Low-level neonatal thimerosal exposure: further evaluation of altered neurotoxic potential in SJL mice.

Ethylmercury in thimerosal-preserved childhood vaccines has been suggested to be neurotoxic and to contribute to the etiology of neurodevelopmental disorders, including autism. Immune system function may be an important factor influencing vulnerability of the developing nervous system to thimerosal. This possibility is based in part on a report by Hornig et al. (2004, Mol. Psychiatry 9, 833-845) of neurodevelomental toxicity in SJL/J mice that develop autoantibodies when exposed to organic mercury. The present study reexamined this possibility by injecting neonatal SJL/J mice with thimerosal, with and without combined HiB and DTP vaccines. Injections modeled childhood vaccination schedules, with mice injected on postnatal days 7, 9, 11, and 15 with 14.2, 10.8, 9.2, and 5.6 mug/kg mercury from thimerosal, respectively, or vehicle. Additional groups received vaccine only or a 10 times higher thimerosal + vaccine dose. Low levels of mercury were found in blood, brain, and kidneys 24 h following the last thimerosal injection. Survival, body weight, indices of early development (negative geotaxis, righting) and hippocampal morphology were not affected. Performance was unaffected in behavioral tests selected to assess behavioral domains relevant to core deficits in neurodevelopmental disorders such as autism (i.e., social interaction, sensory gating, anxiety). In an open-field test the majority of behaviors were unaffected by thimerosal injection, although thimerosal-injected female mice showed increased time in the margin of an open field at 4 weeks of age. Considered together the present results do not indicate pervasive developmental neurotoxicity following vaccine-level thimerosal injections in SJL mice, and provide little if any support for the hypothesis that thimerosal exposure contributes to the etiology of neurodevelopmental disorders.     

Immunosuppressive and autoimmune effects of thimerosal in mice

The possible health effects of the organic mercury compound thimerosal (ethylmercurithiosalicylate), which is rapidly metabolized to ethylmercury (EtHg), have recently been much debated and the effect of this compound on the immune system is largely unknown. We therefore studied the effect of thimerosal by treating A.SW (H-2s) mice, susceptible to induction of autoimmunity by heavy metals, with 10 mg thimerosal/L drinking water (internal dose ca 590 microg Hg/kg body weight/day) for up to 30 days. The lymph node expression of IL-2 and IL-15 mRNA was increased after 2 days, and of IL-4 and IFN-gamma mRNA after 6 and 14 days. During the first 14 days treatment, the number of splenocytes, including T and B cells as well as Ig-secreting cells decreased. A strong immunostimulation superseded after 30 days treatment with increase in splenic weight, number of splenocytes including T and B cells and Ig-secreting cells, and Th2- as well as Th-1-dependent serum immunoglobulins. Antinucleolar antibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin, and systemic immune-complex deposits developed. The H-2s strains SJL and B10.S also responded to thimerosal treatment with ANoA. The A.TL and B10.TL strain, sharing background genes with the A.SW and B10.S strain, respectively, but with a different H-2 haplotype (t1), did not develop ANoA, linking the susceptibility to H-2. Thimerosal-treated H-2s mice homozygous for the nu mutation (SJL-nu/nu), or lacking the T-cell co-stimulatory molecule CD28 (B10.S-CD28-/-), did not develop ANoA, which showed that the autoimmune response is T-cell dependent. Using H-2s strains with targeted mutations, we found that IFN-gamma and IL-6, but not IL-4, is important for induction of ANoA by thimerosal. The maximum added renal concentration of thimerosal (EtHg) and inorganic mercury occurred after 14 days treatment and was 81 microg Hg/g. EtHg made up 59% and inorganic mercury 41% of the renal mercury. In conclusion, the organic mercury compound thimerosal (EtHg) has initial immunosuppressive effects similar to those of MeHg. However, in contrast to MeHg, thimerosal treatment leads in genetically susceptible mice to a second phase with strong immunostimulation and autoimmunity, which is T-cell dependent, H-2 linked and may at least partly be due to the inorganic mercury derived from the metabolism of ethyl mercury.     

