Protective Effects of Ca2+ Channel Blockers against Methyl Mercury Toxicity

Abstract: The protective effects of Ca²⁺ channel blockers against the toxicity of methyl mercury were examined by both in vivo and in vitro experiments. In the in vivo study we first examined the effects of the Ca²⁺ channel blockers (20 mg/ kg/day), flunarizine, nifedipine, nicardipine, and verapamil against the toxic level of methyl mercury treatment (5 mg/kg/ day of methyl mercuric chloride for 12 consecutive days). However, there was a difference in potency of the effects among the reagents. All the Ca²⁺ channel blockers prevented a decrease in body weight and/or the appearance of the symptoms of neurological disorders in the rats treated with methyl mercury. In the next experiment, we examined flunarizine at different levels of supplementation (1,25 and 50 mg/kg/day). Flunarizine in a dose-dependent manner prevented a decrease in body weight, appearance of the symptoms of neurological disorder and mortality in the rats treated with methyl mercury. Flunarizine treatment (25 mg/kg/day) for the first 5 days did not affect mercury distribution among the tissues, suggesting that the mechanism of protection against methyl mercury-induced toxicity may be attributed to its own pharmacological effect. In the in vitro study we examined the effect of flunarizine (0, 0.5, 5 and 50 μM) using primary cultures of cerebellar granular cells in 96-well culture plates. Viable cell numbers were estimated 1 and 3 days after treatment with methyl mercury. The estimated 50% lethal concentration (LC50) of methyl mercury was higher in plates treated with 5 and 50 uM of flunarizine both on days 1 and 3, indicating that flunarizine protected the primary cultured cerebellar granular cells against the toxicity of methyl mercury. As such, Ca²⁺ channel blockers protected against the toxicity of methyl mercury both in vivo and in vitro, suggesting that Ca²⁺ plays an important role in the mechanisms of methyl mercury toxicity.     

Renal functional correlates of methyl mercury intoxication: interaction with acute mercuric chloride toxicity.

To determine the effect of methyl mercury and its possible interaction with mercuric chloride on renal function, male Sprague-Dawley rats were treated with methyl mercuric chloride (1 mg/kg per day × 20 days ip) and/or mercuric chloride (1 mg/kg ip at Day 20) in a 2 × 2 factorial experimental design. Methyl mercury depressed urine osmolality and N-methylnicotinamide (NMN) uptake by renal cortical slices but did not affect the uptake of p-aminohippurate (PAH), blood urea nitrogen concentration (BUN), urine volume, or body weight. Urinary excretion of the lysosomal enzymes, β-galactosidase and acid phosphatase, appeared to be decreased, but excretion of the brush border enzyme, alkaline phosphatase, was not affected. Mercuric chloride treatment increased enzyme excretion, BUN, and uptake of NMN by renal cortical slices, while it decreased PAH uptake and urine osmolality, BUN concentration was further increased by combined treatment, yielding the only significant treatment interaction between methyl mercury and mercuric chloride. Prostaglandin E₂ synthesis and release by renal medullary tissue in vitro was not depressed by methyl mercuric chloride pretreatment nor was renal ammoniagenesis or gluconeogenesis. The effects of methyl mercury upon lysosomal enzyme excretion and NMN accumulation are suggestive of lysosomal and mitochondrial dysfunction. The failure to detect significant interaction between methyl mercury and mercuric chloride indicates that methyl mercury neither potentiates nor protects against acute mercuric chloride toxicity at this time and dose.     

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.     

