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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