Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

(Table 1) Composition of organic matter from sediments of the triple junction area in the Indian Ocean

Bituminous substances in metalliferous sediments from the region of the triple junction in the Indian Ocean were studied. Specific peculiarities of their structure confirming location in "hot conditions" were revealed. Hydrocarbons are genetically connected with hydrothermal matter, and thus they could be considered as a geochemical indicator of hydrothermal processes in the ocean.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

The Elizabeth River Story: A Case Study in Evolutionary Toxicology.

The Elizabeth River system is an estuary in southeastern Virginia, surrounded by the towns of Chesapeake, Norfolk, Portsmouth, and Virginia Beach. The river has played important roles in U.S. history and has been the location of various military and industrial activities. These activities have been the source of chemical contamination in this aquatic system. Important industries, until the 1990s, included wood treatment plants that used creosote, an oil-derived product that is rich in polycyclic aromatic hydrocarbons (PAH). These plants left a legacy of PAH pollution in the river, and in particular Atlantic Wood Industries is a designated Superfund site now undergoing remediation. Numerous studies examined the distribution of PAH in the river and impacts on resident fauna. This review focuses on how a small estuarine fish with a limited home range, Fundulus heteroclitus (Atlantic killifish or mummichog), has responded to this pollution. While in certain areas of the river this species has clearly been impacted, as evidenced by elevated rates of liver cancer, some subpopulations, notably the one associated with the Atlantic Wood Industries site, displayed a remarkable ability to resist the marked effects PAH have on the embryonic development of fish. This review provides evidence of how pollutants have acted as evolutionary agents, causing changes in ecosystems potentially lasting longer than the pollutants themselves. Mechanisms underlying this evolved resistance, as well as mechanisms underlying the effects of PAH on embryonic development, are also described. The review concludes with a description of ongoing and promising efforts to restore this historic American river.

(Table) Content of organic compounds in the bottom sediments of the Volga River estuary

Materials from different spheres of the Earth are ultimately delivered to bottom sediments, which serve as a natural recorder of the functioning of other spheres and originate as a result of the accumulation of their substances. Sedimentary material and species of river-transported elements are subjected to dramatic reworking in marginal filters, where river and sea waters are mixed. These processes are most important for the Caspian Sea, where runoffs of rivers (especially the Volga River) and the intense development and transportation of hydrocarbon fuel by tankers and pipelines (related to the coastal petroleum industry in the Sumgait and Baku ports, Apsheron Peninsula) are potential sources of hydrocarbon pollution. Previously obtained data showed that the total content of hydrocarbon fraction (i.e., the sum of aliphatic hydrocarbons (AHC) and polycyclic aromatic hydrocarbons (PAH)) in bottom sediments varied within 29-1820 µg/g. The content of petroleum hydrocarbons in the northeastern Caspian region varied from 0.052 to 34.09 µg/g with the maximum content in the Tengiz field. The content of six polyarenes in the Volga delta sediments was no more than 40 ng/g. To determine the recent HC pollution of bottom sediments and trends in the functioning of the Volga marginal filter, in summer of 2003 and 2004 we analyzed bottom sediments (58 samples) in the river waterway; Kirovsk channel; Bakhtemir and Ikryanoe branches; tributaries of the Kizan, Chagan, and other rivers; and the Caspian seashore.

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds

This study analysed the levels of androgen receptor antagonist activity in extracts of coastal sediments sampled from estuaries in southern UK and northern France. Anti-androgenic (AA) activity varied between <0.2 and 224.3±38.4μg flutamide equivalents/g dry weight of sediment and was significantly correlated with the total organic carbon and silt content of samples. AA activity was detected in tissues extracts of clams, Scrobicularia plana, sampled from a contaminated estuary, some of which was due to uptake of a series of 4 or 5 ring polycyclic aromatic hydrocarbons (PAHs). Initial studies also indicated that fractionated extracts of male, but not female, clams also contained androgen receptor agonist activity due to the presence of dihydrotestosterone in tissues. This study reveals widespread contamination of coastal sediments of the Transmanche region with anti-androgenic compounds and these contaminants should be investigated for their potential to disrupt sexual differentiation in aquatic organisms.

