Akira Yasutake

Methylmercury transport across the placenta via neutral amino acid carrier

Abstract Methylmercury (MeHg) penetrates the placental barrier to affect developing fetuses in the uterus. However, the mechanism of placental MeHg transport is not well defined. To clarify the MeHg transport system that functions in the placenta, pregnant rats were intravenously administered MeHg on day 18 of gestation. The fetal blood was collected from the umbilical cord at 30 and 60 min after the administration, and its mercury concentration was measured. MeHg was found to be rapidly transported to the fetal blood in a time- and dose-dependent manner, and predominantly distributed in the blood cells there. MeHg transport was effectively suppressed by the co-injection of neutral amino acids, i.e., L-methionine and L-phenylalanine, suggesting that MeHg is actively transported as its cysteine conjugate via the neutral amino acid carrier system. The suppression by methionine was not so marked as by phenylalanine. Since methionine administration caused a rapid increase of the cysteine, which functioned as a predominant carrier in MeHg transport, in the maternal plasma, newly synthesized cysteine seemed to accelerate the mercury uptake. Accordingly, the acceleration by the extra cysteine would compensate partly the competitive effect of methionine as a neutral amino acid.

Balancing the benefits of n-3 polyunsaturated fatty acids and the risks of methylmercury exposure from fish consumption

Fish and shellfish are widely available foods that provide important nutrients, particularly n-3 polyunsaturated fatty acids (n-3 PUFAs), to many populations globally. These nutrients, especially docosahexaenoic acid, confer benefits to brain and visual system development in infants and reduce risks of certain forms of heart disease in adults. However, fish and shellfish can also be a major source of methylmercury (MeHg), a known neurotoxicant that is particularly harmful to fetal brain development. This review documents the latest knowledge on the risks and benefits of seafood consumption for perinatal development of infants. It is possible to choose fish species that are both high in n-3 PUFAs and low in MeHg. A framework for providing dietary advice for women of childbearing age on how to maximize the dietary intake of n-3 PUFAs while minimizing MeHg exposures is suggested.

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

Mechanism of urinary excretion of methylmercury in mice

To elucidate the mechanism by which methylmercury (MeHg) is eliminated from organisms, male C57BL/6N mice were orally administered with MeHg chloride (5 mg/kg) and the chemical forms of its metabolites in plasma, urine and the kidney were determined by column chromatographic analysis. Orally administered MeHg rapidly entered the circulation, accumulated in the kidney and other tissues, and was slowly excreted in the urine. Ultrafiltration and gel filtration analysis revealed that most of plasma MeHg was accounted for by its albumin conjugate. Cell fractionation analysis revealed that about 80% of renal MeHg was recovered from the 15 000 g supernatant fraction of the kidney homogenate. If the kidney was homogenized in the presence of serine-borate complex, a potent inhibitor of γ-glutamyltranspeptidase (γ-GTP), about 50% of the MeHg in the supernatant fraction was recovered as its glutathione S-conjugate while the rest was bound to cytosolic protein(s). The major part of urinary MeHg was accounted for by its cysteine conjugate. However, urinary excretion of its glutathione conjugate increased significantly if animals were pretreated with acivicin, an affinity labeling reagent for γ-GTP. These and other results suggested that MeHg bound to albumin accumulated in the kidney predominantly via some non-filtrating peritubular mechanism, and localized in renal cytosolic compartment as its glutathione- and protein-bound forms. The glutathione S-conjugate of MeHg in the tubule cells might be transferred to the lumenal space, hydrolyzed to the cysteine S-conjugate, and then excreted in urine. These sequential events might constitute an important eliminatory pathway for a hazardous mercurial metabolite in mice.