Dose-response study of thimerosal-induced murine systemic autoimmunity

The organic compound ethylmercurithiosalicylate (thimerosal), which is primarily present in the tissues as ethylmercury, has caused illness and several deaths due to erroneous handling when used as a disinfectant or as a preservative in medical preparations. Lately, possible health effects of thimerosal in childhood vaccines have been much discussed. Thimerosal is a well-known sensitizing agent, although usually of no clinical relevance. In rare cases, thimerosal has caused systemic immune reactions including acrodynia. We have studied if thimerosal might induce the systemic autoimmune condition observed in genetically susceptible mice after exposure to inorganic mercury. A.SW mice were exposed to 1.25-40 mg thimerosal/l drinking water for 70 days. Antinucleolar antibodies, targeting the 34-kDa protein fibrillarin, developed in a dose-related pattern and first appeared after 10 days in the two highest dose groups. The lowest observed adverse effect level (LOAEL) for antifibrillarin antibodies was 2.5 mg thimerosal/l, corresponding to an absorbed dose of 147 microg Hg/kg bw and a concentration of 21 and 1.9 microg Hg/g in the kidney and lymph nodes, respectively. The same LOAEL was found for tissue immune-complex deposits. The total serum concentration of IgE, IgG1, and IgG2a showed a significant dose-related increase in thimerosal-treated mice, with a LOAEL of 5 mg thimerosal/l for IgG1 and IgE, and 20 mg thimerosal/l for IgG2a. The polyclonal B-cell activation showed a significant dose-response relationship with a LOAEL of 10 mg thimerosal/l. Therefore, thimerosal induces in genetically susceptible mice a systemic autoimmune syndrome very similar to that seen after treatment with inorganic mercury, although a higher absorbed dose of Hg is needed using thimerosal. The autoimmune syndrome induced by thimerosal is different from the weaker and more restricted autoimmune reaction observed after treatment with an equipotent dose of methylmercury.     

The role of biotransformation in organic mercury neurotoxicity

Although the clinical patterns of organic and inorganic mercury poisoning are very different, systemic toxicity experiments have shown that the histologicale changes in the kidneys and dorsal root ganglia neurones are identical with the 2 classes of compounds. It has been further suggested that the toxicity of organic mercurials is the result of biotransformation to inorganic mercury. To test this hypothesis, between 10^?7 and 10^?10 mol of mercuric chloride and methyl mercuric acetate were injected directly into the cerebrum of rats. The comparative size of lesions was estimated anatomically and by reference to blood brain barrier dysfunction. Inorganic lesions were only slightly larger than those produced by equimolar amounts of organic mercury. Consequently both organic and inorganic mercury must be regarded as neurotoxic in their own right. Conversion of organic to inorganic mercury certainly occurs but is not the only mechanism by which organic mercury exerts its toxicological effect.     

Effects of dietary lipids on whole-body retention and organ distribution of organic and inorganic mercury in mice.

The effect has been investigated of dietary lipids on the whole-body retention and organ distribution of organic and inorganic mercury in mice. A single oral dose of methylmercury chloride or mercuric chloride labelled with 203Hg was given to female NMRI mice fed semi-synthetic diets containing varying amounts (5, 10, 20 or 50%) of energy derived from lipid (coconut oil, soya oil, or cod liver oil). The whole-body retention and relative organ distribution of mercury depended on diet composition. Thus, a significant reduction of the whole-body retention of mercury was seen in mice fed a diet containing 50% cod liver oil compared with mice fed a diet containing 50% coconut oil. After oral administration of mercuric chloride the relative deposition of mercury in the kidneys increased while that in the liver decreased with increasing concentrations of soya oil or coconut oil in the diet. The whole-body retention of mercury after treatment with methylmercury chloride was significantly decreased in mice fed cod liver oil compared with mice fed coconut oil; there was no difference between mice fed cod liver oil and those fed soya oil. The relative disposition of mercury was significantly higher in all organs of mice fed a diet containing 20% energy from cod liver oil compared with mice fed a diet containing 20% energy from soya oil. The present study demonstrates that diet composition is of major importance to the toxicokinetics of methylmercury and mercuric mercury.     