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds

This study analysed the levels of androgen receptor antagonist activity in extracts of coastal sediments sampled from estuaries in southern UK and northern France. Anti-androgenic (AA) activity varied between <0.2 and 224.3±38.4μg flutamide equivalents/g dry weight of sediment and was significantly correlated with the total organic carbon and silt content of samples. AA activity was detected in tissues extracts of clams, Scrobicularia plana, sampled from a contaminated estuary, some of which was due to uptake of a series of 4 or 5 ring polycyclic aromatic hydrocarbons (PAHs). Initial studies also indicated that fractionated extracts of male, but not female, clams also contained androgen receptor agonist activity due to the presence of dihydrotestosterone in tissues. This study reveals widespread contamination of coastal sediments of the Transmanche region with anti-androgenic compounds and these contaminants should be investigated for their potential to disrupt sexual differentiation in aquatic organisms.

Potential adverse health effects of persistent organic pollutants on sea turtles: Evidences from a cross-sectional study on Cape Verde loggerhead sea turtles

[EN] The Cape Verde nesting population of loggerhead sea turtles (Caretta caretta) is the third largest population of this species in the world. For conservation purposes, it is essential to determine how these reptiles respond to different types of anthropogenic contaminants. We evaluated the presence of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) in the plasma of adult nesting loggerheads from Boa Vista Island, Cape Verde, and studied the effects of the contaminants on the health status of the turtles using hematological and biochemical parameters. All turtles had detectable levels of non-dioxin like PCBs, whereas dioxin-like congeners (DL-PCBs) were detected in only 30% of the turtles.

Exploring hydrocarbonoclastic bacterial communities in the estuarine surface microlayer

Bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) in the estuarine surface microlayer (SML) of the Ria de Aveiro, Portugal—which is chronically polluted with oil hydrocarbons (OH)—were isolated and characterized; Pseudomonas was dominant among the PAH-degrading bacteria. Screening for PAH dioxygenase genes detected almost identical nahAc genes (encoding the alpha subunits of naphthalene dioxygenase) in 2 phylogenetically distinct isolates: Pseudomonas sp. and an unknown species of the family Enterobacteriaceae; this suggested that horizontal transfer of nah genes might be involved in PAH degradation in the SML. We also investigated the effect of PAH contamination on the spatial variability of the bacterioneuston along a gradient of pollution in the estuarine system of the Ria de Aveiro. Culture-independent techniques—fluorescence in situ hy - bridization (FISH) and denaturing-gradient gel electrophoresis (DGGE)—revealed a similar structure among the bacterioneuston communities along the estuary. In contrast, we detected differences in the relative abundance and diversity of organisms of the Gammaproteobacteria, including those of the genus Pseudomonas (which belongs to the Gammaproteobacteria). This is the first insight into the hydrocarbonoclastic bacterial communities in the SML of an estuarine area polluted with hydrocarbons. Our findings highlight the importance of SML-adapted hydrocarbonoclastic bacterioneuston as a potential source of new PAH-degrading bacteria (including new pseudomonads) with potential use in the bioremediation of hydrocarbon-polluted ecosystems.

New N-acylamino acids and derivatives from renewable fatty acids: gelation of hydrocarbons and thermal properties

This work reports the synthesis of new fatty N-acylamino acids and N-acylamino esters from the C16:0, C18:0, C18:1, and C18:1(OH) fatty acid families and demonstrates the activity of these compounds as organogel agents. Compounds were heated and dissolved in various solvents (n-hexane, toluene, and gasoline). Only saturated C16:0 and C18:0 derived from alanine were able to form gels in toluene, and saturated C16:0 derived from phenylalanine showed gelation in n-hexane. This is the first evidence that fatty N-acylamino esters and N-acylamino acid derivatives of l-serine and fatty acids C16:0, C18:0, and C18:1 are able to form gels with hexane. This observation confirms the importance of the hydroxyl group in the segment derivative of l-serine in forming good gels.