Sex and age differences in mercury distribution and excretion in methylmercury‐administered mice

Sex differences in mercury distribution and excretion after single administration of methylmercury chloride (MMC, 5 mg/kg) were studied in mice. A sex difference in urinary mercury excretion was found in sexually mature mice (age of 7 wk) of C57BL/6N and BALB/cA strains. Males showed higher mercury levels in urine than females, though no significant difference was found in fecal mercury levels 24 h post exposure to MMC. The higher urinary excretion rates in males accounted for significant lowering of mercury levels in the brain, liver, and blood, but not in the kidney, which showed higher values. At 5 min, however, these sex difference was found only in the kidney, showing higher levels in males. Changes in mercury distribution with time were studied in C57BL/6N mice. The brain mercury increased in both sexes up to 3 d, and decreased only in males on d 5. Liver and blood mercury decreased with time in both sexes, and these were constantly higher in females than in males. Renal mercury in males decreased to similar levels to females on d 3. The sex differences at various ages were studied with C57BL/6N mice 24 h after dosing. Two-week-old mice, the youngest in this study, did not show significant sex difference in the mercury distribution and excretion, and their urinary mercury levels were much lower as compared to the older mice. Then, urinary mercury excretion in both sexes increased at 4 wk of age and then decreased at 45 wk of age. At 4, 7, 10, and 45 wk of age, males showed higher urinary mercury levels than females. These studies demonstrated sex and age differences in the mercury distribution and urinary excretion after methylmercury administration in mice. From these findings, it has been suggested that urinary mercury excretion may be related to sex hormones, especially androgens.

Relationships between trace element concentrations in chorionic tissue of placenta and umbilical cord tissue: potential use as indicators for prenatal exposure.

The role of the placenta was assessed by comparing the profiles of methylmercury (MeHg), inorganic mercury (I-Hg), lead (Pb), cadmium (Cd), selenium (Se), zinc (Zn), and copper (Cu) in freeze-dried chorionic tissue of the placenta and umbilical cord tissue. The significance of the placenta and cord tissue as predictors of prenatal exposure to these trace elements in pregnant women and newborns was also examined by comparing the element profiles among placenta and cord tissue, and maternal and cord blood red blood cells (RBCs). The samples were collected from 48 mother-child pairs at birth in the general population of Japanese. The concentrations of all elements, except for MeHg, were significantly higher in placenta than in cord tissue. In particular, the Cd showed the highest placenta vs. cord tissue ratio (59:1), followed by I-Hg (2.4:1), indicating that the placental barrier works most strongly against Cd among the examined toxic elements. Contrary to the other elements, the MeHg concentration in cord tissue was significantly higher (1.6 times) than that in placenta, indicating its exceptionally high placental transfer. The MeHg in placenta showed significant correlations with total mercury (T-Hg) in maternal and cord RBCs (rs=0.80 and 0.91, respectively). The MeHg in cord tissue also showed significant correlations with T-Hg in maternal and cord RBCs (rs=0.75 and 0.85, respectively). Therefore, both placenta and cord tissue are useful for predicting maternal and fetal exposure to MeHg. The Se concentration in placenta showed significant but moderate correlations with that in maternal and cord RBCs (rs=0.38 and 0.57, respectively). The Pb, Zn, and Cu concentrations in placenta and cord tissue showed no significant correlations with those in maternal and cord RBCs. As an exception, the Cd concentration in placenta showed a moderate but significant correlation (rs=0.41) with that in maternal RBCs, suggesting that the placenta is useful for predicting maternal exposure to Cd during gestation.