Toxicity of ethylmercury (and Thimerosal): a comparison with methylmercury

Ethylmercury (etHg) is derived from the metabolism of thimerosal (o‐carboxyphenyl‐thio‐ethyl‐sodium salt), which is the most widely used form of organic mercury. Because of its application as a vaccine preservative, almost every human and animal (domestic and farmed) that has been immunized with thimerosal‐containing vaccines has been exposed to etHg. Although methylmercury (meHg) is considered a hazardous substance that is to be avoided even at small levels when consumed in foods such as seafood and rice (in Asia), the World Health Organization considers small doses of thimerosal safe regardless of multiple/repetitive exposures to vaccines that are predominantly taken during pregnancy or infancy. We have reviewed in vitro and in vivo studies that compare the toxicological parameters among etHg and other forms of mercury (predominantly meHg) to assess their relative toxicities and potential to cause cumulative insults. In vitro studies comparing etHg with meHg demonstrate equivalent measured outcomes for cardiovascular, neural and immune cells. However, under in vivo conditions, evidence indicates a distinct toxicokinetic profile between meHg and etHg, favoring a shorter blood half‐life, attendant compartment distribution and the elimination of etHg compared with meHg. EtHg's toxicity profile is different from that of meHg, leading to different exposure and toxicity risks. Therefore, in real‐life scenarios, a simultaneous exposure to both etHg and meHg might result in enhanced neurotoxic effects in developing mammals. However, our knowledge on this subject is still incomplete, and studies are required to address the predictability of the additive or synergic toxicological effects of etHg and meHg (or other neurotoxicants). Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of the interaction of methyl mercury and mercuric chloride with murine macrophages

The toxicity of organic methyl mercury was studied on murine macrophages in cell culture and compared to that of inorganic mercuric chloride. Long-term treatment of macrophage cultures with methyl mercury resulted in decreased cell viability in a concentration-dependent fashion. Experiments showed that 20 M methyl mercury was highly toxic, causing cell death within a few days, while cultures exposed to lower levels were less severely affected. Comparison of the toxicity of organic and inorganic mercury by cell viability showed no difference between equimolar concentrations of methyl mercury and mercuric chloride. Furthermore, protein synthesis (interferon-/ß) was reduced in a concentration dependent manner and had the same reduced magnitude in cells treated with either methyl mercury or mercuric chloride. However, impairment of random migration and phagocytosis of macrophages appeared at lower concentrations in cells exposed to methyl mercury than in cells exposed to mercuric chloride. Electron microscopy of cells exposed to methyl mercury revealed mercury deposits in lysosomes and dispersed in the cytoplasm and nuclei. The present study shows that methyl mercury and mercuric chloride impair cell viability and protein production in cell cultures at equimolar concentrations, while methyl mercury inhibits macrophage functions such as migration and phagocytosis at lower concentrations than mercuric chloride.     

Tissue-specific and dose-related accumulation of arsenic in mouse offspring following maternal consumption of arsenic-contaminated water

The developmental toxicity of arsenic is not as well characterized as other metals such as lead or mercury. Many previous animal studies have used an acute exposure paradigm, which does not model chronic, low-level human exposure. The following study administered 10, 20, 40, 80 or 100 ppm sodium arsenite in drinking water to pregnant C57BL6/J mice. Adipose, blood, brain, breastmilk in stomach, kidney and liver tissues were collected from male and female offspring on postnatal day (PND) 1 and 21 to allow for disposition comparisons between tissues, sexes and across time. The 100 ppm dose was foetotoxic. Significantly fewer female pups were born in litters exposed to 80 ppm, while significantly more male pups were born in litters exposed to 20 ppm. Total arsenic levels differed between tissues with the highest levels in the brain and kidney in PND1 offspring. Levels were higher on PND1 than PND21, and there were few sex differences. The dose-response relationships in PND1 tissues were curvilinear, but in PND21 liver and kidney tissues, arsenic levels in control animals were significantly higher than levels in exposed animals. The tissue and age-specific disposition suggests that common biomarkers such as blood and urinary arsenic are not accurate predictors of levels in sensitive organs such as the brain.     

Validity of methyl mercury hair analysis: mercury monitoring in human scalp/nude mouse model

OBJECTIVE: The grafting of human scalp hair was used as a new application of this method to explore methyl mercury incorporation into human hair and to validate this model for mercury monitoring in hair. METHODS: Human scalp grafts were transplanted to athymic BALB/c nude mice. The animals were exposed to methyl mercury either as a single dose i.p. or continuously for 4 months, using ALZET osmotic pumps. The mercury concentration in hair was determined using x-ray fluorescence (XRF) spectrometry by segmental (2 mm) analysis of a single strand, and tissue concentrations were measured by cold vapor atomic absorption analysis. RESULTS: Human scalp hair grown in nude mice showed long-term persistence of human features including the expression of histocompatibility antigens (KAB 3, W 6/32, SF 1-1.1.1) and normal hair morphometry. The disposition of methyl mercury in nude mice followed a one-compartment model with a whole body elimination half-life of 6.7 days (elimination constant, k = 0.1/day). Autoradiographic studies revealed that methyl mercury was rapidly incorporated into areas of the hair follicle undergoing active keratinization. Methyl mercury concentrations in human hair transplanted onto nude mice were two orders of magnitude higher than in blood and attained a mean hair: blood ratio of 217 : 1, similar to ratios reported only in human studies. CONCLUSIONS: This study demonstrated that human hair grown on nude mice can record the level of exposure to methyl mercury and can serve as a valuable research tool to study mercury incorporation into human hair.     

