Kinetics of cadmium-induced hepatic and renal metallothionein synthesis in the mouse

Probst, Gregory S.; Bousquet, William F.; Miya, Tom S.

doi:10.1016/0041-008x(77)90176-4

Cited by 67 publications

(12 citation statements)

References 22 publications

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“…Liver from mice treated with CdSO4 and ZnSO4 for 6 hr was chosen as the starting material for isolating MT mRNA because the rate of hepatic MT synthesis reaches a maximum 4-6 hr after administration of heavy metals (unpublished observations; ref. 24). Total mRNA was isolated and fractionated on 5-20% sucrose gradients.…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of the mouse metallothionein-I gene.

Durnam¹,

Perrin²,

Gannon³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

180

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Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 38base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The We became interested in studying MT gene regulation because the amount of translatable MT mRNA increases rapidly in response to heavy metals or glucocorticoids (3, 4). MT synthesis occurs predominantly in liver and kidney in vivo, but nearly all cell types are responsive in culture (unpublished observations; refs. 5 and 6). In addition, cell cultures can be made resistant to Cd, and these lines overproduce MT (7, 8). The molecular mechanisms by which heavy metals and steroids regulate MT mRNA production are unknown. Because an understanding of gene regulation depends upon specific probes for RNA and DNA sequences, we have initiated our investigation by isolating a MT-I cDNA clone and using it to select genomic clones containing the MT-I gene.MATERIALS AND METHODS Preparation of RNA. Swiss Webster mice (25 g) were injected subcutaneously with 1.5 ,umol of CdSO4 and 0.5 ,mol of ZnSO4. Six hours later, a 5% (wt/vol) liver homogenate was prepared in NaDodSO4 and proteinase K, and total nucleic acids were extracted. RNA was precipitated with 2 M LiCl; poly(A)-RNA was selected by oligo(dT)-cellulose chromatography and sedimented for 20 hr at 280,000 X g on 12-ml, 5-20% sucrose gradients in 10 mM Hepes (pH 7.5) (9). Fractions (0.5 ml) were collected and assayed directly by translation; those containing MT mRNA were pooled and sedimented on a second, identical gradient. Fractions were assayed for MT mRNA activity with a nuclease-treated rabbit reticulocyte lysate essentially as described (10) except that no exogenous amino acids other than [85S]cysteine (400 Ci/mmol; 1 Ci = 3.7 X 10"0 becquerels) were added and the reaction mixtures contained 5 mM dithiothreitol. After translation, samples (5 ,l) were incubated with 20,Ml of 100 mM iodoacetate in 0.5 M Tris-HCl (pH 8.8) for 1 hr at 37°C, electrophoresed on a NaDodSO4/polyacrylamide gel for 14 hr at 10 mA, and analyzed by fluorography. Purified mouse MT (11) was included as a standard.Preparation of Double-Stranded (ds) cDNA, Insertion into pBR322, and Transformation of X1776. Double-stranded cDNA was prepared (12) and ligated to a 1:1 mixture of HindIII and EcoRI linkers (Collaborative Research, Waltham, MA) prior to being inserted into EcoRI/HindIII-digested pBR322 as described (13). Transformation of E. coli X1776 was performed as described (14) with the following modifications: (i) the 100 mM CaCl2 buffer was replaced by 70 mM MnCl2/30 mM CaCl2/40 mM NaOAc, pH 5.6; (ii) the cells were transformed with plasmid at a concentration of 3 ,ug/ml; and (iii) the transformed cells were incubated at 37°C in L broth f...

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Section: Resultsmentioning

confidence: 99%

Isolation and characterization of the mouse metallothionein-I gene.

Durnam¹,

Perrin²,

Gannon³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

180

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“…There are several independent studies on the kinetics of induction and appearance of cadmiumthionein in the liver Cempel & Webb, 1976;Probst et al, 1977;Shaikh & Smith, 1976). Maximum cadmium-thionein synthesis has been reported (1) to be preceded by a lag period of 2-3 h (Squibb & Cousins, 1974;Webb, 1975), and (2) to continue for approx.…”

