Changes in mitochondrial membrane potential and of ROS generation induced by ethylmercury in Normal Human Astrocytes. Changes in mitotracker and ROS induced DCF levels caused by incubation with increasing concentrations of Thimerosal (b) and the time course of incubation with 14.4 μM Thimerosal (a), with respect to control cultures. Cells were imaged in the centerfield of three independent wells, consisting of an average of 44 cells per field, with a standard deviation of 16.5.

Colocalization of Mitotracker (ΔΨ) and DCF (peroxide) fluorescence in normal human astrocytes. Thimerosal induces oxidative stress at the mitochondrial level. High resolution images of control NHAs and NHAs treated for 60 minutes with 14.4 μM Thimerosal. (a) Mitotracker (red), ROS-induced DCF (green), and nuclear Hoechst staining (blue) of NHAs at × 60 in the absence (left) and presence (right) of 14.4 μM Thimerosal. (b) Images of control and treated cells obtained at × 150 magnification. An orange-colored “horseshoe” shaped signal in the control cell consists of a network of mitochondria which is mirrored in the ROS-induced DCF image. The same is demonstrated in the treated cells by a “lightening bolt” shaped mitochondrial network. (c) Square outlines of the cells from (b) highlighting individual Mitotracker and ROS images, and their overlaid images. (d) Intensity profile of MT, DCF, and Hoechst along the two diagonal lines in (b) (with the MT signal × 4 in the Thimerosal treated image). Red: MT signal, blue: Hoechst signal, green: ROS-generated DCF, black: fit to the ROS signal, based on the amplitudinal changes of MT and Hoechst. The two simulations indicate that four times the amount of DCF is generated by mitochondria in the ethylmercury-treated cells, but background cytosolic rates of generation are the same.

Colocalization of Mitotracker and carbonyls in normal human astrocytes. Thimerosal induces oxidative damage at the mitochondrial level. Control and 14.4 μM Thimerosal-treated cells prepared using MT (red) and Hoechst (blue), with FITC-Avidin/Biotin-Hydrazide carbonyl labeling (green). (a) A large ethylmercury-treated cell showing an increase in green ROS damaged cell contents as a function of distance from the nucleus. (b) Two boxes from (a) highlighting the correlation between MT (red) and carbonyl (green) signals. (c) The two vertical lines in (a) indicate the position of fluorophore intensity profile interrogation. Red: MT, green: carbonyls, blue: Hoechst, black: a simulation of the levels of ROS damage generated by combining fractions of the MT and Hoechst signals. In both samples the simulation is a poor match for the actual ROS-induced signal, with cross-correlations of ROS versus simulation giving R₂ values of only 0.68 and of 0.86, respectively.

Colocalization of carbonyls and mtDNA damage normal human astrocytes. Thimerosal-induced oxidative damage occurs at mitochondrial matrix level. Nuclei (blue), carbonyls (red), and 3′OH DNA ends (green) in (a) and Fpg-labile DNA bases/apurinic and apyrimidinic sites (green) in (b). (c) (insert): A cell that has entered the final stages of apoptosis with large numbers of 3′OH DNA ends derived from nuclear DNA.

Mitochondrial superoxide production correlates with hydroxyl radical generation and mtDNA damage in normal human astrocytes. Thimerosal potentiates Fenton/Haber-Weiss chemistry in the mitochondrial matrix. Control NHAs and NHAs incubated for 1 hour with 14.4 μM Thimerosal. Production of ROS measured with the mitochondrial superoxide probe MitoSox (red), and measurement of HO^• via hydroxyphenyl fluorescein (HPF) (green) in (a), 3′OH DNA ends with ddTUNEL (green) in (b), and blunt-ended DNA breaks (green) in (c).

Summary of observed changes in NHA following incubation with ethylmercury. Bar plot showing the summarized changes observed in NHA following a one-hour exposure to 14.4 μM Thimerosal, with respect to untreated controls. Each value is expressed as mean ± SD of five fields measured in the center field of triplicate experiments. Each of the treated values is statistically different from the controls at P < 0.01. The increase in HO^• is statistically different from the increase in H₂O₂ (H₂DCF-AM) at P < 0.01. Statistical analyses were performed using one-way analysis of variance (ANOVA) with the Holm-Bonferroni post hoc test. The test was performed only when the results of ANOVA were P < 0.05, using Daniel's XL Toolbox, a free, open source add-in for Microsoft Excel.

Proposed mechanism for the toxicity of organomercury. (a) As a lipophilic cation, ethylmercury will become concentrated inside astrocytes, following the plasma membrane potential of 45 mV [37], by a factor of 5.6 fold, and cytosolic ethlymercury will partition into the mitochondria by a factor of 1,000 fold, its accumulation driven by the approximate 180 mV mitochondrial membrane potential [25]. (b) Inside the mitochondria ethylmercury will react and cap thiols/selenols, including the cysteine residues of iron-sulfur centers. The formation of ethylmercuricthiol adducts will not only cause enzyme inhibition, but also increase the levels of free iron inside the mitochondria. (c) The release of iron catalyzes Fenton/Haber-Weiss chemistry leading to the formation of the highly oxidizing HO^•. HO^• has multiple targets, including sensors of the permeability transition complex and also mtDNA. High levels of HO^• cause Mitoposis, leading to cytochrome c release from the mitochondria and the initiation of apoptosis.