Intracellular Demethylation of Methylmercury to Inorganic Mercury by Organomercurial Lyase (MerB) Strengthens Cytotoxicity.

Some methylmercury (MeHg) is converted to inorganic mercury (Hg2+) after incorporation into human and animal tissues, where it can remain for a long time. To determine the overall toxicity of MeHg in tissues, studies should evaluate low concentrations of Hg2+. Although demethylation is involved, the participating enzymes or underlying mechanisms are unknown; in addition, the low cell membrane permeability of Hg2+ makes these analyses challenging. We established model cell lines to assess toxicities of low concentrations of Hg2+ using bacterial organomercury lyase (MerB). We engineered MerB-expressing HEK293 and HeLa cell lines that catalyze MeHg demethylation. These cells were significantly more sensitive to MeHg exposure compared to the parental cells. MeHg treatment remarkably induced metallothioneins (MTs) and hemeoxygenase-1 (HMOX-1) mRNAs and modest expression of superoxide dismutase 1, whereas catalase and glutathione peroxidase 1 mRNAs were not up-regulated. merB knockdown using small interfering RNA supported the induction of MT and HMOX-1 mRNA by MerB enzymatic activity. Pretreatment with Trolox, a water-soluble vitamin E analog, did not inhibit MeHg-induced elevation of MT-Ix and HMOX-1 mRNAs in MerB-expressing cells, suggesting that Hg2+ works independently of reactive oxygen species generation. Similar results were obtained in cells expressing MerB, suggesting that high MTs and HMOX-1 induction and cytotoxicity are common cellular responses to low intracellular Hg2+ concentrations. This is the first study to establish cell lines that demethylate intracellular MeHg to Hg2+ using bacterial MerB for overcoming the low membrane permeability of Hg2+ and exploring the intracellular responses and toxicities of low Hg2+ concentrations.

X-ray structure determination and deuteration of nattokinase.

Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

Region-dependent differences and alterations of protective thiol compound levels in cultured astrocytes and brain tissues.

We examined region-dependent differences and alterations in the levels of protective thiol compounds, glutathione (GSH) and metallothionein (MT)-I and -II, in cultured rat astrocytes under several culture conditions and in brain tissues of rats at postnatal and weaning periods. Regardless of culture conditions, both protein concentrations and mRNA expressions of MT-I and -II were much higher in the cerebral hemisphere than in cerebellar astrocytes, whereas no difference was observed in GSH concentration. In both astrocytes, the GSH concentrations did not change within 12 h but significantly increased 24 h after being maintained in a serum-free defined medium. At 24 h, protein concentrations and mRNA expressions of MT-I and -II also increased in the respective astrocytes, and were further enhanced when maintained in the presence of 50 microM Zn(2+). In the brain tissues, the MT-I/-II protein concentrations were significantly higher in the cerebral cortex (a part of the cerebral hemisphere) than in the cerebellum, whereas the GSH concentration was similar at both postnatal day (P)1 and P35. In addition, the concentrations in the respective regions were significantly higher at P35 than at P1. These results suggest that region-dependent differences in the cellular levels of GSH and MTs in cultured astrocytes might reflect the in vivo differences, and that the levels of the respective thiol compounds in cultured astrocytes increase after serum elimination along with the region-dependent differences.

Acute cytotoxic effects of mercuric compounds in cultured astrocytes prepared from cerebral hemisphere and cerebellum of newborn rats.

We investigated acute cytotoxic effects and Hg accumulation after exposure to methylmercury (MeHg) or Hg(2+) in the presence or absence of serum in cultured astrocytes prepared from the cerebral hemisphere or cerebellum of newborn rats. Dose-related changes in viable cell numbers after exposure to mercuric compounds were not different between astrocytes from both regions under the specified conditions. Accumulation of each compound for 3 h was similar in both astrocytes but that for 24 h became different, especially that of Hg(2+). In both astrocytes, susceptibility to the respective compounds was higher in the order of those exposed immediately after, without, and 24 h after changing the serum-containing medium to a serum-free defined medium (SFDM). Accumulation for 3 h was higher in the respective astrocytes exposed to MeHg or Hg(2+) immediately after being maintained in SFDM than in those exposed 24 h after. These results suggest that accumulation of mercuric compounds up to 3 h strongly correlates with susceptibility, at least when maintained in SFDM. Astrocytic morphology changed to a satellite shape after the medium change to SFDM particularly in cerebellar astrocytes but only a few in cerebral hemisphere astrocytes, and it was reverted to a polygonal shape by MeHg but not Hg(2+) at 3 microM. The present results suggest that although some properties such as morphological changes and Hg accumulation are different between cerebral hemisphere and cerebellar astrocytes, these differences are not simply reflected by susceptibility to the acute cytotoxicity of mercuric compounds.

