Metal-responsive transcription factor 1 (MTF-1) is essential for activating transcription of the metallothionein genes in response to multiple species of heavy metals by binding to the metal-responsive element (MRE) located in the upstream region of the genes. In contrast, MTF-1 binding to the MRE in vitro is induced only by zinc ions, suggesting that MTF-1 acts as a zinc sensor protein. Although the mechanism allowing such a zinc-responsive factor to respond to various metal species in vivo remains ambiguous, recent information regarding the metal response of MTF-1 and the genes expressed downstream of MTF-1 provides important clues to unraveling the intrinsic mechanism and the biological significance of the MTF-1 functions in vivo.

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The role of transferrin in iron delivery to tissues is described. Transferrin-dependent iron uptake by erythroid cells in the bone marrow is essential for the development of erythrocytes, while nontransferrin-bound iron can be taken up in tissues such as liver. On the basis of the evidence that iron distribution in the body is changed by iron saturation of plasma transferrin, the role of transferrin in iron delivery to the brain is reviewed. In the case of transient iron saturation of plasma transferrin, ⁵⁹Fe concentrations in the brain of iron-loaded mice are approximately 40-50% of those of control mice in all brain regions tested except the choroid plexus, in which the ⁵⁹Fe concentration is equal. A similar distribution of ⁵⁹Fe in the brain is also observed in neonatal hypotransferrinemic (HP) mice, which have a splicing defect in the transferrin gene, resulting in < 1% of the normal plasma levels of transferrin. These results suggest that transferrin-bound iron is responsible for the fraction of iron in the circulation that enters the brain. On the other hand, the iron concentration in the brain of HP mice is approximately three times higher than that in nonmutant mice. It is likely that the management of iron is affected in the brain of HP mice. Brain transferrin may be involved in the management of iron in the brain.

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Endothelin-1 (ET-1) is known as a potent stimulator of cell proliferation and as a vasoconstrictor. It is believed that ET-1 contributes to the development of arterial diseases such as atherosclerosis. In this study, we demonstrated the expression of tropoelastin and lysyl oxidase (LO) on gene levels as induced by ET-1 in cultured smooth muscle cells (SMCs). ET-1 stimulated cell proliferation in a dose-dependent manner, and the level of this proliferation increased about 1.3-fold at 100 nM of ET-1. ET-1 suppressed the tropoelastin protein synthesis in a dose-dependent and time-dependent manner. In addition, ET-1 dose-dependently suppressed the tropoelastin and LO mRNA expression. The tropoelastin and LO mRNA levels decreased to about half and 4/5, respectively, at 100 nM of ET-1. The inhibition of elastin synthesis was completely prevented by BQ123, an endothelin receptor A (ET_A) antagonist. These results indicate that ET-1 can modulate the tropoelastin and LO mRNA expression via an ET_A receptor in cultured SMC and that the regulator for elastin expression may play an important role in elastogenesis and SMC proliferation during the development of atherosclerosis.

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The effect of various algae on bone calcification in the femoral-metaphyseal tissues of rats was investigated. Undaria pinnatifida, Sargassum horneri, Eisenia bicyclis, Cryptonemia scmiziana, Gelidium amansii, and Ulva pertusa Kjellman, which were gathered seasonally, were used. Water suspensions (5%) of marine algae powder were orally administered once daily for 7 days. Bone calcium content was significantly increased by the administration of U. pinnatifida, S. horneri, E. bicyclis, or C. scmitziana. Bone alkaline phosphatase activity, which is an enzyme for calcification, was significantly enhanced by the administration of S. horneri or G. amansii. Femoral-metaphyseal tissues were cultured for 48 hr in Dulbecco’s modified Eagle’s medium containing either vehicle or water-solubilized extract (25 and 50 μg/ml) obtained from U. pinnatifida, S. horneri, E. bicyclis, and C. scmitziana. The bone calcium content was significantly elevated in the presence of S. horneri extract (25 and 50 μg/ml). No effect was seen with other extracts. The effect of S. horneri extract in increasing bone calcium content was completely inhibited in the presence of cycloheximide (10^-6 M), an inhibitor of protein synthesis. The present study demonstrates that S. horneri extract has an anabolic effect on bone calcification in vivo and in vitro. The anabolic effect of S. horneri extract may be based on a newly synthesized protein component.