Identificação do transcrito CYP1A no peixe Phalloceros caudimaculatus e resposta para a exposição a beta-naftoflavona e amostras ambientais de sedimento

Contaminantes orgânicos, como os hidrocarbonetos policíclicos aromáticos (HPAs), podem atingir corpos da água e possuem potencial para causar efeitos tóxicos em organismos. A exposição aos HPAs causa indução nos níveis de citocromo P450 1A (CYP1A) em peixes, e portanto, é utilizado como um biomarcador de contaminação ambiental. O guarú Phalloceros caudimaculatus ocorre naturalmente em ambientes aquáticos dulcícolas e mixohalinos na América do Sul. O presente estudo identificou a sequência nucleotídica do transcrito CYP1A de P. caudimaculatus, que codifica uma proteína com 521 aminoácidos, e que apresenta 91% e 70% de identidade com CYP1A de killifish e paulistinha, respectivamente. A partir desta sequência foi possível realizar a avaliação dos níveis de mRNA de CYP1A deste peixe por RTq-PCR. Foi realizada uma caracterização de sua indução órgão- e tempo-dependente frente a exposição ao HPA beta-naftoflavona (BNF) e ao elutriato preparado a partir de sedimento de dois corpos da água possivelmente contaminados com HPAs. Foi constatado um aumento significativo nos níveis de mRNA de CYP1A em fígado, brânquia, intestino, cérebro, nadadeira anal de macho adultos e em alevinos na primeira hora de exposição a 1 µM de BNF, em relação ao grupo controle. O rim e as nadadeiras caudal e dorsal apresentaram indução de CYP1A após duas horas de exposição ao BNF. As maiores induções nos peixes dos grupos expostos ao BNF em relação ao controle foram de 176 no rim e 122 vezes no cérebro, observadas respectivamente após 8 e 48 horas de exposição. Os níveis de mRNA de CYP1A nos órgãos e tecidos de alevino, mantiveramse induzidos pela exposição ao BNF até o final das 96 horas de exposição. A exposição dos peixes ao elutriato produzido a partir dos sedimentos coletados em dois locais potencialmente contaminados causou indução do CYP1A no fígado de 22 e 122 vezes em relação ao controle. Os resultados demonstram que a indução de CYP1A em Phalloceros caudimaculatus ocorre em tempos curtos de exposição, além da variação de acordo com o tempo de exposição e com o órgão analisado. Além disso, foi demonstrado que tecidos externos também podem ser utilizados para tais análises e que o elutriato feito a partir de sedimento de locais que recebem descargas de contaminantes podem causar indução de CYP1A nos organismos.

A importância do uso de métodos complementares para a avaliação da toxicidade de hidrocarbonetos aromáticos policíclicos

Hidrocarbonetos Aromáticos Policíclicos (HAPs) e seus derivados Halogenados (HHAPs) são conhecidos como um conjunto de contaminantes ambientais, classificados como potencialmente tóxicos, mutagénicos e carcinogénicos, sendo um assunto de extrema importância e preocupação para a saúde pública. Diversos estudos têm reportado o potencial efeito tóxico destes compostos após longos períodos de exposição, pelo uso de métodos colorimétricos como o ensaio de brometo de 3-[4,5- dimetil-tiazol-2-il]-2,5-difeniltetrazólio (MTT) e vermelho neutro. Contudo, apesar de estes métodos clássicos de avaliação da citotoxicidade in vitro permitirem, de forma rápida, identificar as propriedades nocivas dos compostos, não permitem compreender os mecanismos moleculares subjacentes responsáveis pela sua toxicidade. Deste modo, no presente estudo, para avaliar os efeitos à exposição aos compostos Pireno (Pir) e seu derivado resultante da desinfeção de águas por bromação, 1-BromoPireno (1-BrPir), foram utilizados outros métodos complementares como avaliação do stress oxidativo e vários fenómenos associados à morte celular programada. Os resultados obtidos comprovam não só a existência de efeitos citotóxicos dos poluentes quando acumulados, como também possíveis efeitos genotóxicos. Assim, pelos métodos complementares, foi possível a identificação de alvos moleculares passiveis de ação farmacológica, o que pode constituir o primeiro passo para o desenho de estratégias terapêuticas que visem prevenir ou tratar os danos provocados por estes poluentes no Homem.