Luteolin and thiosalicylate inhibit HgCl(2) and thimerosal-induced VEGF release from human mast cells

HgCl2 is a known environemental neurotoxin, but is also used as preservative in vaccines as thimerosal containing ethyl mercury covalently linked to thiosalicylate. We recently reported that mercury choloride (HgCl(2)) can stimulate human mast cells to release vascular endothelial growth factor (VEGF), which is also vasoactive and pro-inflammatory. Here we show that thimerosal induces significant VEGF release from human leukemic cultured LAD2 mast cells (at 1 microM 326 ± 12 pg/106 cells and 335.5 ± 12 pg/106 cells at 10 microM) compared to control cells (242 ± 21 pg/106 cells, n=5, p less than 0.05); this effect is weaker than that induced by HgCl2 at 10 microM (448 ± 14 pg/106 cells) (n=3, p less than 0.05). In view of this finding, we hypothesize that the thiosalicylate component of thimerosal may have an inhibitory effect on VEGF release. Thimerosal (10 microM) added together with the peptide Substance P (SP) at 2 microM, used as a positive control, reduced VEGF release by 90 percent. Methyl thiosalicylate (1 or 10 microM) added with either SP or HgCl2 (10 microM) inhibited VEGF release by 100 percent, while sodium salicylate or ibuprofen had no effect. Pretreatment for 10 min with the flavonoid luteolin (0.1 mM) before HgCl2 or thimerosal compeletly blocked their effect. Luteolin and methyl thiosalicylate may be useful in preventing mercury-induced toxicity.     

Thimerosal induces apoptotic and fibrotic changes to kidney epithelial cells in vitro

Thimerosal is an ethyl mercury‐containing compound used mainly in vaccines as a bactericide. Although the kidney is a key target for mercury toxicity, thimerosal nephrotoxicity has not received the same attention as other mercury species. The aim of this study was to determine the potential cytotoxic mechanisms of thimerosal on human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposed for 24 h to thimerosal (0–2 µM), and assessed for cell viability, apoptosis, and cell proliferation; expression of proteins Bax, nuclear factor‐κB subunits, and transforming growth factor‐β1 (TGFβ1); mitochondrial health (JC‐1, MitoTracker Red CMXRos); and fibronectin levels (enzyme‐linked immunosorbent assay). Thimerosal diminished HK2 cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion. This is the first report about kidney cell death and pro‐fibrotic mechanisms promoted by thimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic pathway, and (2) thimerosal is a potential pro‐fibrotic agent in human kidney cells. We suggest that new evidence on toxicity as well as continuous surveillance in terms of fibrogenesis is required concerning thimerosal use. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1423–1433, 2015.     

BASOLATERAL UPTAKE OF MERCURIC CONJUGATES OF N-ACETYLCYSTEINE AND CYSTEINE IN THE KIDNEY INVOLVES THE ORGANIC ANION TRANSPORT SYSTEM

