Browsing by Author "Antunes, Alexandra M.M."
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- Chlorinated Polycyclic Aromatic Hydrocarbons Associated with Drinking Water Disinfection: Synthesis, Formation under Aqueous Chlorination Conditions and Genotoxic EffectsPublication . Pinto, Miguel; Rebola, Marlene; Louro, Henriqueta; Antunes, Alexandra M.M.; José, Silvia S.; Rocha, Maria; Silva, Maria João; Cardoso, Ana SofiaPolycyclic aromatic hydrocarbons (PAHs) are among the most persistent and toxic organic micropollutants present in water and several of them are mutagenic and carcinogenic. Although it has been shown that chlorinated derivatives of PAHs (Cl-PAHs) may be formed during the water chlorination procedure, little is known about their potential genotoxic and carcinogenic effects. The objectives of the present work were to prepare and characterize the major chlorinated derivatives of benzo[a]pyrene (BaP) and fluoranthene (Fluo), to develop an analytical methodology for their quantification in water samples and to analyse their potential genotoxicity. Chlorinated standards were prepared by a newly developed two phase method (water/n-hexane) using sodium hypochlorite. 6-Chloro-benzo[a]pyrene was selectively obtained from BaP, while 1,3- dichloro-fluoranthene and 3-chloro-fluoranthene were obtained from Fluo. All products were isolated and characterized by nuclear magnetic resonance and mass spectrometry. The formation of BaP- and Fluo-chlorinated derivatives under aqueous chlorination conditions was observed using a SPE-HPLC-FLD methodology. In addition, the cytotoxic and genotoxic activities of the three chlorinated derivatives were analyzed in comparison to their parent compounds, in a human-derived hepatoma cell line using the neutral red uptake and comet assays, respectively. The results showed 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, suggesting a more potent genotoxic effect. In contrast, neither Fluo nor its chlorinated derivatives were genotoxic in the same cell line. The identification of new and possibly hazardous water chlorination by-product from PAHs emphasizes the need to minimize total organic carbon content of raw water and the implementation of safer water disinfection methods.
- Formação de subprodutos de desinfeção da água e avaliação do seu potencial genotóxico: o caso dos hidrocarbonetos aromáticos policíclicosPublication . Pinto, Miguel; Rebola, Marlene; Louro, Henriqueta; Antunes, Alexandra M.M.; José, Sílvia S.; Rocha, M. Raquel; Silva, M. João; Cardoso, Ana Sofia
- Integration of cellular and molecular endpoints to assess the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell linePublication . Morgado, Patrícia I.; Jose, Sílvia; Wanke, Riccardo; Antunes, Alexandra M.M.; Sofia Cardoso, Ana; Jordao, LuisaPolycyclic 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.