Quantitative immunohistochemistry of metallothionein in rat placenta

The presence of metallothionein (MT) was demonstrated in placentae from cadmium-exposed and control rats by an immunohistochemical technique, using peroxidase as label and the diaminobenzidine procedure for the staining reaction. The protein was found in different regions of the placenta, i.e. in trophoblastic labyrinth, in spongiotrophoblast and in visceral yolk sac. Cytophotometric analysis of the final reaction product revealed that the amount of MT was increased in the placental labyrinth of cadmium-exposed rats. Increases were found in both nuclei and cytoplasm of trophoblast cells in the labyrinth region. Possible roles of MT in the transport of zinc and in the carbohydrate metabolism are discussed.     

The volume of reinforcement and the structure of rat behavior in a radial labyrinth

In rats choice reaction was elaborated in 12 passages radial labyrinth with different volumes of food only in six of twelve passages. Then the conditions were modified: the first group of rats in the beginning of the experiment was placed to another labyrinth sector, and in the second group the food volume was changed in passages. In new situations in animals of both groups the reaction efficiency temporarily decreased, simultaneously the correction of behaviour structure took place. The character of behavioural tactics (tendencies to visit passages with larger food volume at the beginning of the experiment, spatial-motor asymmetry etc.) is described in dynamics of adaptation to new conditions. Factors determining the rats behaviour structure are discussed.     

Sex and restraint stress differences in rat metallothionein and Zn levels

Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and female rats. Whereas it was found that female rats had higher hepatic MT levels than male rats in basal, unstressed conditions, no significant differences were found in serum MT levels. Restraint stress increased both serum and liver MT in both sexes. The increase in serum MT was greater in male than in female rats, whereas no significant differences between sexes were found in liver MT content after restraint stress. It is suggested that MT regulation might be sex-dependent and that MT might play some extrahepatic function during stress.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Comparison of serotonin and 5-methoxytryptamine immunoreactivity in rat raphe nuclei

We studied the immunoreactivity of 5-methoxytryptamine (MT) and 5-hydroxytryptamine (HT) in the raphe region of rats using specific polyclonal antibodies and the peroxidase/anti-peroxidase (PAP) technique. Overall, the patterns of the specific staining for these two antibodies were found to be the same in this region of the rat brain. The staining reaction was considerably less intense for MT than for HT. Specificity tests were performed using HT, MT and tryptamine (T) conjugates at concentrations of 5 X 10(-8) M for antibodies to HT and 2.5 X 10(-9) M for antibodies to MT. Although the distribution of HT-like and MT-like immunoreactivity broadly overlapped, the results obtained from adsorption-specificity tests confirmed the presence of specific MT staining in the rat raphe.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Localization of metallothionein in the brain of rat and mouse.

Metallothionein (MT) is a low molecular mass protein inducible by heavy metals such as cadmium (Cd), zinc, and copper, and having high affinity for these metals. In the present study, we investigated the immunohistological localization of MT in the brains of rats and mice. In adult rat brain, almost no MT immunostaining was observed, whereas in adult mouse brain strong MT immunostaining was found in the ependymal cells, some glial cells, arachnoid, and pia mater. No immunostaining was detected in neurons and endothelial cells. In younger rats (1-3 weeks old), strong MT immunostaining was observed in ependymal cells, choroid plexus epithelium, arachnoid, and pia mater. The overall MT concentration in adult mouse brain appeared higher than that of the brains of young and adult rats. When adult rats were administered Cd, MT was induced not only in some glial cells, ependymal cells, arachnoid, and pia mater but also in endothelial cells. Although Cd treatment resulted in an increase in the MT immunostaining in the specific cells described above, the MT induction was not great enough to significantly affect the overall MT level in the brain. The present result suggest a possible link of MT with cell growth of choroid plexus epithelium and ependymal cells, as well as a detoxifying role of MT in the blood-brain barrier and the cerebrospinal fluid-brain barrier.     

Comparison of serotonin and 5-methoxytryptamine immunoreactivity in rat raphe nuclei

Summary We studied the immunoreactivity of 5-methoxytryptamine (MT) and 5-hydroxytryptamine (HT) in the raphe region of rats using specific polyclonal antibodies and the peroxidase/anti-peroxidase (PAP) technique. Overall, the patterns of the specific staining for these two antibodies were found to be the same in this region of the rat brain. The staining reaction was considerably less intense for MT than for HT. Specificity tests were performed using HT, MT and tryptamine (T) conjugates at concentrations of 5×10^–8 M for antibodies to HT and 2.5×10^–9 M for antibodies to MT. Although the distribution of HT-like and MT-like immunoreactivity broadly overlapped, the results obtained from adsorption-specificity tests confirmed the presence of specific MT staining in the rat raphe.     

LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH¹ was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.     