Isothiocyanates Reduce Mercury Accumulation via an Nrf2-Dependent Mechanism during Exposure of Mice to Methylmercury

Background: Methylmercury (MeHg) exhibits neurotoxicity through accumulation in the brain. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) plays an important role in reducing the cellular accumulation of MeHg. Objectives: We investigated the protective effect of isothiocyanates, which are known to activate Nrf2, on the accumulation of mercury after exposure to MeHg in vitro and in vivo. Methods: We used primary mouse hepatocytes in in vitro experiments and mice as an in vivo model. We used Western blotting, luciferase assays, atomic absorption spectrometry assays, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and we identified toxicity in mice based on hind-limb flaccidity and mortality. Results: The isothiocyanates 6-methylsulfinylhexyl isothiocyanate (6-HITC) and sulforaphane (SFN) activated Nrf2 and up-regulated downstream proteins associated with MeHg excretion, such as glutamate-cysteine ligase, glutathione S-transferase, and multidrug resistance–associated protein, in primary mouse hepatocytes. Under these conditions, intracellular glutathione levels increased in wild-type but not Nrf2-deficient primary mouse hepatocytes. Pretreatment with 6-HITC and SFN before MeHg exposure suppressed cellular accumulation of mercury and cytotoxicity in wild-type but not Nrf2-deficient primary mouse hepatocytes. In comparison, in vivo administration of MeHg to Nrf2-deficient mice resulted in increased sensitivity to mercury concomitant with an increase in mercury accumulation in the brain and liver. Injection of SFN before administration of MeHg resulted in a decrease in mercury accumulation in the brain and liver of wild-type, but not Nrf2-deficient, mice. Conclusions: Through activation of Nrf2, 6-HITC and SFN can suppress mercury accumulation and intoxication caused by MeHg intake.

Induction by mercury compounds of brain metallothionein in rats: Hg0 exposure induces long-lived brain metallothionein.

Abstract Metallothionein (MT) is one of the stress proteins which can easily be induced by various kind of heavy metals. However, MT in the brain is difficult to induce because of blood-brain barrier impermeability to␣most heavy metals. In this paper, we have attempted to induce brain MT in rats by exposure to methylmercury (MeHg) or metallic mercury vapor, both of which are known to penetrate the blood-brain barrier and cause neurological damage. Rats treated with MeHg (40 μmol/kg per day × 5 days, p.o.) showed brain Hg levels as high as 18 μg/g with slight neurological signs 10␣days after final administration, but brain MT levels remained unchanged. However, rats exposed to Hg vapor for 7 days showed 7–8 μg Hg/g brain tissue 24 h after cessation of exposure. At that time brain MT levels were about twice the control levels. Although brain Hg levels fell gradually with a half-life of 26 days, MT levels induced by Hg exposure remained unchanged for >2␣weeks. Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT. Hybridization analysis showed that, despite a significant increase in MT-I and -II mRNA in brain, MT-III mRNA was less affected. Although significant Hg accumulation and MT induction were observed also in kidney and liver of Hg vapor-exposed rats, these decreased more quickly than in brain. The long-lived MT in brain might at least partly be accounted for by longer half-life of Hg accumulated there. The present results showed that exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT.

Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor.

This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg(0)) vapor. Both strains were exposed to Hg(0) vapor at 5.5-6.7 mg/m(3) for 3 h during late gestation. Twenty-four hours after exposure to Hg(0) vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg(0) vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer.

Methylmercury Poisoning in Common Marmosets—A Study of Selective Vulnerability Within the Cerebral Cortex

Neuropathological lesions found in chronic human Minamata disease tend to be localized in the calcarine cortex of occipital lobes, the pre- and postcentral lobuli, and the temporal gyri. The mechanism for the selective vulnerability is still not clear, though several hypotheses have been proposed. One hypothesi s is vascular and postulates that the lesions are the result of ischemia secondary to compression of sulcal arteries from methylmercury-induced cerebral edema. To test this hypothesis, we studied common marmosets because the cerebrum of marmosets has 2 distinct deep sulci, the calcarine and Sylvian fi ssures. MRI analysis, mercury assays of tissue specimens, histologic and histochemical studies of the brain are reported and discussed. Brains sacrifi ced early after exposure to methylmercury showed high contents of methylmercury and edema of the cerebral white matter. These results may explain the selective cortical degeneration along the deep cerebral fi ssures or sulci.