - CAT, glutathione-S-transferase - GST; reduced glutathione - GSH and metallothionein - MT) evaluations in Eisenia andrei earthworms exposed to a Brazilian leachate for 77 days. The leachate sample contained high organic matter (COD - 10,630 mg L−1) and ammoniacal nitrogen (2398 mg L−1), as well as several metals, including Ca, Cr, Fe, Mg, Ni and Zn. Leachate exposure resulted in SOD activity alterations and increased CAT activity and MT levels. Decreased GST activity and GSH levels were also observed. Antioxidant system alterations due to leachate exposure led to increased malondialdehyde levels as a result of lipid peroxidation after the 77 day-exposure. An inflammatory process was also observed in exposed earthworms, evidenced by increased amoebocyte density, and DNA damage was also noted. This study demonstrates for the first time that sublethal effect assessments in leachate-exposed earthworms comprise an important tool for solid waste management.

RESUMO A relação entre exposições ocupacionais, compostos químicos e doenças é conhecida há muito tempo e está no cerne do campo saúde-trabalho-ambiente. A pobreza presente na vida de numerosos trabalhadores brasileiros cria um cenário de vulnerabilidade social, gerando riscos à saúde. Este estudo tem por objetivo discutir a importância dos fatores socioeconômicos na avaliação da exposição ocupacional e a relação com o processo de saúde/doença dos trabalhadores, por meio de uma revisão exploratória da literatura. Diversos fatores estão ligados à forma como o trabalhador se relaciona com a exposição, como as condições de vida, moradia, acesso a serviços de saúde e educação, nível salarial e escolar. ABSTRACT The relationship between occupational exposures, chemical compounds and diseases has been known for a long time and is at the heart of the health-work-environment field. The condition of poverty in the lives of many Brazilian workers creates a scenario of social vulnerability, generating risks to health. This study aims to discuss the importance of socioeconomic factors in the evaluation of occupational exposure and the relationship with workers´ health/illness process, through an exploratory review of the literature. Several factors are related to how the worker relates to exposure, such as living conditions, housing, access to health and education services, salary levels and schooling.

Oxidative stress can be induced by mercury (Hg) exposure, including through fish consumption (diet), leading to health risks. The objective of this study was to evaluate the association between oxidative stress biomarkers and dietary Hg exposure levels in riverine children and adoluiaescents at Madeira River (RO/Brazil). Population from three riverine local communities presenting different fish consumption frequencies was sampled. Hg was determined in blood (ICP-MS) and glutathione (GSH), glutathione S-transferases (GST) and malondialdehyde (MDA) were determined in serum (spectrophotometry). Statistical analyses were performed using parametric and non-parametric tests. Multiple linear regression models and generalized additives models were also used to estimate the relationships between oxidative stress biomarkers and blood Hg. The juvenile riverine population from Cuni&atilde, RESEX presented the highest levels of oxidative stress and Hg levels in blood (GST = 27.2 (4.93) U/L, MDA = 1.69 (0.27) &micro, mol/L, Hg = 20.6 (18.0) &micro, g/L). This population also presented the highest frequency of fish consumption. The positive relation between Hg and GST and MDA, adjusted for individual characteristics, suggests an oxidative effect. This study shows the importance of oxidative stress biomarkers in the evaluation of dietary Hg exposure since initial and reversible metabolic changes were observed, enriching health risk assessments.

Environmental and occupational exposure to benzene from fuels is a major cause for concern for national and international authorities, as benzene is a known carcinogen in humans and there is no safe limit for exposure to carcinogens. The objective of this study was to evaluate the genotoxic effects of chronic occupational exposure to benzene among two groups of workers: filling station workers (Group I) and security guards working at vehicles entrances (Group II), both on the same busy highway in Rio de Janeiro, Brazil. Sociodemographic data on the workers were evaluated, the concentration of benzene/toluene (B/T) in atmospheric air and individual trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (S-PMA) were measured, oxidative stress was analyzed by catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD), thiol groups (THIOL) and malondialdehyde (MDA), genotoxicity was measured by metaphases with chromosomal abnormalities (MCA) and nuclear abnormalities, comet assay using the enzyme formamidopyrimidine DNA glycosylase (C-FPG), and methylation of repetitive element LINE-1, CDKN2B and KLF6 genes. Eighty-six workers participated: 51 from Group I and 35 from Group II. The B/T ratio was similar for both groups, but Group I had greater oscillation of benzene concentrations because of their work activities. No differences in ttMA and S-PMA, and no clinical changes were found between both groups, but linearity was observed between leukocyte count and ttMA, and 15% of workers had leukocyte counts less than 4.5 &times, 109 cells L&minus, 1, demanding close worker&rsquo, s attention. No differences were observed between the two groups for THIOL, MDA, MCA, or nuclear abnormalities. A multiple linear relationship was obtained for the biomarkers MCA and C-FPG. A significant correlation was found between length of time in current job and the biomarkers C-FPG, MCA, GST, and MDA. Although both populations had chronic exposure to benzene, the filling station workers were exposed to higher concentrations of benzene during their work activities, indicating an increased risk of DNA damage.

