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Teresa Binstock
Researcher in Developmental & Behavioral Neuroanatomy
May 18, 2009
Comment: Drs. Geier, King, and Geier are to be congratulated for an important new study wherein in vitro cell cultures were used to compare toxicities of thimerosal and other metals (1). This methodology has already been used for other metals and compounds such as methylmercury and reinforces the neuronal genotoxicity findings of Baskin et al (2) and the lymphocyte genotoxicity findings of Westphal et al (3).
1. Mitochondrial dysfunction, impaired oxidative-reduction activity, degeneration, and death in human neuronal and fetal cells induced by low-level exposure to thimerosal and other metal compounds
Geier DA, King PG, Geier MR.
Toxicological & Environmental Chemistry 2009, 1–15, iFirst
http://www.informaworld.com/smpp/content~content=a910652305~db=all~order=pubdate
Thimerosal (ethylmercurithiosalicylic acid), an ethylmercury (EtHg)-releasing compound (49.55% mercury (Hg)), was used in a range of medical products for more than 70 years. Of particular recent concern, routine administering of Thimerosal-containing biologics/childhood vaccines have become significant sources of Hg exposure for some fetuses/infants. This study was undertaken to investigate cellular damage among in vitro human neuronal (SH-SY-5Y neuroblastoma and 1321N1 astrocytoma) and fetal (nontransformed) model systems using cell vitality assays and microscope-based digital image capture techniques to assess potential damage induced by Thimerosal and other metal compounds (aluminum (Al) sulfate, lead (Pb)(II) acetate, methylmercury (MeHg) hydroxide, and mercury (Hg)(II) chloride) where the cation was reported to exert adverse effects on developing cells. Thimerosal-associated cellular damage was also evaluated for similarity to pathophysiological findings observed in patients diagnosed with autistic disorders (ADs). Thimerosal-induced cellular damage as evidenced by concentration- and time-dependent mitochondrial damage, reduced oxidative–reduction activity, cellular degeneration, and cell death in the in vitro human neuronal and fetal model systems studied. Thimerosal at low nanomolar (nM) concentrations induced significant cellular toxicity in human neuronal and fetal cells. Thimerosal-induced cytoxicity is similar to that observed in AD pathophysiologic studies. Thimerosal was found to be significantly more toxic than the other metal compounds examined. Future studies need to be conducted to evaluate additional mechanisms underlying Thimerosal-induced cellular damage and assess potential co-exposures to other compounds that may increase or decrease Thimerosal-mediated toxicity.
2. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.
Baskin DS, Ngo H, Didenko VV.
Department of Neurosurgery, Baylor College of Medicine
Toxicol Sci. 2003 Aug;74(2):361-8. {free online}
http://toxsci.oxfordjournals.org/cgi/content/full/74/2/361
Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.
3. Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes.
Westphal GA et al.
Georg-August-University, Gottingen, Germany.
Arch Toxicol. 2003 Jan;77(1):50-5.
Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 micro g/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 micro g/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.
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