The influence of selinium on methyl mercury toxicity in rat hepatoma cells, human embryonic fibroblasts and human lymphocytes in culture

The effect of methyl mercury and two selenium compounds have been studied in cell cultures. Methyl mercury in concentrations above 1 microM had a pronounced inhibiting effect on the growth of rat Morris hepatoma cells. Glucose and lactate uptake in relation to cell protein was appreciably stimulated by the organic mercury compound. Selenite in low concentration (0.5 microM) and seleno-di-N-acetyl glycine in thousandfold higher concentrations offered considerable protection against these effects of methyl mercury. The same selenite concentration (0.5 microM), which did not affect cell growth, caused an appreciable protection against methyl mercury (6 microM), even if it was added 3 days after methyl mercury. The methyl mercury inhibited the growth of human embryonic fibroblasts and the DNA-synthesis in the human lymphocytes. However, no protective effect of selenite were observed in these cell types. These results suggest that selenium compounds exert their protective effect through cell specific processes rather than by a direct chemical reaction between selenite and methyl mercury.     

The Influence of Selenium on Methyl Mercury Toxicity in Rat Hepatoma Cells,Human Embryonic Fibroblasts and Human Lymphocytes in Culture

Abstract The effect of methyl mercury and two selenium compounds have been studied in cell cultures. Methyl mercury in concentrations above 1 μM had a pronounced inhibiting effect on the growth of rat Morris hepatoma cells. Glucose and lactate uptake in relation to cell protein was appreciably stimulated by the organic mercury compound. Selenite in low concentrations (0.5 μM) and seleno-di-N-acetyl glycine in thousandfold higher concentrations offered considerable protection against these effects of methyl mercury. The same selenite concentration (0.5 μM), which did not affect cell growth, caused an appreciable protection against methyl mercury (6 μM), even if it was added 3 days after methyl mercury. The methyl mercury inhibited the growth of human embryonic fibroblasts and the DNA-synthesis in the human lymphocytes. However, no protective effect of selenite were observed in these cell types. These results suggest that selenium compounds exert their protective effects through cell specific processes rather than by a direct chemical reaction between selenite and methyl mercury.     

Thimerosal distribution and metabolism in neonatal mice: comparison with methyl mercury

Thimerosal, which releases the ethyl mercury radical as the active species, has been used as a preservative in many currently marketed vaccines throughout the world. Because of concerns that its toxicity could be similar to that of methyl mercury, it is no longer incorporated in many vaccines in the United States. There are reasons to believe, however, that the disposition and toxicity of ethyl mercury compounds, including thimerosal, may differ substantially from those of the methyl form. The current study sought to compare, in neonatal mice, the tissue concentrations, disposition and metabolism of thimerosal with that of methyl mercury. ICR mice were given single intramuscular injections of thimerosal or methyl mercury (1.4 mg Hg kg(-1)) on postnatal day 10 (PND 10). Tissue samples were collected daily on PND 11-14. Most analysed tissues demonstrated different patterns of tissue distribution and a different rate of mercury decomposition. The mean organic mercury in the brain and kidneys was significantly lower in mice treated with thimerosal than in the methyl mercury-treated group. In the brain, thimerosal-exposed mice showed a steady decrease of organic mercury levels following the initial peak, whereas in the methyl mercury-exposed mice, concentrations peaked on day 2 after exposure. In the kidneys, thimerosal-exposed mice retained significantly higher inorganic mercury levels than methyl mercury-treated mice. In the liver both organic and inorganic mercury concentrations were significantly higher in thimerosal-exposed mice than in the methyl mercury group. Ethyl mercury was incorporated into growing hair in a similar manner to methyl mercury. The data showing significant kinetic differences in tissue distribution and metabolism of mercury species challenge the assumption that ethyl mercury is toxicologically identical to methyl mercury.     

Increased acetaminophen-induced hepatotoxicity after chronic ethanol consumption in mice

The effect of chronic ethanol consumption on acetaminophen (200, 400, and 600 mg/kg) toxicity was determined by maintaining mice for 10 days on diets consisting of chow and one of the following drinking solutions: 10% ethanol + 10% sucrose, 8% sucrose, or tap water. Toxicity as manifested by mortality, liver enlargement, and liver congestion was greatest in the ethanol-treated group. We suggest that the greater mortality was a result of the increased liver congestion and consequent hypovolemia. Despite the increased levels of cytochrome(s) P-450, covalent binding of [³H]acetaminophen reactive metabolite(s) to liver protein was not higher in ethanol-treated animals. This can be explained by the higher initial glutathione concentration and/or ability to replenish glutathione in the ethanol-treated group. We suggest that the enhancement of acetaminophen toxicity by ethanol is the result of an effect of ethanol on hepatocyte membranes which renders the cells more susceptible to toxic injury.     