Renal uptake and disposition of administered inorganic mercury were studied in rats that had undergone an acute bilateral ureteral ligation shortly before being injected intravenously with a nontoxic 0.5 mumol/kg dose of inorganic mercury with or without 2 mumol/kg N-acetylcysteine or cysteine. Bilateral ureteral ligation was performed in an attempt to reduce glomerular filtration to negligible levels, which in turn permitted the study of the basolateral uptake of inorganic mercury. The disposition of mercury was studied in the kidneys, liver, and blood 1 h after treatment. In rats given only mercuric chloride, the renal burden of mercury was approximately 20% of the administered dose of mercury. This confirms previous observations implicating a basolateral mechanism in the renal uptake of inorganic mercury. Coadministration of inorganic mercury with either N-acetylcysteine or cysteine caused a significant increase in the renal uptake of mercury 1 h after treatment, particularly in the rats treated with inorganic mercury plus N-acetylcysteine. The enhanced uptake of mercury in the kidneys was due to increased uptake of mercury in the renal cortex and outer stripe of the outer medulla. Interestingly, the rate of uptake of mercury was so great in the rats treated with inorganic mercury plus N-acetylcysteine that the renal burden of mercury was virtually equivalent to that generally detected in normal animals administered the same dose of inorganic mercury as mercuric chloride. Pretreatment with para-aminohippuric acid (PAH) (which is a potent inhibitor of the organic anion transport system) caused significant reductions in the renal uptake and burden of inorganic mercury in all the rats administered inorganic mercury, regardless of whether the inorganic mercury was coadministered with N-acetylcysteine or cysteine. Overall, the findings from the present study provide additional evidence that there is basolateral uptake of inorganic mercury in the kidneys, and that the primary or sole mechanism is dependent on the activity of the organic anion transporter. The present findings also show that cysteine and N-acetylcysteine enhance the basolateral uptake of mercuric ions in the kidney when they are coadministered with inorganic mercury, presumably in the form of mercuric conjugates. Moreover, it appears that mercuric conjugates of N-acetylcysteine are taken up more avidly at the basolateral membrane than mercuric conjugates of cysteine. Furthermore, the basolateral uptake of mercuric conjugates of N-acetylcysteine or cysteine in the kidney is due primarily to a mechanism involving the organic anion transport system.     

Alteration of the spontaneous systemic autoimmune disease in (NZB x NZW)F1 mice by treatment with thimerosal (ethyl mercury)

Inorganic mercury may aggravate murine systemic autoimmune diseases which are either spontaneous (genetically determined) or induced by non-genetic mechanisms. Organic mercury species, the dominating form of mercury exposure in the human population, have not been examined in this respect. Therefore, ethyl mercury in the form of thimerosal, a preservative recently debated as a possible health hazard when present in vaccines, was administered in a dose of 0.156-5 mg/L drinking water to female (NZB x NZW)F1 (ZBWF1) mice. These mice develop an age-dependent spontaneous systemic autoimmune disease with high mortality primarily due to immune-complex (IC) glomerulonephritis. Five mg thimerosal/L drinking water (295 microg Hg/kg body weight (bw)/day) for 7 weeks induced glomerular, mesangial and systemic vessel wall IC deposits and antinuclear antibodies (ANA) which were not present in the untreated controls. After 22-25 weeks, the higher doses of thimerosal had shifted the localization of the spontaneously developing renal glomerular IC deposits from the capillary wall position seen in controls to the mesangium. The altered localization was associated with less severe histological kidney damage, less proteinuria, and reduced mortality. The effect was dose-dependent, lower doses having no effect compared with the untreated controls. A different effect of thimerosal treatment was induction of renal and splenic vessel walls IC deposits. Renal vessel wall deposits occurred at a dose of 0.313-5 mg thimerosal/L (18-295 microg Hg/kg bw/day), while splenic vessel wall deposits developed also in mice given the lowest dose of thimerosal, 0.156 mg/L (9 microg Hg/kg bw/day). The latter dose is 3- and 15-fold lower than the dose of Hg required to induce vessel wall IC deposits in genetically susceptible H-2s mice by HgCl2 and thimerosal, respectively. Further studies on the exact conditions needed for induction of systemic IC deposits by low-dose organic mercurials in autoimmune-prone individuals, as well as the potential effect of these deposits on the vessel walls, are warranted.     

A toxicokinetic model for predicting the tissue distribution and elimination of organic and inorganic mercury following exposure to methyl mercury in animals and humans. II. Application and validation of the model in humans

The objective of this study was to develop a biologically based dynamical model describing the disposition kinetics of methyl mercury and its inorganic mercury metabolites in humans following different methyl mercury exposure scenarios. The model conceptual and functional representation was similar to that used for rats but relevant data on humans served to determine the critical parameters of the kinetic behavior. It was found that the metabolic rate of methyl mercury was on average 3 to 3.5 times slower in humans than in rats. Also, excretion rates of organic mercury from the whole body into feces and hair were 100 and 40 times smaller in humans, respectively, and urinary excretion of organic mercury in humans was found to be negligible. The human transfer rate of inorganic mercury from blood to hair was found to be 5 times lower than that of rats. On the other hand, retention of inorganic mercury in the kidney appeared more important in humans than in rats: the transfer rate of inorganic mercury from blood to kidney was 19 times higher than in rats and that from kidney to blood 19 times smaller. The excretion rate of inorganic mercury from the kidney to urine in humans was found to be twice that of rats. With these model parameters, simulations accurately predicted human kinetic data available in the published literature for different exposure scenarios. The model relates quantitatively mercury species in biological matrices (blood, hair, and urine) to the absorbed dose and tissue burden at any point in time. Thus, accessible measurements on these matrices allow inferences of past, present, and future burdens. This could prove to be a useful tool in assessing the health risks associated with various circumstances of methyl mercury exposure.     