Resumo Introdução: trabalhadores de postos de combustíveis estão expostos às diversas substâncias químicas presentes no ambiente de trabalho, destacando-se entre elas o benzeno, devido às suas propriedades carcinogênicas. Objetivo: avaliar os danos genotóxicos relacionados à exposição ocupacional ao BTEX (benzeno, tolueno, etilbenzeno, xilenos) em trabalhadores de cinco postos de combustíveis do município do Rio de Janeiro, RJ. Metodologia: foram analisadas concentrações de BTEX no ar; atividades das enzimas catalase e glutationa S-transferase; e ensaio cometa em amostras de sangue total de 97 trabalhadores. Resultados: as concentrações de BTEX estavam dentro dos valores preconizados pela NR 15, incluindo Anexo 13-A. Entretanto, uma oscilação nos resultados de ensaio cometa foi observada entre os trabalhadores dos diferentes postos de combustíveis, principalmente em trabalhadores de postos com menores concentrações de benzeno. Discussão: esse resultado está de acordo com a literatura científica atual, que indica uma curva dose-resposta supralinear para o benzeno, observando-se em baixas concentrações um aumento não linear do risco de leucemia, provavelmente relacionado à maior metabolização do benzeno e à maior produção de seus metabólitos tóxicos nessas concentrações. Conclusão: os resultados deste estudo sugerem que a exposição ao BTEX, mesmo em baixas concentrações, contribui para o risco genotóxico à saúde humana.

Outra Agência Introdução: a avaliação de uma exposição mensura sua intensidade, frequência e duração, podendo detectar danos precoces que, se ignorados, podem evoluir para um quadro nocivo. Nos campos da saúde ambiental e ocupacional, os biomarcadores de genotoxicidade tem sido largamente utilizados para essa avaliação. Objetivo: identificar, descrever e discutir os principais bioindicadores de genotoxicidade e seu uso conjunto com técnicas de avaliação de expressão gênica em estudos de exposição ocupacional ao benzeno em postos de revenda de combustíveis (PRC). Métodos: revisão bibliográfica de trabalhos publicados entre 1995 e 2015. Resultados: as técnicas identificadas foram: ensaio cometa, estresse oxidativo, micronúcleos, aberrações cromossômicas, polimorfismos, adutos de DNA e proteínas, fatores epigenéticos e expressão gênica. Foi observado que testes de danos genéticos e epigenéticos são utilizados em frentistas de PRC que participam de programas de saúde do trabalhador ou de pesquisas, embora um baixo número de publicações sobre o tema tenha sido identificado. Esse fato talvez possa ser explicado pelos poucos países onde a profissão persiste e pelas limitações para o desenvolvimento de pesquisas nesses países. Conclusão: os bioindicadores de genotoxicidade e as técnicas de expressão gênica são úteis na detecção de dano precoce desta exposição ocupacional e devem ser avaliados em conjunto. Introduction: an exposure evaluation measures its intensity, frequencyand duration, detecting premature damage that, if ignored, might develop into a harmful framework. On environmental and occupational health fields, genotoxicity biomarkers have been widely used for this evaluation. Objective: to identify, describe and discuss main genotoxicity biomarkers and their use together with gene expression evaluation techniques in studies concerning occupational exposure to benzene in gas stations (GS). Methods: bibliographical review of studies published between 1995 and 2015. Results: the following techniques were identified: comet assay, oxidative stress, micronuclei, chromosomal aberrations, polymorphisms, DNA and protein adducts, epigenetic factors and gene expression. We observed that genetic and epigenetic damage tests are used in gas station attendants who participate in worker’s health programs or in researches, although a short number of publications on the theme have been identified. This can be explained by the small number of countries where such job still exists and by the limitations for developing research in such countries. Conclusion: genotoxicity biomarkers and gene expression techniques are useful for detecting the premature damage resulting from this occupational exposure and must be jointly evaluated.