N-acetylpenicillamine potentiation of biliary excretion of methyl mercury: influence of glutathione depletors

The effect of 1-chloro-2,4-dinitrobenzene (DNB), sulfobromophtalein (BSP) and cyclohexene oxide (cho) on N-acetylpenicillamine (NAPA) potentiated biliary excretion of methyl mercury in rats pretreated with cho for liver glutathione (GSH) depletion, has been tested. DNB, BSP and cho depressed NAPA potentiation of methyl mercury excretion in bile, while simultaneously biliary sulfhydryl concentration increased, due mainly to a relatively large proportion of unchanged NAPA, being excreted in bile. The fact that relatively large amounts of unchanged NAPA are excreted in bile, without affecting mercury excretion, indicates that NAPA per se cannot carry methyl mercury from liver to bile. In addition to unchanged NAPA, bile collected after administration of NAPA and DNB or BSP contained relatively large amounts of GSH conjugates of DNB or BSP, respectively, together with smaller amounts of GSH and another methyl mercury carrying component. The data are interpreted as suggesting that NAPA potentiation of methyl mercury excretion in bile can be due to an increased availability of GSH. However, it cannot be excluded that methyl mercury carrying components other than GSH and NAPA appearing in bile upon NAPA administration could play a role in NAPA potentiation of methyl mercury excretion in bile.     

Effects of methyl mercury exposure on the growth of juvenile common loons

We conducted a dose–response laboratory study to quantify the level of mercury exposure associated with negative effects on the development of common loon chicks reared in captivity from hatch to 105 days. A dose regimen was implemented that provided exposure levels that bracketed relevant exposure levels of methyl mercury found in loon chicks across North America. We observed no overt signs of mercury toxicosis and detected no significant effect of dietary mercury exposure on growth or food consumption. However, asymptotic mass was lower in chicks that hatched from eggs collected from nests on low pH lakes relative to eggs from neutral pH lakes. Rapid excretion of methyl mercury during feather growth likely provides loon chicks protection from methyl mercury toxicity and may explain the lack of convincing toxicological findings in this study. Lake-source effects suggest that in ovo exposure to methyl mercury or other factors related to lake pH have consequences on chick development.     

The Effect of N-acetylhomocysteine and its Thiolactone on the Distribution and Excretion of Mercury in Methyl Mercuric Chloride Injected Mice

Abstract: The distribution and excretion of mercury was studied in mice given a single intravenous dose of 5 μmol/kg of methyl mercuric chloride. Intravenous treatment with N-acetylhomocysteine (10 mmol/kg) increased the urinary excretion of mercury. The corresponding thiolactone mixed into the feed of the mice turned out to be more effective in removing mercury from the body. The toxicity of the thiolactone seemed to be remarkably low compared to other sulphur containing agents. Mercury deposited in the brain was mobilized by oral administration of the thiolactone even if the treatment was delayed until 5 days after the injection of methyl mercury. The results indicate that the formation of a N-acetylhomocysteine-methyl-mercuric-complex is responsible for this effect.     

The Effect of Selenium on the Biliary Excretion and Organ Distribution of Mercury in the Rat after Exposure to Methyl Mercuric Chloride

