Noriko Otaki

The isolation of metallothionein and its protective role in cadmium poisoning

Abstract People that have been subjected to cadmium poisoning show marked calcified tissue and kidney disturbances. In rats fed a cadmium-containing, low-calcium-vitamin D-deficient diet, the major portion of the cadmium accumulated in the liver and kidneys. Despite the fact that only a small amount (2.8 ppm) of cadmium completely inhibits the in vitro enzymic 1-hydroxylation reaction of 25-hydroxycholecalciferol, the in vivo 1-hydroxylation proceeded without appreciable inhibition even in the rats loaded with large amounts of oral cadmium. No light-microscopic morphological changes could be found in the kidneys of cadmium-fed rats. Most of the cadmium that accumulated in the kidneys was in a form bound to the protein, metallothionein, and therefore was not toxic to that organ. On the other hand, only 20% of the cadmium present in bone appears to be protein bound. The data strongly suggest that the protective effect of metallothionein in the kidney is serendipitous when involved in cadmium poisoning and that cadmium ion acts directly on bone rather than by an indirect action through a functional disturbance of the kidney.

Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes

In primary cultured adult rat hepatocytes stimulated by epidermal growth factor and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concentrated in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was observed 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggests that primary cultured hepatocytes are suitable for examining the relation between subcellular localization of metallothionein and cell growth.

Prevention by metallothionein of cadmium-induced inhibition of vitamin D activation reaction in kidney

Several observations have indicated preferential accumulation of cadmium in the liver and kidney when animals were administered the metal by either peroral or parenteral route [ 1,2]. Most of the cadmium thus accumulated in the organs exists bound to a low molecular weight protein [3,4] . The cadmium binding protein has several characteristics which suggest the protein to be closely related to metallothionein originally detected in the horse kidney [5] . It is also remarkable that the protein appeared to be induced rapidly in the liver upon cadmium loads [6] . However, the biological significance of those observations has not been established yet. It is generally accepted that vitamin D must be hydroxylated on C-25 position in the liver and subsequently on C-l position in the kidney before it can function on bone and intestine [7]. We found that I-hydroxylation reaction of 25OHDs in chick kidney mitochondria was completely inhibited in the presence of 0.1 mM of cadmium in vitro [S] , but that the I-hydroxylation reaction occurred in vivo even in the animals loaded with large amounts of oral cadmium [ 14,151.

Rabbit Liver Metallothionein. Tentative Amino Acid Sequence of Metallothionein-B

The primary structure of equine renal metallothionein-1B was first reported by Kojima et al. (1). Most recently, Kissling and Kagi (2) have elucidated the amino acid sequence of human hepatic metallothionein-II and compared it to the known structure of the former. Huang et al. (3) have also demonstrated the complete sequence of hepatic metallothionein-I obtained from the liver of mouse administered with cadmium. We report the amino acid sequence of liver metallothionein-B isolated from rabbits exposed to cadmium.

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.

Copper-metallothionein induction in the liver of LEC rats

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.

Blocking effect of anti-mouse interleukin-6 monoclonal antibody and glucocorticoid receptor antagonist, RU38486, on metallothionein-inducing activity of serum from lipopolysaccharide-treated mice

Although there is much evidence to suggest that lipopolysaccharide (LPS)-induced elevation of hepatic metallothionein (MT) contents is mediated by cytokines, the presence of MT-inducing activity in the serum of LPS-treated animals has not been examined. It was found that serum from LPS-treated mice stimulated MT induction in a hepatoma cell culture. The MT-inducing activity in serum was highest 2 h after LPS injection. Tumor necrosis factor and interleukin (IL)-6 levels in the serum were highest 1 and 2 h, respectively, after LPS injection. Anti-mouse IL-6 monoclonal antibody neutralized MT-inducing activity in serum obtained from mice 2 h after LPS injection. The MT-inducing activity in serum was blocked by the glucocorticoid antagonist, RU38486. A similar requirement for glucocorticoid was also observed in an IL-6-stimulated culture. These results show that the LPS-induced elevation of hepatic MT is mediated by IL-6, and the expression of the stimulating activity of IL-6 is dependent on the presence of glucocorticoid.

Metallothionein in Developing Human Brain

Localization of metallothionein (MT) in the developing human brain was investigated by immunohistochemical techniques. Fetal brain at 21 weeks showed no MT expression. In 35-week-old fetuses, glial cells in the gray matter showed MT expression in the nucleus and perinuclear cytoplasm, but glial cells in the white matter showed MT not only in the nucleus and perinuclear cytoplasm but also in glial processes. At 40 weeks, glial cells in gray and white matter expressed immunoreactive MT in the nucleus, cytoplasm and glial processes. Blood vessels were positive for MT. In the infant brain, there were clear differences in glial cells between protoplasmic and fibrous astrocytes; their nuclei, cytoplasm and processes were positive for MT, but the MT-positive glial processes of protoplasmic astrocytes were fine. In the child, typical protoplasmic astrocytes in gray matter and fibrous astrocytes in white matter were observed and their nuclei, cytoplasm and glial processes were positive for MT. Pia mater and blood vessels expressed MT in infants and children.

Localization of metallothionein in aged human brain.

The localization of metallothionein (MT), a small molecular weight heavy metal binding protein in aged human brain, was investigated by immunohistochemical techniques. The amount of MT and heavy metals (Zn, Cu) were also assayed by radioimmunoassay and atomic absorption spectrophotometry, respectively. Immunohistochemically, MT was found in the pia mater, ependymal cells, protoplasmic astrocytes nand glial processes neuropil of the gray matter and fibrous astrocytes of the white matter of the telencephalon, whereas oligodendroglia and microglia did not show any positive immunostaining for MT. Cytoplasm, glial processes and some nuclei of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Vascular feet and adventitia were also positive for MT immunostaining. Moreover, the pia mater, astrocytes and ependyma in the diencephalon, mesencephalon, pons, medulla oblongata and spinal cord showed the same positive immunostaining for MT as the telencephalon. In the cerebellum, Bergmanns glia, protoplasmic astrocytes of the granular layer and fibrous astrocytes of the white matter showed strongly positive immunostaining for MT.

Metallothionein in human seminal plasma.

Metallothionein (MT) concentrations were measured in the seminal plasma of 4 fertile and 35 infertile mbn and in the hydrocele and spermatocele fluids. The relationship between MT content and sperm density, total number of sperm per ejaculate, sperm motility and abnormal form rates, leukocyte count and zinc levels in seminal plasma, as well as the relationship between MT and serum follicle‐stimulating hormone, luteinizing hormone, testosterone, and prolactin were examined. MT was not detected in the hydrocele and spermatocele fluids. MT levels were related to zinc levels and to the leukocyte count in seminal plasma, but there was no correlation between MT and the other factors examined. This study supported previous findings that MT was secreted predominantly from the prostate and induced by inflammation of the prostate gland or seminal vesicles; the findings suggest that MT binds mainly to zinc and is one of the zinc‐binding proteins in seminal plasma.