The acetylcholinesterasic activity of exposed animals
The acetylcholinesterasic activity of exposed animals, after treatment with diverse concentrations of uranium and following distinct recovery periods, remained unaltered for all species. This finding suggests that uranium, in spite of being a water-soluble metal, does not exert any effect on the selected biomarker. In comparative terms, fish SAR131675 can be more sensitive to uranium exposure than other organisms, such as earthworms or freshwater clams (Labrot et al., 1998). However, freshwater fish species can be of moderate sensitivity when compared to other aquatic organisms, as shown by Sheppard et al. (2005). However, and despite this favourable comparative sensitivity, our data are not in line with the conclusions obtained by Barillet et al. (2011), which showed the cholinesterasic impairment caused by uranium on Danio rerio. This study described that short term exposures to several concentrations of uranium could elicit a significant, albeit not dose-dependent inhibitory response; on the contrary, longer periods of exposure could cause a significant increase in cholinesterasic activity of exposed organisms. The here-obtained trend is in agreement with what has been previously reported by other authors (Frasco et al., 2005) for other metallic compounds. In addition, the activity of ocular cholinesterases from the shrimp Palaemon serratus was not altered following exposure to mercury chloride, giving additional evidences that some metals may not exert any inhibitory effects on these enzymes, as referred by Frasco et al. (2008). Several metals, such as zinc, copper and cadmium, did not elicit any change in the cholinesterasic activity of digestive gland of the snail Helix aspersa, as described by de Souza Dahm et al. (2006). AChE activity and the metal body burden were also not correlated in the fish Lipophrys pholis, after exposure to effluents from urban and industrialised sites, as shown by Solé et al. (2008). In fact, another study by Cunha et al. (2007) showed that the in vivo exposure of Nucella lapillus to cadmium resulted in an enhancement of cholinesterasic activity, which is an unprecedented result. On the other hand, the same study refers that in vitro exposure of Nucella lapillus and Monodonta lineata to copper showed impairment of cholinesterasic activities. The study conducted by Stefano et al. (2008) showed that the metal cadmium was capable, after in vitro exposure, to significantly inhibit the cholinesterasic activity of the clam species Pecten jacobaeus. However, in vivo results do not always corroborate observations made using in vitro test systems. This is an interesting finding, which may show that cholinesterasic activity impairment by metallic species may only result from direct contact between metals and reagent particles that are added to the medium (tissue homogenate) for the development of the enzymatic quantification, as suggested by Frasco et al. (2005). The drawbacks of using the Ellmann’s method are considerable, since the intrinsic reactivity of 5,5‘-dithiobis-2-nitrobenzoic acid (DTNB) can yield a significant incidence of false positive results. In fact, DTNB is prone to promptly react with −SH containing proteins or peptides. Considering that some peptides are in fact expressed when organisms are in the presence of metallic species, it is likely that the augmented levels of −SH containing peptides react with DTNB, indicating a reduction in cholinesterasic activity (Puzon et al., 2011); however, this situation corresponds to an artefact of the methodology, rather than being an indication of a true enzyme inhibition by metals. According to the study by Frasco et al. (2005) metals tend to chemically react with the products resulting from the main reaction of the Ellman’s method. This a major interference of such methodology, and these interferences by metals are difficult to identify and/or quantify. From all tested metals (nickel, copper, zinc, cadmium and mercury), only nickel was not causative of false positives. According to this study, metallic species are likely to react with the thiol groups of TNB, the metabolite of DTNB (Ellman‘s reagent). This hypothesis may justify why in vitro studies present inhibitory effects (due to the direct contact of metals with DTNB, resulting in less colour developed along the time course of the enzymatic assay), and the generic absence of inhibitory effects reported for in vivo studies (in which the uptaken metals never reach levels to sustain this same effect). In fact, only with extremely unrealistic levels it is possible to attain significant in vivo inhibitory effects, while inhibition after in vitro exposures is rather common (e.g. for the metal cadmium; ; de Stefano et al., 2008). In fact, in vivo assays, using concentrations in the μM range (thus ecologically relevant) showed the absence of anticholinesterasic effects of the metals zinc, copper, cadmium, and mercury (de Souza Dahm et al., 2006, Frasco et al., 2008). On the contrary, there are even studies reporting a paradoxical effect of cholinesterase induction, as in Nucella lapillus previously exposed to cadmium (Cunha et al., 2007). Our data also support the absence of in vivo effects in ChEs in three species.