Abstract The influence of selenium compounds on the biliary excretion and the organ distribution of mercury after injection of methyl mercuric chloride (4 μmol/kg) have been tested. Selenite, seleno-di-N-acetylglycine and seleno-methionine strongly inhibited the biliary excretion of mercury. Selenite even in a molar dose of 1/40 of the methyl mercury dose inhibited the biliary excretion of mercury. The less toxic seleno-di-N-acetylglycine was needed in larger molar doses and did not act as rapidly as selenite. Biliary excreted methyl mercury is known to be partly reabsorbed in the gut. Subsequently a part of it is deposited in the kidneys since drainage of the bile lowered the kidney content of mercury. Rats given selenium compounds in combination with bile drainage showed further reduction of the kidney mercury content than bile duct drainage alone. Thus the demonstrated lowering effect of selenium compounds on the kidney mercury content cannot be completely explained by an inhibition of biliary excretion of mercury. The mercury concentration in the brain was increased by the selenium compounds; the effect being dependent of the selenium dose reaching a maximum at an equimolar selenite - to methyl mercury dose ratio. The mechanisms by which selenium influences the methyl mercury kinetics are discussed.     

Toxicological interactions of chlorpyrifos and methyl mercury in the amphipod, Hyalella azteca.

The mechanism of interaction between chlorpyrifos, an organophosphate insecticide, and methyl mercury, an organometal, was assessed utilizing the amphipod, Hyalella azteca. Previous studies have demonstrated that chlorpyrifos and methyl mercury interact additively, with survival as the endpoint. In addition, exposure to chlorpyrifos and methyl mercury increased the accumulation and decreased the elimination of methyl mercury. To elucidate the mechanism responsible for these interactions, biochemical mechanisms indicative of chlorpyrifos and methyl mercury toxicity were assessed in H. azteca. Biochemical endpoints that were evaluated include the inhibition of acetylcholinesterase enzyme and indicators of oxidative stress such as glutathione-S-transferase activity, lipid peroxidation, protein oxidation, and glutathione content. Methyl mercury antagonized the effects of chlorpyrifos in vivo on acetylcholinesterase inhibition. Methyl mercury did not induce oxidative damage; however, chlorpyrifos decreased glutathione-S-transferase activity. Additional studies demonstrated that methyl mercury did not affect the in vitro bioactivation of chlorpyrifos or the subsequent inhibition of acetylcholinesterase enzyme activity. Chemical-chemical interactions were examined utilizing chromatographic techniques. Results of thin layer chromatography suggested the formation of a chlorpyrifos-methyl mercury complex. The formation of this complex may result in increased accumulation of methyl mercury, apparent additive toxicity, and protection against chlorpyrifos mediated acetylcholinesterase inhibition.     

The effect of selenium on the biliary excretion and organ distribution of mercury in the rat after exposure to methyl mercuric chloride.

The influence of selenium compounds on the biliary excretion and the organ distribution of mercury after injection of methyl mercuric chloride (4 mumol/kg) have been tested. Selenite, seleno-di-N-acetylglycine and seleno-methionine strongly inhibited the biliary excretion of mercury. Selenite even in a molar dose of 1/40 of the methyl mercury dose inhibited the biliary excretion of mercury. The less toxic seleno-di-N-acetylglycine was needed in larger molar doses and did not act as rapidly as selenite. Biliary excreted methyl mercury is known to be partly reabsorbed in the gut. Subsequently a part of it is deposited in the kidneys since drainage of the bile lowered the kidney content of mercury. Rats given selenium compounds in combination with bile drainage showed further reduction of the kidney mercury content than bile duct drainage alone. Thus the demonstrated lowering effect of selenium compounds on the kidney mercury content cannot be completely explained by an inhibition of biliary excretion of mercury. The mercury concentration in the brain was increased by the selenium compounds; the effect being dependent of the selenium dose reaching a maximum at an equimolar selenite--to methyl mercury dose ratio. The mechanisms by which selenium influences the methyl mercury kinetics are discussed.     

