Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action

摘要

The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500 mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10 mg L-1) were treated under different ultrasound operating conditions (frequency at 582 and 862 kHz, electric power density at 133 and 167 W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/center dot O-2(-), nitric oxides such as NO2- and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1 h to 5, 10 and 100 ng L-1 and 1, 10 and 20 mu gL(-1) of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10 mu M L-NAME, a NO synthase inhibitor and 10 mu M DPI, a NADPH oxidase inhibitor. According to the results, 350 mL of 2 mg L-1 DCF showed higher degradation (> 50%) under 167 W electric power density and frequency at 862 kHz for 120 min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of center dot O-2(-) and NO2- generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase and NO synthase in mussel hemocytes, could be significantly diminished after partial US degradation of DCF, at least under optimized operating conditions currently tested. (C) 2016 Elsevier B.V. All rights reserved.