Resumo Introdução: trabalhadores estão sujeitos a riscos decorrentes da exposição ambiental e ocupacional ao benzeno, situação ainda mais crítica quando associada a fatores como desigualdade social, múltipla exposição, suscetibilidade individual, degradação ambiental e às possíveis interações entre os contaminantes ambientais. Além disso, nem sempre as regulamentações são estabelecidas e/ou atualizadas apenas a partir de achados científicos, pois são processos permeados por conflitos de interesses. Objetivo: realizar análise crítica sobre a identificação de risco e avaliação da exposição ao benzeno. Métodos: revisão bibliográfica e documental do marco normativo brasileiro e internacional sobre exposição ao benzeno, com relação aos aspectos toxicológicos, de exposição ocupacional e ambiental e de avaliação de risco. Discussão: o controle da exposição ao benzeno permeia o campo da disputa técnico-política de saúde e segurança, constituindo-se em marco regulatório resultante de consenso de entendimentos. Esses entendimentos são marcados pela disputa entre a proteção à saúde e a gestão empresarial das condições de trabalho, em detrimento das evidencias científicas. Em tal contexto, os Limites de Exposição Ocupacional podem variar significativamente entre os países e agências, apesar do reconhecimento universal de que não há limites seguros para exposição ao benzeno, por ser um composto carcinogênico. Abstract Introduction: workers are subject to risks from environmental and occupational exposure to benzene. This situation is even more critical when combined with social inequality, multiple exposure, individual susceptibility, environmental degradation, and possible interactions between environmental contaminants. In addition, regulations are not always established and/or updated only from scientific evidences, because they are processes permeated by conflicts of interest. Objective: to produce a critical analysis on risk identification and benzene exposure evaluation. Methods: literature and documentary review of the Brazilian and international regulatory standards on benzene exposure, toxicological aspects, occupational and environmental exposure, and risk evaluation. Discussion: benzene exposure control permeates the technical-political dispute of health and safety, resulting in a regulatory standard from consensus of understandings. These understandings are shaped by the dispute between health protection and business management of working conditions, to the detriment of scientific evidence. In this context, the Occupational Exposure Limits can vary significantly between countries and agencies, despite the universal recognition that there are no safe limits for benzene exposure, because it is a carcinogenic compound.

Resumo Introdução: trabalhadores de postos de revenda de combustíveis estão expostos a compostos orgânicos voláteis, dos quais se destaca o benzeno. O manejo clínico do amplo espectro de sinais e sintomas desses trabalhadores ainda é um desafio para assistência em saúde. Objetivo: apresentar proposta de classificação clínico-laboratorial baseada na análise de hemograma e na avaliação clínica dos trabalhadores, para orientar a conduta dos profissionais de saúde. Métodos: a classificação clínico-laboratorial foi definida a partir de critérios de hierarquização dos resultados das avaliações clínicas e de vigilância à saúde de modo a possibilitar a organização dos procedimentos e fluxos de atenção à saúde. Resultados: a proposta define o manejo clínico com base na classificação dos expostos ao benzeno em quatro níveis de alterações clínico-laboratoriais que determinam os procedimentos compatíveis com o grau da exposição e de seus efeitos à saúde: 1) sem alterações significativas; 2) alterações inespecíficas ou incompletas, 3) alterações clínicas e/ou laboratoriais; 4) alterações sugestivas de benzenismo. Conclusão: a proposta de classificação dos achados clínico-laboratoriais é potencialmente capaz de orientar a conduta médica na avaliação das condições de saúde e dos riscos da exposição ao benzeno e de subsidiar o monitoramento sistemático e contínuo necessário para a definição de rotinas assistenciais para os coletivos de trabalhadores. Abstract Introduction: gas stations workers are exposed to volatile organic compounds, especially benzene. Clinical management of the broad spectrum of symptoms and signs observed in these workers is still a challenge for health care. Objective: to present a clinical-laboratory classification, based on hemogram analysis and clinical evaluation of workers exposed to benzene, as guidelines for health care professionals. Methods: the clinical-laboratory classification was defined based on priority-setting criteria for the results of clinical evaluations and health surveillance in order to allow the organization of health care flows and procedures. Results: the proposal defines the clinical management based on classification of workers exposed to benzene in four levels of clinical and laboratory alterations that determine procedures according to level of exposure and health effects: 1) without significant alterations; 2) unspecific or incomplete alterations; 3) clinical and/or laboratory alterations; and 4) alterations suggesting benzene poisoning. Conclusion: the proposal for classification of clinical and laboratory findings is potentially able to guide medical practice in the assessment of health conditions and risks of benzene exposure, and subsidize the systematic and continuous monitoring necessary for establishing health care practices offered to workers.

