Noncooperative cadmium(II) binding to human metallothionein 1a

The two-domain (βα) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain βα-rhMT and the isolated β-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd₃-β-rhMT and Cd₇-βα-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd₄-β- and Cd₈-βα-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.     

Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes.

An isolated rat hepatocyte preparation was used to study the cellular toxicity of cadmium and the protective effects of metallothionein on cadmium-induced toxicity. Exposure of primary suspension cultures of isolated rat hepatocytes to Cd2+ (0-35.7 microM) for 15 min resulted in a dose-dependent reduction in the synthesis of cellular proteins during a subsequent 6 h incubation. Such inhibition could not be correlated with cellular lethality or gross membrane damage. Pre-induction of metallothionein in hepatocytes by zinc treatment in vivo of donor rats protected hepatocytes in vitro from cadmium-induced inhibition of protein synthesis. The protective effects in zinc-pre-induced hepatocytes are not due to alterations in the level of total cellular cadmium, but could be accounted for by the redistribution of intracellular cadmium in the presence of high levels of zinc-metallothionein. The data suggest that metallothionein exerts its protective effect by a kinetic detoxification mechanism, i.e. a decrease in reactive intracellular cadmium.     

Studies on isolated cloned populations from irradiated human embryonic cell cultures.

The recovery of substrains with stable chromosome aberrations from irradiated fibroblast culture are reported. Four human fetal cell strains were exposed to 600 rad of gamma rays at 200 rad/min. The efficiency of recovering viable cloned subpopulations was approximately 87%, and the frequency of clones with abnormal chromosomes was 40/100 colonies. G-band chromosome analyses for 34 abnormal substrains are described. Karyotypes of some of the clones with complex rearrangements are also presented. Analyses of a total of 47 aberrant events in the 34 abnormal substrains revealed at 7:1 and a 9:1 translocation-inversion and translocation-deletion ratios, respectively. Five of the abnormal substrains were continuously cultured; all except one showed signs of sensecence toward the end of 44 +/- 10 doublings. Unusual prolonged proliferation capacity was observed in substrain FFS-1-9. The significance of this finding is discussed.     

Induction of metallothionein in a human trophoblast cell line by cadmium and zinc

The induction of metallothionein (MT) in a cell line derived from a malignant trophoblastic tumor (JAr cells) was demonstrated using the Cd/heme radioassay following exposure to Cd or Zn. Cd at an optimal concentration of 1 microM produced a 30-fold increase in MT following a 24 hr incubation. Induction by Cd was both time and dose dependent, with a significant increase in MT noted as early as 3 hr, with levels continuing to increase up to 24 hr. Zn was also quite effective in inducing MT synthesis in this cell line. Exposure to 80 microM Zn for 24 hr produced a 70-fold increase in MT. Although Cd was a more potent inducer of MT, exposure to Zn resulted in a greater magnitude of induction. The magnitude of MT induction in JAr cells was much greater than that seen in cultured trophoblasts from term chorion laeve. The degree of induction seen in this cell line makes it an interesting model for the study of MT's role in trophoblast function. MT induction in trophoblasts may reflect a protective mechanism against heavy metal toxicity and/or an integral aspect of normal zinc homeostasis.     

Isolation and partial characterization of metallothionein in the liver of the red-eared turtle (Trachemys scripta) following intreperitoneal administration of cadmium

Cadmium was distributed predominantly in the liver (42% of the body burden) after intraperitoneal injection with 10 mg Cd/kg/day for 6 days, although the kidney, spleen, heart, gonads and shell also contained substantial amounts of the metal. The major cadmium-containing fraction of the liver cytosol eluted in the position of mammalian metallothionein on a Sephadex G-75 column and was further resolved into two isoforms by reverse-phase HPLC. The two isoforms had high cysteine contents (17–22 residues/molecule) and lacked aromatic amino acids, a composition similar to that of other vertebrate metallothioneins. The turtle metal-binding protein had other properties characteristic of vertebrate metallothioneins including heat stability (85°C for 10 min), a relatively high absorbance at 245 nm, a low absorbance at 280 nm and a high metal content (approximately 6 nmol cadmium/nmol protein).     

Infectivity of Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum (Trematoda: Diplostomidae) cercariae following exposure to cadmium

The effect of cadmium (Cd) exposure on infectivity of cercariae of 2 trematode species (Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum) to their second intermediate fish host was evaluated. Individual fathead minnows (Pimephales promelas) were exposed to cercariae that had been treated with Cd solutions at concentrations of 0, 2, 20, or 200 microg/L for 2 hr. Two weeks later, the numbers of encysted metacercariae in the brain (O. ptychocheilus) and body cavity (P. minimum) of the fish were evaluated. ANOVA analyses indicated a strong negative effect of Cd concentration on cercaria infectivity. The species x Cd concentration interaction was not significant, indicating that the magnitude of Cd-induced reduction in infectivity was similar between O. ptychocheilus and P. minimum. The results show that short-term exposure to Cd, even at low concentrations, interferes with transmission processes that affect the recognition and penetration of cercariae, the migration and survival of metacercariae within the second intermediate host, or both.     

Subcellular distribution of metallothionein and cadmium in the liver and kidneys of bank voles (Clethrionomys glareolus) exposed to dietary cadmium

Metallothionein (MT) and cadmium (Cd) contents were determined in the subcellular fractions of the liver and kidneys of bank voles exposed for 6 weeks to elevated levels of dietary Cd-40 and 80 g g^-1 dry weight. Hepatic and renal MT was detected exclusively in the cytosol, while Cd was found in the cytosol (73–79% of the total content), nuclei (14–18%) and particulates (4–9%). The concentration of MT in the cytosol as well as Cd content in the particular subcellular fractions appeared to be a dose-dependent. The absence of MT in the nuclear and particulate fractions implied that Cd present in these compartments was not bound to the protein that is considered to provide protection against the toxic metal. Therefore, it is assumed that this component of intracellular Cd could be responsible for the histopathological changes that occurred in the liver (granuloma and focal hepatocyte swelling) and kidneys (focal degeneration of proximal tubules) of bank voles exposed to the higher level of dietary Cd.     

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure

Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtail Orchesella cincta by determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0 μmol Cd/dry food. Cadmium is primarily located within the gut of O. cincta and isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115 μg metallothionein/g fresh gut. The O. cincta metallothionein gives the possibility of using this protein as a biomarker for heavy metal exposure in soil insects.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.     

Methylation of the alphafetoprotein gene in cell populations isolated from rat livers during carcinogenesis.

We examined the methylation pattern and organization of the AFP gene in whole livers and in isolated cell populations purified from livers of rats fed a carcinogenic diet which interferes with DNA methylation. Using restriction endonuclease digestion, we find no differences in methylation pattern and overall organization of the AFP gene in oval cells (AFP-producers) and hepatocytes (non-producers) isolated at the early stages of carcinogenesis. Our studies indicate that in cell populations which produce AFP as well as in cells which are not active in AFP synthesis, the majority of the CCGG sites of the AFP gene are extensively methylated. In addition, we describe the existence of polymorphism in the AFP and albumin genes of Sprague-Dawley rats.