Influence of LIF and BMP-2 on differentiation and development of glial cells in primary cultures of embryonic rat cerebral hemisphere.

Cells prepared from the cerebral hemisphere of embryonic Day 18 rats were maintained for 2 days in serum-free modified Bottenstein-Sato (mBS) medium containing thyroid hormone (TH), with or without leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP)-2, and these influences on the differentiation and development of glial cells were investigated using the cells maintained in mBS medium containing TH as controls. The levels of glial fibrillary acidic protein (GFAP) expression and the number of GFAP-positive astrocytes increased markedly with the addition of LIF or BMP-2, and were enhanced further with the addition of both LIF and BMP-2. The number of O1-positive oligodendrocytes increased with the addition of LIF, whereas it decreased with the addition of BMP-2. The number did not change with the addition of both cytokines. Using antibody against platelet-derived growth factor (PDGF), we then excluded indirect effects of these cytokines through PDGF, which would increase by accelerated astrocyte development. When PDGF was neutralized, the number of oligodendrocytes increased under all conditions examined. As a result of the neutralization, the effect of BMP-2 on oligodendrocyte differentiation was eliminated, although LIF remained effective. These results suggest that the differentiation of oligodendrocytes was delayed partially by PDGF even in control cultures. It is also suggested that LIF and BMP-2, each of which accelerates the differentiation and development of astrocytes, would seem to have different effects on oligodendrocyte differentiation, i.e., LIF would directly affect oligodendrocyte differentiation, whereas BMP-2 would affect it mainly through PDGF.

Selective disappearance of an axonal protein, 440-kDa ankyrinB, associated with neuronal degeneration induced by methylmercury.

The 440-kDa isoform of brain ankyrin, 440-kDa ankyrinB, is a neuron-specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 microM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440-kDa ankyrinB diminished drastically, whereas that of microtubule-associated protein-2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440-kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440-kDa ankyrinB and an increase of GFAP in a dose-dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3-1.0 microM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440-kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440-kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low-density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high-density culture without TH. This suggests that, in low-density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte-inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell-cell communications.

Attenuation of both apoptotic and necrotic actions of cadmium by Bcl-2.

We examined the effects of cadmium on the bcl-2 family of proteins--bcl-2, bax, bad, and bcl-xS/L--in cadmium-induced cytotoxicity. Addition of 10 microM cadmium to cultured porcine kidney LLC-PK(1) cells caused apoptosis. Western blot analyses revealed that cadmium markedly increased endogenous bcl-2 protein (to 3-4 times the level in wild-type cells) earlier than metallothionein induction, but that the metal did not enhance the induction of bax, bad, or bcl-xS proteins. Cadmium also induced the transcript of bcl-2, with the amount of bcl-2 reaching a maximum at 1-2 hr of exposure; this increase occurred earlier than cadmium-induced increase in the protooncogene such as c-myc. A cadmium-induced increase in endogenous bcl-2 protein was also seen in rat primary thymocytes. Overexpression of the bcl-2 protein by gene transfection prevented cadmium-induced apoptosis. Following the detection of apoptosis, lactate dehydrogenase release in the culture medium (a marker of necrosis) was observed, and this release was also inhibited by overexpression of bcl-2. Electron microscopic observations also supported the fact that cadmium induced apoptotic chromatin condensation at an early stage of exposure, followed by necrotic features of the cells, both of which were also inhibited by overexpression of bcl-2 proteins. Thus, our data demonstrated that both apoptotic and necrotic actions of cadmium were attenuated by bcl-2.