Browsing by Issue Date, starting with "2013-09-16"
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- DNA and Chromosome Damaging Effects in Mice Exposed to an Estuary Sediment ExtractPublication . Pinto, Miguel; Sacadura, Joana; Louro, Henriqueta; Costa, Pedro Manuel; Lavinha, João; Silva, Maria JoãoPrevious studies have shown that an extract of a sediment sample collected in a fishing area of Sado Estuary, impacted by the urban and industrial pollution from the city of Setúbal, was able to induce cytotoxicity and genotoxicity in a human cell line (HepG2) and in local aquatic species, probably due to the presence of PAHs and metals. However, the assessment of the potential hazard of those contaminants to humans, through extrapolation of the in vitro data, is difficult and thereby in vivo studies are crucial to better reflect human exposure and effects. This study aims to assess the in vivo DNA and chromosome damaging potential of a contaminated sediment sample from a local fishing area of the Sado Estuary. Groups of mice (n=6) were exposed, through drinking water, to two concentrations of a sediment extract (1 and 2 g SEQ/ml), as well as to solvent control, for a 28 day period. The DNA and chromosome damaging effects were monitored at 7 day intervals by the comet assay (coupled with DNA repair endonucleases FPG and ENDO III) and the micronucleus assays, performed in blood leukocytes and immature erythrocytes, respectively. After sacrifice, DNA lesions, oxidative damage and histopathological biomarkers (apoptosis and inflammation) were analysed in liver, spleen and kidney cells. Preliminary results showed that at days 7, 14 and 21, mice exposure to the highest extract dose caused a significant induction on the frequency of micronuclei comparatively to the unexposed group (p < 0.001), while exposure to the lowest dose raised the micronucleus frequency only at day 14 (p = 0.003). No induction of DNA damage or oxidative DNA damage was observed in blood cells, at the 2 first timepoints. The results of DNA damage and micronucleus quantification at the 28 days timepoint, together with data from histopathological biomarkers, will be integrated and discussed in view of the contaminants present in the sediment sample under study. Overall, the results obtained in the present in vivo study are expected to reflect the potential hazard to human health associated to the contamination of estuary water and sediments and to contribute to risk assessment.
- Formation of emerging disinfection byproducts in water and evaluation of potential genotoxic effects: the case of halogenated polycyclic aromatic hydrocarbonsPublication . Pinto, M.; Antunes, A.M.M.; José, S.S.; Alves, A.C.; Louro, H.; Silva, M.J.; Cardoso, A.S.Disinfection byproducts (DBPs) are formed when disinfectants used in water treatment plants (WTPs) react with natural (or anthropogenic) organic matter present in the source water. Many studies have addressed health risks posed by a life-time exposure to DBPs through chlorinated drinking water or through dermal or inhalation exposure routes. Experimental studies have revealed genotoxic and carcinogenic effects of some DBPs and epidemiological studies evidenced potential associations between chlorinated drinking water and bladder or colorectal cancer. In addition, a possible link between chlorinated drinking water and reproductive/developmental effects has been hypothesized. Many DBPs have been identified in chlorinated water, which justifies the growing concern about the potential health effects of emerging unregulated DBPs, some of which appear to be more genotoxic, in some assays, than the regulated DBPs. Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent contaminants detected in environmental samples such as river sediments and tap water. A few studies have already proven that water disinfection can lead to the formation of halogenated derivatives of PAHs, such as chlorinated (Cl-PAHs) and brominated PAHs (Br-PAHs). The available toxicological studies have shown that these compounds possess, in general, greater mutagenicity than the corresponding parent PAHs. Our investigation group have also showed that exposure of HepG2 cells to a dose-range of 6-Cl-benzo[a]pyrene (6-Cl-BaP) and BaP resulted in cytotoxicity above 50 µM and that, at the equimolar doses of 100 and 125 µM, 6-Cl-BaP was able to induce a significantly higher level of DNA damage than BaP. The present study had two main objectives: 1) identification of the major chlorinated and brominated derivatives of benzo[a]anthracene (BaA) and pyrene (Pyr) formed as disinfection by-products and 2) evaluation of their potential hazard to humans, through the characterization of their potential cytotoxic and genotoxic effects in a human cell line. To synthesize Cl-PAHs and Br-PAHs the method of Mitchell was developed for BaA and Pyr. 1-Cl-Pyr and 1-Br-Pyr were obtained as the major chlorinated and brominated derivatives of Pyr, and 7-Cl-BaA and 7-Br-BaA as the reaction products of BaA. Cell viability and DNA integrity of those derivatives were assessed by the neutral red uptake (NR) and the comet assay, respectively, allowing the comparison of their genotoxic potential. Although health risks of DBPs are small compared to the health risks of waterborne diseases, the formation of hazardous halogenated-PAHs in chlorinated water water emphasizes the need of development of new and safer water disinfection methods.
- Evaluation of Genotoxicity of Sediments from the Sado-River Estuary Using Solvent Extractions of Diferent PolaritiesPublication . Vicente, Ana Margarida; Sacadura, Joana; Pinto, Miguel; Louro, Henriqueta; Costa, Pedro Manuel; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoLocated in the western coast of Portugal, our case study, the river Sado Estuary, is affected by various sources of pollution, such as heavy-industry, urbanism, mining, agriculture and maritime traffic. Recent studies showed that sediment samples were contaminated with a mixture of different groups of contaminants related to the different anthropogenic pressures along the estuary. The present study aims to assess the cytotoxic and genotoxic potential of these sediments following a fractioning method, in order to elucidate whether their toxicity can be attributed to a particular group of contaminants, or is rather the result of the complex interaction of contaminants. Sediment samples were collected from four distinct and contaminated sites of the Sado Estuary: sites C and P from the urban/industrial area and sites E and A from the riverine/agriculture area. Organic and inorganic contaminants were extracted with solvents of increasing polarity n-hexane < dichloromethane < methanol and a mixture (DCM:methanol), and recovered in DMSO. Cytotoxicity and genotoxicity were evaluated through the neutral red and comet (coupled with FPG) assays, respectively, in HepG2 cells. Cells were exposed for 48h to concentrations of each extract ranging from 1 to 200 mg SEQ/ml. Cytotoxicity was only observed for extracts PDCM/met, EDCM/met and Pmet. Sediment sample C failed to induce genotoxicity. A significant increase in the level of DNA damage was observed for sub-cytotoxic concentrations of PDCM/met, EDCM/met and Pmet. DNA damage was accentuated following treatment with FPG, suggesting oxidative DNA damage, mainly for DCM/met extractions of all samples except C, as well as Phex and Pmet. Complex toxicant mixtures are present in estuarine sediments which often makes the association between surveyed contaminants and toxicity difficult to establish. In this study, genotoxic effects were observed in extracts obtained with the mixture of solvents (DCM:met). Nevertheless, when different extractions were performed with solvents of different polarities, the effects of the different fractions were mainly diluted, or more weakly expressed, suggesting that the interaction between contaminants, and not a set of particular contaminants, might be responsible for the observed effects. Also, we suggest that oxidative DNA damage, revealed by the FPG enzyme, might be a common effect of the exposure to these environmental contaminants. Together with contamination analysis, these results are expected to disclose the genotoxic potential of sediment sample extracts in a human cell line, in order to derive a potential risk to human health.