CYTO-GENOTOXIC EFFECTS AND PROTEIN ALTERATIONSINDUCED BY SOME PHARMACEUTICAL COMPOUNDS ANDILLICIT DRUGS ON NON-TARGET ORGANISMS

Abstract In the last decades the aquatic environment has been continuously loaded by several new classes of pollutants and a lot of monitoring studies have been conducted worldwide in order to study their widespread environmental distribution. Among them, pharmaceuticals and personal care products (PPCPs) as well as illicit drugs received particular attention from scientific community. Indeed, their huge consumption and indiscriminate use led their environmental levels in the aquatic environments at not negligible and sometimes also alarming concentrations, which could compromise the health of the biocoenosis. Notwithstanding their presence in the environment is now a well-established issue, for most of these chemicals their possible adverse effects toward aquatic biocoenosis still remain unknown. Taking into account that these emerging environmental pollutants have physicchemical characteristics similar to those of harmful xenobiotics, they could be potentially dangerous to aquatic non-target organisms, as they are exposed to contaminants over their whole life span. However, especially for what concern illicit drugs, there is a lack of ecotoxicological studies about their sub-lethal effects on non-target organisms or, when they are available, they are inadequate since they consider only the possible acute effects. Actually, these substances are present in environment at concentrations that difficultly could give acute effects, while chronic ones and sub-lethal effects are more probable. In order to partially fill this gap, our research group applied a step-wise approach to evaluate possible adverse effects of these molecules on the freshwater bivalve Dreissena polymorpha (zebra mussel), trying also to understand their possible mechanism of toxic action. This mussel was chosen as biological model because the invertebrates constitute more than the 90% of living species. Moreover, they play an important role in freshwater ecosystems and are particularly susceptible to environmental stressors. Additionally, previous studies have revealed that D. polymorpha is an useful and sensible organism able to highlight sub-lethal effects when exposed to synthetic chemicals. We applied a suite of biomarkers to first investigate the cyto-genotoxic effects of some PPCPs: trimethoprim (TMP), triclosan (TCS), paracetamol (PCM), diclofenac (DCF) and ibuprofen (IBU). Our results highlighted that triclosan showed highest cyto-genotoxicity and thanks to the application of the biomarker response index we draw a scale of toxicity of these PPCPs, integrating data obtained from different biomarkers end-points: TCS>TMP>IBU>DCF>PCM. Moreover, the analysis of zebra mussels antioxidant enzymes activity gave us the possibility to infer a plausible mechanism of action of TCS, that probably exert its toxicity both by the induction of oxidative stress but especially through a direct action on DNA, since it can act as a DNA adduct and/or DNA intercalant. Our results confirmed that these kinds of molecules could have a negative impact on the aquatic ecosystem, and their adverse effects on non-target organisms should not be underestimated. A similar approach has been employed also to test, for the first time, cytogenotoxicity of the emerging pollutants cocaine (CO) and benzoylecgonine (BE). Our preliminary results, obtained by the analysis of crucial biomarkers end-points to test cytogenotoxicity of two CO environmental concentrations, suggested a hazard of this molecule toward Dreissena polymorpha and probably also toward the entire aquatic biocoenosis. Taking into account these findings and since CO is rapidly metabolized into BE once assumed, we then decided to test BE effects on zebra mussel after a long-term exposure, considering also that BE’s environmental concentrations are often higher than those of the parental compound. The analysis of biomarkers end-points pointed out a clear cytogenotoxicity of this new aquatic pollutant on Dreissena polymorpha at environmental concentrations, highlighting its action as oxidative stressor. To better understand BE mechanism of action, we then applied both classic proteomic and redox proteomic approach that confirmed the role of BE to induce oxidative stress in zebra mussel and also to compromise the energetic metabolism of our biological model. This research represents the first effort to invetsigate the ecotoxicity of this new class of environmental pollutants, and we hope that it could be the starting point to a more in-depth study on the potential environmental risk for this kind of contaminants, especially considering the occurrence of measurable concentrations of several other illicit drugs in freshwaters and their possible high biological activity.