Remoção de hidrocarbonetos aromáticos policíclicos em águas utilizando subprodutos da indústria da cortiça

Os Hidrocarbonetos Aromáticos Policíclicos (HAPs) são contaminantes persistentes em meio aquoso. Estes compostos são conhecidos pelas suas propriedades carcinogénicas, mutagénicas e genotóxicas. O principal objetivo deste trabalho consistiu na avaliação das potencialidades de subprodutos da indústria corticeira, como adsorventes alternativos para a remoção de cinco HAPs em meio aquoso: benzo(a)pireno, benzo(ghi)perileno, benzo(b)fluoranteno, benzo(k)fluoranteno e indeno(1,2,3-cd)pireno. A metodologia analítica para quantificar os HAPs envolveu a preparação das amostras, através da técnica de extração em fase sólida (SPE), e a quantificação dos compostos analisados por cromatografia líquida com detetor de fluorescência (LC-FLD). O método foi otimizado e validado, obtendo-se limites de quantificação de 0,004 μg/L para todos os HAPs. Os estudos incidiram na utilização de uma amostra de cortiça, pó de aglomerado de cortiça expandida (PACE), obtida por aglutinação de cortiça em condições hidrotérmicas, a qual nos estudos preliminares revelou desempenho semelhante aos carvões ativados. Com exceção do benzo(ghi)perileno, os resultados mostram que o processo de adsorção dos HAPs na amostra PACE segue uma cinética de pseudo-segunda ordem e as isotérmicas ajustam-se ao modelo de Langmuir.

Firefighters' exposure biomonitoring: Impact of firefighting activities on levels of urinary monohydroxyl metabolites

The concentrations of six urinary monohydroxyl metabolites (OH-PAHs) of polycyclic aromatic hydrocarbons, namely 1-hydroxynaphthalene, 1-hydroxyacenaphthene, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 1-hydroxypyrene (1OHPy), and 3-hydroxybenzo[a]pyrene, were assessed in the post-shift urine of wildland firefighters involved in fire combat activities at six Portuguese fire corporations, and compared with those of non-exposed subjects. Overall, median levels of urinary individual and total OH-PAHs (ΣOH-PAHs) suggest an increased exposure to polycyclic aromatic hydrocarbons during firefighting activities with ΣOH-PAH levels in exposed firefighters 1.7-35 times higher than in non-exposed ones. Urinary 1-hydroxynaphthalene and/or 1-hydroxyacenapthene were the predominant compounds, representing 63-98% of ΣOH-PAHs, followed by 2-hydroxyfluorene (1-17%), 1-hydroxyphenanthrene (1-13%), and 1OHPy (0.3-10%). A similar profile was observed when gender discrimination was considered. Participation in fire combat activities promoted an increase of the distribution percentage of 1-hydroxynaphthalene and 1-hydroxyacenaphthene, while contributions of 1-hydroxyphenanthrene and 1OHPy decreased. The detected urinary 1OHPy concentrations (1.73×10(-2) to 0.152μmol/mol creatinine in exposed subjects versus 1.21×10(-2) to 5.44×10(-2)μmol/mol creatinine in non-exposed individuals) were lower than the benchmark level (0.5μmol/mol creatinine) proposed by the American Conference of Governmental Industrial Hygienists. This compound, considered the biomarker of exposure to PAHs, was the less abundant one from the six analyzed biomarkers. Thus the inclusion of other metabolites, in addition to 1OHPy, in future studies is suggested to better estimate firefighters' occupational exposure to PAHs. Moreover, strong to moderate Spearman correlations were observed between individual compounds and ΣOH-PAHs corroborating the prevalence of an emission source.