海马内微量注射褪黑素的免疫调节及机制研究

本文采用海马内微量注射褪黑素（ｉｈ,ＭＴ）的技术,发现ｉｈＭＴ具有明显的免疫调节作用。进一步发现,ｉｈ ＭＴ能提高海马内去甲肾上腺素水平,通过α２受体,交感神经系统调节免疫功能。本研究还发现,脾功能能产生ＭＴ,脾细胞中可能存在着ＭＴ合成的关键酶。ｉｈＭＴ能通过交感神经系统去甲肾上腺素能神经,促进脾细胞产生ＭＴ,由此诱导β－Ｅｎｄ调节免疫功能,ｉｈＭＴ还能通过海马去甲肾上腺素作用α２受体,反馈抑制松     

Elevation of hepatic levels of metallothionein and zinc in mice bearing experimental tumors.

A tumor growth-dependent elevation in the hepatic levels of Zn and metallothionein (MT), without a change in the level of Cu, was found in mice and rats bearing solid tumors in the inguinal region. The levels of Zn and MT thus elevated gave a significant correlation (r = 0.95) between them. Nevertheless, when tumor-bearing mice and rats were fed a Zn-deficient diet, the hepatic levels of Zn and MT did not increase. In mice in which inflammation was induced at the same region, on the other hand, hepatic levels of Zn and MT increased transiently after the injection of turpentine or carrageenan even when they were fed the Zn-deficient diet. These results suggest that the elevation of MT and Zn levels can be a helpful marker for detecting malignancy.     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ＾－和Ｏ＾－２自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ;同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１０     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ－和Ｏ自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１００倍,清除Ｏ自由基的能力分别是ＧＳＨ和ＳＯＤ的２５和０．０１倍。即ＭＴ是一种很好的ＯＨ自由基清除剂。以ＯＨ对核酸（ＤＮＡ）的损伤为例,研究了ＭＴ对核酸损伤的保护作用,其变化规律与上述结果相一致。     

Inducibility and amounts of metallothionein as influenced by cadmium and zinc in the developing rat

Information on the accumulation and/or depletion of Zn in metallothionein (MT) of rat fetus, rat pup, and maternal rat liver at various ages was obtained with pregnant rats fed a basal casein diet or this diet plus either 100 ppm Zn or 50 ppm Cd. Rats fed each of the respective diets were sacrificed on 12, 16, and 20 d of gestation and 0, 7, 14, and 28 d post-partum. No Cd was detected in the placenta or fetal tissue and the Cd did not affect the accumulation of Zn in the fetal MT, but it did increase the Zn content in liver MT of the dams. Very little Zn in MT was found on day 12 of gestation, but Zn rapidly increased in MT to a maximum at time of birth. The accumulation of Zn in MT was independent of the diet for the fetuses, but the Zn accumulation in the dam and pup tissues was diet dependent. In order to study age-dependent difference in the inducibility of MT, newborn, 5-week-old, or 24-week-old rats were injected with zinc at the levels of 0, 3, 6, or 9 mg/kg and 5 h later injected with³⁵S-cystine. In rats sacrificed 1 h later, the amount of radioactivity in liver MT demonstrated that this protein in older animals was more readily induced by Zn than in younger animals.     

Effects of cadmium exposure on glycogen phosphorylase activity in rat placenta as demonstrated by histochemical means

Glycogen phosphorylase (PHO) activity was demonstrated histochemically in unfixed cryostat sections of placentae from cadmium-treated and control rats with the use of the semipermeable membrane technique. Staining of the newly synthesized glycogen was performed by lugol. A high activity was present in glycogen cells, spongiotrophoblast and visceral yolk sac from cadmium-treated and control animals. A low but distinct activity could be demonstrated in placental labyrinth from control rats in late pregnancy. Cadmium-exposed rats showed a considerably higher activity in the labyrinth during this period of pregnancy. The elevated PHO activity and concomitant higher glycogen content indicate a disturbance by exposure to cadmium of placental carbohydrate metabolism from day 18 onwards.     

Training of rats of the Wistar and Krushinskii-Molodkinaia lines in a radial maze

The experiments were conducted on rats of Wistar (W) and Krushinsky--Molodkina (KM) (with audiogenic epilepsy) lines in 12-rays radial-symmetrical labyrinth. The trained rats of W line made less erroneous (repeated) visits to the labyrinth corridors than the rats of KM line. The corridors repeatedly visited by the rats of W line were more frequently situated near the unvisited corridors. No definite sequence of corridors' visiting by the rats of both lines was observed; still there was a tendency to choose corridors the most remoted from each other. This tendency intensified in the process of training. This is considered as one of tactics of rats' behaviour, providing for adequate reactions in definite surrounding conditions.     

Metallothionein metabolism in the liver and kidney of the streptozotocin-diabetic rat

1. Elevated levels of metallothionein (MT)-I and -II were identified in the liver and kidney of insulin-deficient diabetic rats. 2. The relative rate of MT synthesis and the turnover of cytoplasmic MT were both accelerated in the liver of diabetic rats. 3. The rate of synthesis of MT, but not its cytoplasmic turnover, was increased in diabetic kidney. 4. Maximal relative rates of MT synthesis in liver and kidney were first observed at 4 and 10 days, respectively, after inducing the diabetic condition. 5. The altered metabolism of hepatic MT in diabetic rats was attributed primarily to disturbances in endocrine status, while the altered metabolism of renal MT was largely due to accumulation of excessive dietary copper in the kidney.