Toxicity assessment of polycyclic aromatic hydrocarbons: the importance of the use of complementary methods

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.Polycyclic Aromatic Hydrocarbons (PAHs) and their halogenated derivatives (HPAHs) are known as a set of environmental contaminants classified as potentially toxic, mutagenic and carcinogenic, being a matter of utmost importance and concern to public health. Several studies have reported the potential toxic effects of these compounds after long periods of exposure by the use of colorimetric methods such as 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red. Nonetheless, despite these classic in vitro cytotoxicity assays allow, in a faster way, the identification of the harmful properties of the compounds, they fail in elucidating the underlying molecular mechanisms responsible for their toxicity. Taking into account these mentioned aspects, in the present work, other complementary methods were used in order to evaluate the effects of the exposure to Pyrene (Pyr) and to a brominated-pyrene derivative (resulted from water disinfection), 1-BromoPyrene (1-BrPyr). Effectively, by the evaluation of the oxidative stress and programmed cell death induced by PAHs it was not only possible to verify the cytotoxic effects of PAHs when accumulated but also their potential genotoxic effects. In conclusion, through the use of other tests than the classic cytotoxicity assays, it was possible to identify potential druggable molecular targets which can be the first step towards the development of new therapeutic strategies either to prevent or treat the damage caused by these compounds in Humans.Este trabalho foi desenvolvido no âmbito do projeto RECI/QEQMED/ 0330/2012, financiado pela Fundação para a Ciência e a Tecnologia (FCT)

Environmental biofilms, consequences for health and disease

Ornamental waters of easy access and populated with animals are quite attractive and can hide threats to human health. Here we evaluated, during one year, the microbiota of ornamental waters in a Lisbon park in order to assess the risk for human health. A total of 26 different bacterial species were identified during the study. Among these are potential human pathogens such as Aeromonas, E. coli, K. pneumoniae, Pseudomonas and Raoutella. Aeromonas sobria is a resident bacterium whereas other species such as A. hydrophila and A. veronii were sporadically identified. A possible explanation for this result is the presence of a fish population. The presence of E. coli indicates water contamination with fecal materials. We first isolate this bacterium in June when the water temperature increased and then after it was a resident despite the natural temperature drop in the autumn. This might be explained by the replace of the fish population by ducks in October which lead to an increase in water tubirdity and total carbon content. Pseudomonas fluorescens, P. putida and P. oryzihabitans have been considered low-virulence bacteria but recently have been identified as ethiological agents of healthcare assotiated infections (HAI) mostly in immunosupressed individuals. Another recognized HAI agent present is K. pneumoniae. The same bacteria species were isolated from biofilm and water suggesting the existence of an equilibrium between planktonic and biofilm organized bacteria. The role played by biofilm assembly on the emergence of antibiotic resistance is still ongoing for the isolated bacteria, as well as the possible interaction between water chemical composition and bacteria isolates. Potential pathogens (biofilm or planktonic) represent a significant cause of acute bacterial infections mainly in specific populations (paediatric and geriatric). These results support a periodic control of ornamental water microbiota as simple preventive measure to avoid potential health issues.info:eu-repo/semantics/publishedVersio

Pathogens in ornamental waters: a follow up study

Ornamental waters of easy access and populated with animals are quite attractive and can hide threats to human health. Here we evaluated the microbiota of ornamental waters in a Lisbon park. Water and biofilm samples where collected, in 2 lakes (L1-L2) and ornamental fountains (L3-L4) in February/2015. In May/2015 and monthly during a year (starting March/2016) samples from L4 where collected. Microbiota identification was performed as described previouslya. Biofilm assembly was monitored by crystal violet assay and SEMb and antibiotic susceptibility was performed by conventional methods. The results of the first water sampling (Feb/2015) revealed the presence of Enterobactereaceae and non-fermentative oxidase-positive bacteria. Fountains and lakes presented different microbota being the highest diversity found in L1 hosting a duck population. This result suggested the existence of an interplay between animal inhabitants and microbiota which was confirmed by the second sampling of L4 (May/2015). Between the 2 sampling events a fish population was introduced and the microbiota was completely altered with the appearance of a typical fish pathogen (Aeromonas spp). This tendency was also confirmed over 2016. K. pneumoniae and Aeromonas spp., present as planktonic and biofilm organized bacteria in 2015 showed an enhanced ability to assemble biofilms in vitro at 25 °C than at 37 °C. Bacteria recovered from biofilm showed an increased antibiotic resistance compared to planktonic counterparts. The pilot study conducted during 2015 and the follow up study (still in progress) support a periodic control of ornamental water microbiota as simple preventive measure to avoid potential health issues.Fundação para a Ciência e Tecnologia for the grant PEst-OE/CTM/UI0084/2011N/