Methyl mercury: acute toxicity, tissue distribution and decay profiles in the guinea pig

Acute toxicity studies with methyl mercuric chloride showed that the guinea pig was quite susceptible to methyl mercury intoxication. LD50 values were 5.5 mg Hg/kg ip and 16.5 mg Hg/kg po. One to 2 weeks after dosing, several animals began to display signs of neurotoxicity.Tissue distribution and pharmacodynamic studies with radiolabeled methyl mercuric chloride ([²⁰³Hg]CH₃HgCl) at 1 and 10 mg Hg/kg revealed that while most tissues decreased in mercury concentration from day 1 to day 7, cerebrum, cerebellum and muscle showed a delayed uptake of the alkyl mercurial. In CNS tissue the concentration of mercury decreased in the order cerebrum > cerebellum > spinal cord. Kidney and liver consistently contained the highest levels of mercury, and plasma the lowest, during the 49-day sampling period. One week after dosing the blood: brain ratios were less than 1. The tissue concentration of mercury was generally directly proportional to the dose administered; however, mercury levels in the gall bladder were significantly higher than anticipated on 5 of the 7 sacrifice days.Most of the tissues displayed a biphasic decay profile with a half-life of 2–3 days for the initial rapid phase of decline. This initial phase was followed by a slower tissue excretion rate for which the mean half-life for mercury was 15 ± 0.9 days and 15 ± 0.8 days for the low and high dose, respectively. The similarity of these values again indicates no dose-related effects.     

The assessment of the contribution of hair to methyl mercury excretion

Due to its ability to avidly accumulate methyl mercury from blood, scalp hair has been widely used as a biological monitor for human exposure. The question arises that hair may also be an important route of elimination of methyl mercury from the body. Taking original publications and reviews on the physiology of hair (including growth by weigh and density) and on the deposition parameters for methyl mercury in the body (including the hair to blood concentration ratio of methyl mercury), one can calculate the rate of elimination of methyl mercury in hair. The result indicates that hair accounts for only a small fraction, less than 10%, of the total elimination of methyl mercury from the body. This relationship is expected to be maintained at every level when the dominant form of mercury is methyl. Other species of mercury I eliminated by hair even at a lower rate.     

The modifying effect of multiple generation selection and dietary cadmium on methyl mercury toxicity in Japanese quail

Japanese quails were exposed to dietary methyl mercury chloride in graded amounts from 2–8 ppm through a series of multiple-generation experiments with the experimental periods lasting from 6–12 weeks, starting with the experimental diets when the birds were 6 weeks old. Cadmium chloride was added to diets with and without mercury and fed to groups in three of the five experiments.Hatchability was depressed at 8 ppm mercury. The mortality of chicks from 8 ppm exposed parents was 100% in the first two generations, while chick mortality at the 4 ppm level in the same experiments was 54–63%. After six generations mortality in chicks hatched in 8 ppm group was reduced to about 50%.Cadmium supplementation at a level of 5 ppm counteracted the mercuryinduced toxicity but failed to be effective in preventing the effects of mercury toxicity when added at 15 ppm level.Significant toxic effects of cadmium alone did not occur until the level was raised to 60 ppm. The morphology of mercury-induced encephalopathy was similar to the brain lesions reported in other bird species, with the injuries pre-dominantly being localized to the cerebellar cortex and medulla.     

Potentiation of ethanol toxicity by cyanamide in relation to acetaldehyde accumulation

The possibility that acetaldehyde accumulation potentiates the acute toxicity of ethanol was studied by pretreating rats with cyanamide, an aldehyde dehydrogenase inhibitor. At 30 min after administration of ethanol (7 to 9 g/kg, po), the levels of acetaldehyde in femoral venous blood of cyanamide-treated rats were increased from 10 to 20 to 600 mumol/liter and at death the concentrations of acetaldehyde in heart blood and cerebrospinal fluid were still 7 to 9 and 4 to 9 times higher, respectively, than in rats given ethanol only. The cyanamide pretreatment (25 mg/kg) significantly increased the mortality of rats given 6.5 to 7.0 g/kg ethanol and decreased the LD50 of ethanol from 7.3 to 5.9 g/kg. Cyanamide increased the late mortality, possibly because of sustained acetaldehyde accumulation. Although administration of the alcohol dehydrogenase inhibitor, 4-methylpyrazole (4-MP, 10 mg/kg), prevented the accumulation of acetaldehyde, it only partly counteracted the effect of cyanamide on mortality. After coadministration of cyanamide and 4-MP, the LD50 of ethanol was 6.5 g/kg, and after 4-MP alone, 6.7 g/kg. 4-MP by itself seemed to increase the early mortality of rats to ethanol poisoning. The results suggest that the potentiating effect of cyanamide on ethanol toxicity can partly be explained by acetaldehyde accumulation and that 4-MP can be used to inhibit this accumulation providing its central depressant effect is taken into account.     