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To study the origin of chloroform in vegetables, a simple and sensitive method was established using a gas chromatograph equipped with an electron capture detector. The method includes solvent trapping of chloroform, and gas chromatographic separation with a detection system. The average concentrations in vegetables, tea leaves and frozen vegetables were 30.9, 62.9 and 15.7 μ/kg, respectively. Tea leaves contained the highest amount, followed by vegetables. The former was 2 times the latter. There was no significant difference between chloroform levels in vegetables grown in tap water (chloroform : 19.7-25.0 μg/L) and those in boiled distilled water. On the other hand, there was a high correlation between the number of cultivation days and chloroform levels in vegetables grown in both tap water and boiled distilled water.

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The maternal and fetal toxicity of dimethyltin chloride (DMTC) was examined in two teratological studies in pregnant Wistar rats. In one study, the animals were treated by oral gavage with DMTC at doses of 0, 5, 10, 15, or 20 mg/kg/day on gestational days 7-17. In the second study, animals were treated by oral gavage at doses of 0, 20 or 40 mg/kg/day DMTC on two or three consecutive days at one of four different periods of gestation (gestational days 7-9, 10-12, 13-15 or 16-17). Caesarean sections were performed on day 20 of gestation in both studies. In the first study, vaginal bleeding, tremor and convulsions were observed in animals treated at 20 mg/kg/day after day 15 of gestation. Of ten dams treated with 20 mg/kg/day DMTC, two died and one exhibited total resorption. While a increase in the incidence of cleft palate was found in the fetuses of animals treated with 20 mg/kg/day DMTC, the dams so treated exhibited severe clinical signs of toxicity. Animals treated in the second study with doses of 20 or 40 mg/kg/day at one of four periods of gestation had a reduction in the adjusted body weight gain but not in gravid uterus weight and did not show any evidence of teratogenic effect at either dose and period tested. These studies suggest that DMTC did not produce teratogenic effects at dose levels where no maternal toxicity was observed. It was suggested that under the conditions of the first study, since no signs of maternal or fetal toxicity could be detected up 10 mg/kg/day DMTC, this dose was chosen to represent the no-observed-adverse-effect level (NOAEL).

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Benzo[a]pyrene (BaP), a major environmental pollutant, is metabolized in vivo and produces many hydroxy derivatives. The estrogenic/antiestrogenic activities of twelve monohydroxy derivatives of BaP (1-through 12-OH species) were investigated using competition binding to human estrogen receptor (hER)α and hERβ, and the gene expression assay of the yeast two-hybrid system. BaP and 5-OH BaP did not bind to either hER. The other monohydroxy derivatives bound to both hERs. These compounds bound more strongly to hERβ than to hERα. Using the yeast two-hybrid assay system, 1-, 2-, 3-, and 9-OH BaP induced β-galactosidase with hERβ but not with hERα. This suggested that these compounds were estrogenic. In the presence of 10^-9 M 17β-estradiol, 8-OH BaP inhibited the induction of β-galactosidase. Because 8-OH BaP did not affect cell growth, it appeared to be an antiestrogen. The present study shows that most of the monohydroxy derivatives of BaP bind to estrogen receptors (ERs), and several of them have estrogenic or antiestrogenic activity.

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The ion constituents contained in fresh snow immediately after snowfall were examined at 5-10 km intervals over 50 km from the coast of Hakui city in Ishikawa Prefecture leeward of the northwestern seasonal wind to the plain in Toyama city in Toyama Prefecture. The concentrations of sodium ions and sea-salt sulfate ions that originated from the Sea of Japan decreased exponentially from Hakui city in Ishikawa Prefecture to Toyama city. The concentration of non sea-salt sulfate ions, which probably originate from human activities, also decreased exponentially, but it was lower than the concentration of sea-salt sulfate ions until 25 km from Hakui city and higher in the farther regions. We considered that this was due to additive effects of non sea-salt sulfate ions transported from a distance and local sulfate ions. The concentration of non sea-salt sulfate ions transported from the Asian Continent was estimated in each sampling station. Its contribution was 0.98 of the total at the coast of Hakui city and 0.65 at Toyama city, respectively, and showed a pattern similar to those of sodium ions and sea-salt sulfate ions. The concentrations of ammonium ions and non sea-salt calcium ions were slightly higher in the plain in Toyama than on the coast of Hakui city, suggesting that they mostly originated from the sampling regions in this study. The concentration of nitrate ions was almost constant in the regions from the coast to the inland areas, suggesting also that they mostly originated from the sampling regions.