Genotoxicity of thimerosal in cultured human lymphocytes with and without metabolic activation sister chromatid exchange analysis proliferation index and mitotic index

Thimerosal is an antiseptic containing 49.5% of ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. In this study, we evaluated the genotoxic effect of thimerosal in cultured human peripheral blood lymphocytes using sister chromatid exchange analysis in culture conditions with and without S9 metabolic activation. This study is the first report investigating the genotoxic effects of thimerosal in cultured human peripheral blood lymphocyte cells using sister chromatid exchange analysis. An analysis of variance test (ANOVA) was performed to evaluate the results. Significant induction of sister chromatid exchanges was seen at concentrations between 0.2 and 0.6 μg/ml of thimerosal compared with negative control. A significant decrease (p < 0.001) in mitotic index (MI) and proliferation ındex (PRI) as well as an increase in SCE frequency (p < 0.001) was observed compared with control cultures. Our results indicate the genotoxic and cytotoxic effect of TH in cultured human peripheral blood lymphocytes at tested doses in cultures with/without S9 fraction.     

The comparative toxicology of ethyl- and methylmercury

Neurotoxicity and renotoxicity were compared in rats given by gastric gavage five daily doses of 8.0 mg Hg/kg methyl- or ethylmercuric chloride or 9.6 mg Hg/kg ethylmercuric chloride. Three or 10 days after the last treatment day rats treated with either 8.0 or 9.6 mg Hg/kg ethylmercury had higher total or organic mercury concentrations in blood and lower concentrations in kidneys and brain than methylmercury-treated rats. In each of these tissues the inorganic mercury concentration was higher after ethyl than after methylmercury.Weight loss relative to the expected body weight and renal damage was higher in ethylmercury-treated rats than in rats given equimolar doses of methylmercury. These effects became more severe when the dose of ethylmercury was increased by 20%. Thus in renotoxicity the renal concentration of inorganic mercury seems to be more important than the concentration of organic or total mercury. In methylmercury-treated rats damage and inorganic mercury deposits were restricted to the P₂ region of the proximal tubules, while in ethylmercury-treated rats the distribution of mercury and damage was more widespread.There was little difference in the neurotoxicities of methylmercury and ethylmercury when effects on the dorsal root ganglia or coordination disorders were compared. Based on both criteria, an equimolar dose of ethylmercury was less neurotoxic than methylmercury, but a 20% increase in the dose of ethylmercury was enough to raise the sum of coordination disorder scores slightly and ganglion damage significantly above those in methylmercury-treated rats.In spite of the higher inorganic mercury concentration in the brain of ethylmercurythan in the brain of methylmercury-treated rats, the granular layer damage in the cerebellum was widespread only in the methylmercury-treated rats. Thus inorganic mercury or dealkylation cannot be responsible for granular layer damage in alkylmercury intoxication. Moreover, histochemistry demonstrated no inorganic mercury deposits in the granular layer.     

Methyl mercury-induced encephalopathy in mice

An animal model of methyl mercury-induced encephalopathy was developed in mice to enable a study of the effects of various newly synthesized chelating agents on methyl mercury toxicity. Administration of methyl mercury to mice via drinking water produced neuropathological and behavioral responses similar to those seen in humans following chronic exposure to organic mercurials. Changes in cellular morphology were seen in the cerebellar cortex of mice exposed to 50 and 10 μg of Hg as CH₃HgCl per milliliter of drinking water after 8 days of exposure. At this time, posterior paresis began to develop in mice exposed to 50 μg of Hg/ml of drinking water. Histopathological changes were similar in both exposure groups, although paraplegia did not develop in the low dose group until 24 weeks of exposure. Development of overt clinical signs of methyl mercury toxicity correlated well with blood and whole-body mercury concentrations. There was a lack of correlation between blood and whole-body mercury concentrations and among morphological changes in the cerebellar cortex.     