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC₅₀) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.     

Comparing toxicity endpoints on Lecane quadridentata (Rotifera: Monogononta) exposed to two anticholinesterases pesticides

Toxicity tests were performed on the freshwater rotifer Lecane quadridentata exposed to the pesticides carbaryl and methyl parathion (lethal, sublethal, and chronic) to compare the sensitivity between different endpoints: (a) 48‐h mortality; (b) 30‐min in vivo inhibition of esterase activity; (c) 5‐day inhibition of the instantaneous growth rate. The emphasis of this work was to find the most appropriate endpoint to evaluate the toxicity of these pesticides in view of their sensitivity, duration, and ecological relevance. The comparison between the three toxicity tests show that the 5‐day chronic tests have the lowest EC50 (2.22 and 6.6 mg/L), lowest‐observed‐effect concentration (2.5 and 2.5 mg/L), and no‐observed‐effect concentration (1.0 and 1.2 mg/L) values for carbaryl and methyl parathion, respectively. This indicates that the estimate of the instantaneous rate of natural increase r is the most sensitive endpoint regarding the toxicity of these pesticides. This sensitivity might be due to the effect on reducing the growth potential form the first generation on. Lethal and sublethal tests are closely related, suggesting that the immediate effect after inhibition of esterases is death. In general, the sensitivity of L. quadridentata is similar to other species of rotifers exposed to methyl parathion. Therefore, the 5‐day chronic toxicity test with the freshwater rotifer L. quadridentata should be considered a good candidate to evaluate the effect of anticholinesterase pesticides, due to its high sensitivity and ecological relevance. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Accumulation and tissue distribution of mercury in the guinea pig during subacute administration of methyl mercury

Female guinea pigs were dosed po daily for 71 days with 0.4, 4, 40, or 400 μg Hg/kg given as radiolabeled methyl mercuric chloride. The accumulation of total mercury was followed in 10 tissues at 6 time intervals. After dosing ceased, the decay profiles of mercury were followed for an additional 35 days. The accumulation pattern for mercury was similar for each dose level, and the tissue mercury concentration on day 71 increased in the following order: blood < cerebellum < hypothalamus < calcarine cortex < frontal lobe < occipital lobe < caudate nucleus < muscle < liver < kidney. Mercury accumulation in all tissues, except kidney at the 4-, 40-, and 400-μg/kg dose levels, approached steady-state values in the 35–71 -day dosing period. The accumulation curves could be fitted by an exponential equation incorporating the mercury half-life obtained from the decay profiles. As the dose level increased, tissue mercury concentrations increased to a greater extent than anticipated. Although doses increased 1000-fold from 0.4 to 400 μg Hg/kg, kidney concentrations increased 3300-fold after 71 days of dosing. At this time, inorganic mercury (Hg²⁺) comprised 42% of the total kidney mercury and 5% of the total liver mercury at the 400 μg/kg dose.Clinical signs of methyl mercury intoxication were induced in guinea pigs after dosing daily for 9 days at 5 mg Hg/kg. The activities of 6 enzymes were monitored and cholinesterase (serum), choline acetylase (caudate nucleus) and carboxylesterase (liver) were significantly lower than control values. The total mercury concentration in whole brain was 28 μg/g (wet weight). Animals dosed at 400 μg Hg/kg for 71 days showed no decrease in the activities of the selected enzymes, there was no change in weight gain when compared to the control and there were no signs of methyl mercury toxicity. The highest brain mercury concentration after 71 days dosing was 11 μg/g (wet weight) in the caudate nucleus.