The toxicologically most relevant mercury (Hg) species for human exposure is methylmercury (MeHg). Thiomersal is a common preservative used in some vaccine formulations. The aim of this study is to get further mechanistic insight into the yet not fully understood neurotoxic modes of action of organic Hg species. Mercury species investigated include MeHgCl and thiomersal. Additionally HgCl2 was studied, since in the brain mercuric Hg can be formed by dealkylation of the organic species. As a cellular system astrocytes were used. In vivo astrocytes provide the environment necessary for neuronal function. In the present study, cytotoxic effects of the respective mercuricals increased with rising alkylation level and correlated with their cellular bioavailability. Further experiments revealed for all species at subcytotoxic concentrations no induction of DNA strand breaks, whereas all species massively increased H2O2-induced DNA strand breaks. This co-genotoxic effect is likely due to a disturbance of the cellular DNA damage response. Thus, at nanomolar, sub-cytotoxic concentrations, all three mercury species strongly disturbed poly(ADP-ribosyl)ation, a signalling reaction induced by DNA strand breaks. Interestingly, the molecular mechanism behind this inhibition seems to be different for the species. Since chronic PARP-1 inhibition is also discussed to sacrifice neurogenesis and learning abilities, further experiments on neurons and in vivo studies could be helpful to clarify whether the inhibition of poly(ADP-ribosyl)ation contributes to organic Hg induced neurotoxicity.
In order to reveal the time-depending mercury species uptake by human astrocytes, a novel approach for total mercury analysis is presented, which uses an accelerated sample introduction system combined on-line with an inductively coupled plasma mass spectrometer equipped with a collision/reaction cell. Human astrocyte samples were incubated with inorganic mercury (HgCl2), methylmercury chloride (MeHgCl), and thimerosal. After 1-h incubation with Hg(2+), cellular concentrations of 3 μM were obtained, whereas for organic species, concentrations of 14-18 μM could be found. After 24 h, a cellular accumulation factor of 0.3 was observed for the cells incubated with Hg(2+), whereas the organic species both showed values of about 5. Due to the obtained steady-state signals, reliable results with relative standard deviations of well below 5 % and limits of detection in the concentration range of 1 ng L(-1) were obtained using external calibration and species-unspecific isotope dilution analysis approaches. The results were further validated using atomic fluorescence spectrometry.
Mercury (Hg) is an environmental contaminant. Whereas within terrestrial food sources it is mostly found as inorganic Hg, in fish and seafood it is largely present in form of methylmercury. Because of its antibacterial/antifungal properties the organic Hg compound thiomersal is used as a preservative in medical preparations. However, exposure to organic Hg promotes primarily neurological effects. The tolerable weekly intake (TWI) of 1.6 mg/kg body weight for methylmercury has been recently reevaluated by the EFSA in 2012. Based on epidemiological studies a new TWI of 1.3 mg/kg body weight has been established EFSA Panel on Contaminants in the Food Chain, 2012. Up to date the transfer of Hg compounds into the brain and the mechanisms of Hg species induced neurotoxicity are not clearly understood. Here we apply an in vitro model of the blood-cerebrospinalfluid (CSF) barrier to identify the transfer mechanisms of different Hg species in the brain and to characterize their effects on the barrier properties. In first studies effects of mercury chloride (HgCl 2 ), methylmercury (MeHgCl) and thiomersal (THI) on the barrier integrity have been analyzed. Quantitative analysis of the total Hg amount in aliquots of both, blood and brain side of the in vitro barrier system, will give information about the Hg-species dependent transfer properties. The respective method to quantify Hg via ICP-MS/MS has already been established. Our studies indicate that the barrier system is significantly more sensitive towards organic Hg species as compared to inorganic compounds. These results are in agreement with our studies concerning the cytotoxicity of the Hg compounds in the barrier building porcine epithelial cells of the Plexus choroideus.In order to identify potential target cells of Hg mediated neurotoxicity, cytotoxic effects of the Hg species have been performed in different cell lines. Using the neutral red uptake assay based on the lysosomal integrity, differentiated human neurons (LUHMES) seem to be more sensitive towards organic and inorganic Hg species compared to human astrocytes. In both cell lines inorganic Hg shows a lower cytotoxicity compared to the organic species.
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