Effects of lipopolysaccharide and chelator on mercury content in the cerebrum of thimerosal-administered mice

Thimerosal is one of the best-known preservative agents for vaccines in the world but a relationship between its use and autism has long been suspected so that its effects on the brain need more detailed research. We here examined the influence of lipopolysaccharide injury to the blood–brain barrier on the penetration of mercury from thimerosal into mouse cerebrums, as well as the effect of chelator of heavy metals on cerebrum mercury content. Mercury can be expected to be detected in the cerebrum of normal mice, because the metal is present in standard mouse chow. When 60 μg/kg of thimerosal was subcutaneously injected into the mouse, the mercury content in the cerebrum was significantly higher 48 h after the thimerosal injection with a maximum peak after 72 h. In addition, mercury content in the cerebrum was still higher on day 7 than in the control group. When lipopolysaccharide was pre-injected into mice to induce damage on blood–brain barrier, the mercury content in the cerebrum was significantly higher at 24 and 72 h after the injection of 12 μg/kg of thimerosal compared to the control group, this dose alone does not cause any increase. The mercury content in the cerebrums of mice was decreased to the control group level on day 7 when a chelator, dimercaprol, was administered once a day from days 3 to 6 after a 60 μg/kg, s.c. injection. In addition, d-penicillamine as a chelator decreased the mercury contents in the cerebrum after the high dose administration. In conclusion, a physiological dose of thimerosal did not increase the content of mercury in the cerebrum, but levels were increased when damage to the blood–brain barrier occurred in mice injected with thimerosal. In addition, a chelator of heavy metals may be useful to remove mercury from the cerebrum.     

Distribution and Biotransformation of Methyl Mercuric Chloride in Different Tissues of Mice

Abstract: The distribution of ²⁰³Hg radioactivity has been studied in various organs of adult male and female mice from one hour to 21 days after treating with ²⁰³Hg-labeled methyl mercuric chloride (MMC). The amount of methyl mercury (MeHg) and inorganic mercury (Hg) has also been determined by injecting single doses of non-radioactive MMC, and subsequently measuring total, organic and inorganic Hg content by atomic absorption technique. In addition, photoemulsion histochemical method (PEHM) was used to demonstrate localization of Hg grains in various cellular compartments of organs and tissues. The highest levels of radioactivity were attained at 7 hours post-treatment in all organs except for brain and testis. The testis showed the highest radioactivity at one day and the brain at two days post-treatment. MeHg persisted in brain over a longer period though the level was not as high. The content of MeHg and inorganic Hg was maximum in kidneys as compared to other organs. The brain and the reproductive organs contained the least amount of inorganic Hg. By PEHM, Hg grains were most prominently observed in the sinusoids, Kupfer cells, hepatic cells and bile duct epithelium of liver; in the lumen of blood vessels, convoluted and collecting tubules of kidneys; and in the gastrointestinal epithelium. The pattern of uptake and distribution of MeHg correlated well with the morphological demonstration of Hg grains in tissue sections.     

Effects of coadministered low-molecular-weight thiol compounds on short-term distribution of methyl mercury in the rat

Following iv administration to the rat, methyl mercury was rapidly deposited in the liver, kidneys, and cerebrum. Methyl mercury concentrations in cerebrum 5 min after injection were about equal to the levels attained at 60 min post-treatment. At 5 min after dosing, concentrations of methyl mercury in plasma and tissues were directly proportional to the dose level of methyl mercury. Coadministration of equimolar amounts of l-cysteine increased short-term accumulation of methyl mercury in liver, kidneys, and cerebrum while reducing the levels of methyl mercury in plasma from those found after administration of methyl mercury alone. Equimolar doses of d-cysteine or d-penicillamine lowered plasma methyl mercury levels below those produced by injection of methyl mercury alone, but coadministered N-acetyl-l-cysteine or l-penicillamine did not. Each of these low-molecular-weight thiol compounds given in combination with methyl mercury increased deposition of methyl mercury in liver and kidneys. In addition, l-penicillamine also increased accumulation of methyl mercury in the cerebrum. These results suggest a critical role of plasma methyl mercury levels in the control of short-term methyl mercury distribution. Modification of the distribution pattern by coadministered low-molecular-weight thiol compounds further suggests that methyl mercury-thiol complexes may play a role in the tissue deposition process.