Decephring the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell line

Polycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effect of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr) and three HPAHs (1-ClPyr; 1-BrPyr and 7-ClBaA) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as accessed by the classic MTT and neutral red showed a mild toxic effect in response to single or multiple dose exposure for up to 72h; except for multiple dose exposure to BaA and 7-ClBaA (cumulative concentration of 4 µM) and single exposure to 10 µM BaA. Furthermore, a selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. In order to understand the underlying molecular mechanisms responsible for this effect, ROS production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation and apoptosis mediators were evaluated after exposure to single PAHs doses. All compounds were able to trigger oxidative stress after 24h as measured by catalase activity and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase and MTT and neutral red assays, respectively. The evaluation of cell death mediators showed that caspase-3/7 but not anexin-V pathways were involved in toxicity triggered by the studied compounds. In conclusion, the studied PAHs, especially 1-BrPyr and BaA, exhibit cytotoxic effects when accumulated, and may have adverse effects to humans after long periods of exposure.FCT grant RECI/QEQ-MED/330/2012 (128642)N/

Pathogens in Ornamental and Recreational Waters: A Risk Assessment Study

Ornamental and recreational waters of easy access are quite attractive and can hide threats to human health. Here we evaluated, the microbiota of ornamental waters in a Lisbon park (1 year), as well as, the microbiota of two dams with nautical recreational activities and fluvial beach to assess the risk for human health. In the microbiota, we identified faecal indicator bacterium, E.coli, and other potential human pathogens such as A.baumannii, Aeromonas, E.cloacae, K.pneumoniae, Pseudomonas and Raoutella. The identification of known healthcare associated infections agents such as K. pneumoniae, as well as species of Pseudomonas (P.fluorescens, P.putida and P.oryzihabitans) that recently have been identified as etiological agents in immunosuppressed individuals is of particular relevance. The same bacterial species were isolated from biofilm and water suggesting the existence of an equilibrium between planktonic and biofilm organized bacteria. The role played by biofilm assembly on the emergence of antibiotic resistance is still ongoing for the isolated bacteria, as well as the possible interaction between water chemical composition and bacteria isolates. Potential pathogens (biofilm or planktonic) represent a significant cause of acute bacterial infections mainly in specific populations (paediatric and geriatric). These results support periodic control of ornamental water microbiota as a preventive messure.N/

Integration of cellular and molecular endpoints to assess the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell line

Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effect of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr) and three halogenated derivatives of these compounds (1-ClPyr; 1-BrPyr and 7-ClBaA) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as assessed by the classic MTT and neutral red assays showed a mild toxic effect in response to single or multiple dose exposure for up to 72h; except for multiple dose exposure to BaA and 7-ClBaA (1μM per day during 4 days) and single exposure to 10 μM BaA. Furthermore, selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. In order to understand the underlying molecular mechanisms responsible for this effect ROS production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation, early and late apoptosis mediators were evaluated after exposure to single doses of the compounds. All compounds were able to trigger oxidative stress after 24h as measured by catalase activity and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase and MTT and neutral red assays, respectively. The evaluation of cell death mediators showed that caspase-3/7 but not annexin-V pathways were involved in toxicity triggered by the studied compounds. The integration of all results showed that 1-BrPyr and BaA have a higher toxicity potential.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) [RECI/QEQ-MED/0330/2012] and Sílvia José work was supported by INSA [BRJ-DSA/2012].info:eu-repo/semantics/publishedVersio