Section: Cadmium-thionein Synthesismentioning

confidence: 99%

Induction of cadmium-thionein in isolated rat liver cells

Hidalgo¹,

Koppa²,

Bryan³

1978

Biochemical Journal

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The uptake of cadmium by isolated liver cells was linearly related to the cadmium concentration to which the cells were exposed in the medium. Cadmium-treated cells synthesized proteins de novo with the characteristics of cadmium-thionein induced in the liver ofcadmium-treated animals. Thionein from liver cells incorporated cadmium and[35S]cysteine, had a Vl/Vo (Sephadex G-50) of 1.8-1.9, and was separated into two subfractions by DEAE-cellulose ion-exchange chromatography. Cycloheximide and actinomycin D when added after a cadmium exposure prevented the synthesis ofthionein. However, addition of actinomycin D after synthesis had started only decreased the total amount of thionein synthesized. The concentration of cadmium to which the cells were exposed affected the amount of cadmium-thionein synthesized in 6h. The maximum response occurred when cells were exposed to 0.5,ug of cadmium/ml; at higher metal concentrations the total amount of cadmium-thionein synthesized declined. The system described in the present paper can be used to study the mode of metal toxicity and the mechanism of cadmium-thionein synthesis.

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“…Metallothionein biosynthesis attains its maximum at 4 to 6 hours after Cd administration. It has been found in mice that metallothionein concentration in the liver increases proportionally to cadmium intake (Probst et al 1977). It has been demonstrated that binding to this protein makes cadmium harmless because it can no longer adversely affect cell organelles or enter biochemical processes.…”

Section: Discussionmentioning

confidence: 99%

Distribution of Heavy Metals and Their Ultracytochemical Determination in the Organs of Calves

Horký¹,

Illek²,

Pechová³

1998

Acta Vet. Brno

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Horky D., 1. Illek, A. Pechova: DistributionoJHeavyMetalsandTheirUltracytochemical Determination in the Organs oJCalves. Acta vet. Bmo 1998,67: 51-58.The distribution of Cd, Zn, Se and Cu in the tissues of animals receiving different total doses of these metals for different time periods was studied at the sub microscopical level. In experiment I, 32 calves of both sexes were given heavy metals singly (Cd and Zn) or in combination (Cd, Zn; Cd, Se) from 2 to 14 weeks of age. Experiment II included 21 bulls which received Cd. or Cd and Cu or Cd and Zn in a diet from 3 to 15 months of age. For ultrahistochemical examination, samples of liver. kidney, pancreas. testis or ovary and diaphragm were collected in experiment I and those of liver. kidney. pancreas and testis in experiment II.The ultrahistochemical method used was based on the detection of metal sulphides using a hydroquinone developer with silver nitrate added. The foci of reduced silver on sulphide molecules were identified as dark granules in cell cross-sections.In experiment I, after combined administration of Cd and Zn, the highest amounts of reduced silver granules were seen in the cytoplasm ofhepatocytes and cells of the proximal and distal renal tubules and the lowest amounts were found in glandular cells of the pancreas. Administration of Cd and Se resulted in the presence of large numbers of granules in the nuclei and nucleoli of spermatogonies. In experiment II, ingestion of Cd and Zn in feed led to the appearance of highest amounts of granules in the nucleoli, nuclei and cytoplasm of cells in testes, kidneys and pancreas. Following Cd intake. the highest accumulation of granules was observed in the nucleoli of hepatocytes and cells of the proximal and distal renal tubules. Combined Cd and Cu produced the highest numbers of granules in cells of the proximal and distal renal tubules and in the nucleoli and nuclei of germinal epithelium. Xenobiotics, Cd, Zn, Se and Cu administration, detection. distribution, cellsThe content of heavy metals in and their effects on animal and human tissues have been assessed first by biochemical, histological and histochemical methods and later by techniques working at the ultrastructural and ultrahistochemicallevels. These methods have been able to reveal changes in the morphology, structure, including those at the subcellular level, and biochemistry of tissues and organs and have permitted quantitative assessments of heavy metals in the specimens examined but failed to localize heavy metals in the cell. The circulation of xenobiotics in the environment and some of their effects on living organisms have been reviewed in the monograph by Cibulka et al. (1991). Gradually, histochemical methods for localization of heavy metals in cells have been developed; the first of them, published by Ti mm in 1958, was subsequently modified by several authors (Danscher 1981; Da nscher et al. 1994) to be eventually adapted to suitthe requirements

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Kinetics of cadmium-induced hepatic and renal metallothionein synthesis in the mouse

Cited by 67 publications

References 22 publications

Isolation and characterization of the mouse metallothionein-I gene.

Isolation and characterization of the mouse metallothionein-I gene.

Induction of cadmium-thionein in isolated rat liver cells

Distribution of Heavy Metals and Their Ultracytochemical Determination in the Organs of Calves

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