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In order to confirm the participation of peroxynitrite in potassium bromate (KBrO₃)-induced oxidative stress and kidney damage in mice, we investigated effects of administration of nitric oxide (NO) synthase inhibitor on them. Cytoplasmic glutathione peroxidase (GPx) activity remarkably decreased within 3 hr after KBrO₃ administration, and then oxidative stress started to occur. However, kidney damage occurred 24 hr after KBrO₃ administration. Pre-administered N^G-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, suppressed KBrO₃-induced oxidative stress and kidney damage. However, no effect of L-NMMA was observed on the KBrO₃-induced reduction of cytoplasmic GPx activity. These results suggest that reduction of cytoplasmic GPx activity resulted from the KBrO₃ administration initiates oxidative stress and that NO also participates in the promotion of KBrO₃-induced oxidative stress and kidney damage.

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Phenanthraquinone (PQ) is a component of diesel exhaust particles which inhibits nitric oxide synthase (NOS) activity by shunting electrons away from the normal catalytic pathway of the enzyme and which can oxidize proximal protein sulfhydryls. In the present study, we examined the possibility that PQ may also modify critical thiol residues on endothelial NOS (eNOS), leading to a disruption of catalytic activity. PQ and the thiol-modifying agent N-ethylmaleimide (NEM) suppressed NO formation from L-arginine by the total membrane fraction of bovine aortic endothelial cells in a concentration-dependent manner. The dithiol agent dithiothreitol (DTT) completely blocked NEM-mediated inhibition of eNOS activity. In contrast. PQ-inhibited eNOS activity was reduced by DTT, but not by the monothiol agent glutathione. These results suggest that PQ-mediated suppression of eNOS activity involves not only uncoupling of the electron transport of this enzyme, but also modification of presumably the proximal protein sulfhydryls that play an important role in the maximal catalytic activity.

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The ability of a dioxin-like toxic compound, coplanar polychlorinated biphenyl, 3,3',4,4',5-penta-chlorobiphenyl (PCB126) to reduce the protein level of hepatic class I alcohol dehydrogenase (ADH), which plays an important role in the metabolism of ethanol, was studied. Male Wistar rats received PCB126 25 mg/kg i.p. At this dose the compound induces a wasting syndrome. PCB126 administration resulted in a significant suppression of the protein level of class I ADH, whereas the difference between free- and pair-fed controls was slight. These results suggest that dioxins also reduce class I ADH without involvement of decreased food consumption. These data offer new insights into the toxicity of dioxins via a marked decrease in the level of class I ADH.

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The effect of nijiru, which is a by-product of the processing of soybeans to make the fermented soybeans called natto, on circulating blood chemistry levels related to calcium and bone metabolism in healthy individuals was investigated. Twelve volunteers (six men and six women) were received nijiru twice a day for 60 days at a dose of 1500 mg (6 tablets) per day. The serum γ-carboxylated osteocalcin concentration was significantly increased by the intake of nijiru in both men and women to about 2-fold that in the control group. The serum calcium concentration was significantly decreased by nijiru supplementation in women, and the serum inorganic phosphorus concentration was significantly reduced in both men and women. However, the intake of, nijiru did not have a significant effect on serum glucose, nitrogen urea, albumin, free cholesterol, triglyceride, high-density lipoprotein cholesterol, and γ-glutamyltranspeptidase concentrations in men or women, indicating that liver and renal function is not affected by nijiru supplementation. The results of the present study suggest that the intake of isoflavone- and saponin-containing nijiru can stimulate the γ-carboxylation of osteocalsin, which plays an important role in bone formation and mineralization, in healthy individuals.

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Two isoforms of brain ankyrin, 440 kD and 220 kD ankyrin_B, and neuronal cell adhesion molecule L1, which can bind to brain ankyrin, are both expressed in human neuroblastoma NB-1 cells. To examine the potential importance of the interaction between brain ankyrins and L1 in neuronal cell functions, an expression vector encoding a cytoplasmic domain of L1 has been constructed and transfected in NB-1 cells. The results obtained show that the expressed L1 cytoplasmic domain is ineffective in perturbing the interaction between brain ankyrins and L1, possibly because it exists in a monomeric form.

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Metallothionein gene is transcriptionally regulated by heavy metals through cis-acting metal responsive elements (MREs). Two proteins, metal-regulatory transcription factor-1 (MTF-1) and zinc regulatory factor (ZRF), have been isolated and cloned from human cells as MRE-binding transcriptional factor (MREBT). These proteins are almost identical to each other, except for only one base substitution at codon 185 that causes an amino acid change from histidine to tyrosine. This single amino acid difference has been reported to influence zinc-responsive transcriptional activities. In this study, we determined the nucleotide sequence of the region containing codon 185 in DNA samples obtained from normal Japanese (n = 30) and three human-derived cultured cell lines. The findings indicate that all subjects have the same sequence identical to ZRF, suggesting that ZRF is the original MREBT gene in normal humans, and MTF-1 is its minor variant.

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