DNA is continuously under attack, from environmental chemicals and radiation and also from intrinsic damaging agents, notably reactive oxygen species. In the case of the latter, potential damage is kept under control by antioxidant defences. The damage that does occur is mostly removed by efficient repair pathways, but damage that remains when cells replicate DNA can lead to mutations and possible cell transformation. Oxidative damage to DNA is elevated in various human diseases, including cancers, though this does not necessarily indicate a causal relationship; oxidative stress resulting from the disease could cause DNA damage as a secondary effect. The comet assay is a sensitive and popular method for measuring DNA damage. The underlying principle is that, after cell lysis and removal of nuclear membranes and histones, DNA remains attached to a matrix in the form of supercoiled loops; a strand break in one loop relaxes supercoiling in that loop; on subsequent electrophoresis (normally at high pH), relaxed loops are pulled towards the anode, forming the tail of a comet-like image viewed by fluorescence microscopy. The percentage of DNA in the tail is proportional to break frequency. Oxidised bases are measured by including, after lysis, a digestion with an enzyme with endonuclease activity specific for oxidised pyrimidines (EndoIII or Nth) or for 8-oxoguanine and other products of purine oxidation (formamidopyrimidine DNA glycosylase, Fpg). The comet assay with Fpg has been shown to be more accurate than chromatographic techniques at measuring low levels of 8-oxoguanine, and it is the method of choice in human biomonitoring studies which aim to detect effects of occupational or environmental exposure, or of nutritional supplementation, e.g. with antioxidant-rich foods. A major application of the comet assay is in testing chemicals for genotoxic effects. OECD guidelines exist for use of the comet assay in in vivo experiments, and it is also widely used in in vitro testing. The inclusion of Fpg in the in vitro assay greatly increases the sensitivity of detection of various types of DNA-damaging agent (not only those that induce oxidation damage). In this chapter we provide a detailed protocol, covering all steps from embedding cells in agarose to scoring comets, and including the use of mini-gels (12 per slide) to increase throughput.

Genotoxic effects of occupational workers exposed to asbestos can be evaluated using different biomarkers as oxidative stress enzymes in conjuction with comet assay. This study assessed changes to oxidative stress enzymatic parameters and genotoxic damage in workers occupationally exposed and non-exposed to chrysotile asbestos, who attended the outpatient Clinic of the Center for Worker Health Studies and Human Ecology (CESTEH/ENSP/FIOCRUZ) in Brazil. Chest radiography and spirometry were performed to assess clinical progression of symptoms. The traditional visual score comet assay in peripheral whole blood cells was used to assess DNA damage, and oxidative stress was evaluated by measuring catalase (CAT) and glutathione S-transferase (GST) activities. Respiratory alterations were observed in 53% of workers exposed, as determined by pulmonary function and bronchodilation, and 6 workers were diagnosed with asbestosis. The comet assay was statistically significantly higher in the exposed group and individuals with asbestosis compared to the non-exposed group and individuals without asbestosis, respectively. Linear regression analysis showed that 28,4% and 50,5% of comet assay results were increased by exposure to asbestos and developed asbestosis. The results of CAT and GST were not difference between the groups. These results supports the association of genotoxic damage and the onset of asbestosis by chrysotile asbestos exposure in workers of this study.

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.