Hepatic and renal metallothionein induction in the rat and mouse after treatment with furosemide.

Induction of hepatic and and renal metallothionein by furosemide was studied in the rat and mouse. Treatment of mice with 200 and 300 mg/kg furosemide elevated hepatic metallothionein by 117% and 366%, while renal metallothionein was induced by 29% and 380%, respectively. In the rat the drug was less potent i.e. liver metallothionein was increased by 167% and 217% following injection of 300 and 400 mg/kg furosemide, respectively, whereas kidney was not significantly changed by this treatment. The mouse hepatic and renal metallothionein was identified as zinc-containing thionein by Sephadex G-75 gel filtration (Ve/Vo = 2.0). In both species maximal induction was observed 24 hours post exposure. However, the mouse hepatic and renal metallothionein content declined after additional 24 hours whereas the rat metalloprotein was not reduced even after 72 hours of treatment. It is suggested that alterations in metal homeostasis may be responsible for hepatic and renal metallothionein induction caused by furosemide.     

Effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat

The effects of alterations in sulfur metabolism on hepatic and renal metallothionein and glutathione metabolism were studied in the adult rat using inhibition of two enzymes of these pathways, hepatic cystathionase and renal gamma-glutamyl transpeptidase. Rats were fed a diet containing both methionine (0.66%) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, a selective cystathionase inhibitor, at various doses (2.5–375 μmol/kg). When hepatic cystathionase was inhibited greater than 90% (≥50 μmol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. On the other hand, renal glutathione was increased twofold with a concomitant decrease in renal gamma-glutamyl transpeptidase activity (50% of control). In another experiment, when renal gamma-glutamyl transpeptidase was inhibited greater than 90% with three consecutive daily injections of acivicin, a selective gamma-glutamyl transpeptidase inhibitor (10 mg/kg IP), renal glutathione content was unaltered while hepatic glutathione was decreased. Renal and hepatic metallothionein were not changed. Thus, the cysteine pools for metallothionein and glutathione appear unrelated under the present experimental conditions. In addition, following either propargylglycine or acivicin injections, renal and hepatic glutathione pools appear to be altered differently. These results suggest that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased.     

Effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat.

The effects of alterations in sulfur metabolism on hepatic and renal metallothionein and glutathione metabolism were studied in the adult rat using inhibition of two enzymes of these pathways, hepatic cystathionase and renal gamma-glutamyl transpeptidase. Rats were fed a diet containing both methionine (0.66%) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, a selective cystathionase inhibitor, at various doses (2.5-375 mumol/kg). When hepatic cystathionase was inhibited greater than 90% (greater than or equal to 50 mumol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. On the other hand, renal glutathione was increased two-fold with a concomitant decrease in renal gamma-glutamyl transpeptidase activity (50% of control). In another experiment, when renal gamma-glutamyl transpeptidase was inhibited greater than 90% with three consecutive daily injections of acivicin, a selective gamma-glutamyl transpeptidase inhibitor (10 mg/kg IP), renal glutathione content was unaltered while hepatic glutathione was decreased. Renal and hepatic metallothionein were not changed. Thus, the cysteine pools for metallothionein and glutathione appear unrelated under the present experimental conditions. In addition, following either proparglyglycine or acivicin injections, renal and hepatic glutathione pools appear to be altered differently. These results suggest that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

Laser photo-CIDNP NMR of pyridoxal phosphate and pyridoxyllysine residues: an extrinsic probe for non-aromatic amino acid residues in proteins

The interferon inducing agents, polyriboinosinic: polyribocytidylic acid and tilorone, and Freund's complete adjuvant cause a marked depression of several components of the hepatic mixed-function oxidase system. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation by fluorescence gel scanning of different molecular weight species of cytochrome P-450 indicate that the depressant effect of these agents on the apoprotein moieties of cytochrome P-450 is of a specific nature.     

Protective effect of betulinic acid on Freund's complete adjuvant‐induced arthritis in rats

The purpose of the experiment was to study the effects of betulinic acid (BA) on adjuvant‐induced arthritis in rats. The rat model of rheumatoid arthritis (AA) was established by Freund's complete adjuvant. Arthritis index, joint pathology, toe swelling, hemorheology, related cytokines and ROCK/NF‐κB signaling pathway were measured in rats. BA can significantly inhibit the arthritis index, improve joint pathology, reduce toe swelling, improve blood rheology, improve synovial cell apoptosis, and restore related cytokine negative regulation of ROCK/NF‐κB signaling pathways. BA has an obvious therapeutic effect on joint inflammation of toes in AA model rats, which may be due to the regulation of ROCK/NF‐κB signaling pathway.     

Cd-metallothionein in liver and kidney of goldfish (Carassius auratus): effects of temperature and salinity.

Treatment of goldfish with Cd, by intraperitoneal injection, resulted in Cd-metallothionein (Cd-MT) synthesis mainly in liver and kidney. The relative amount of Cd sequestered by liver metallothionein was always greater in fish maintained at 20 degrees C compared to those reared at 10 degrees C, indicating a temperature dependence of metallothionein biosynthesis; in the kidney this dependence was not so clearly evident. Changes in MT levels induced by adapting fish to different salinities did not correlate with the salinity change.     

Radioimmunoassay of rat liver metallothionein

A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of ¹⁰⁹Cd(II) to a specific activity of 1–2 × 10⁶ cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.     

Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture

Primary cultures of adult rat liver parenchylmal cells, isolated by the collagenase perfusion technique and maintained as a monolayer,l were used to investigate the characteristics of hepatic cadmium accumulation and metabolism. Cadmium accumulation was found to be temperature- and concentration-dependent process that required sulfhydryl groups and was significantly stimulated by the addition of dexamethasome to the medium. Once taken up, cadmium was less available for exit-exchange processes than its biologically required congener, zinc. Moreover, cadmium influx enhanced zinc efflux. While most of the intracellular cadmium was located in the cytosol, its distribution within this fraction was altered with time. Initially the metal was bound to both high molecular weight species (>50 000) and metallothionein. As the incubation period increased, the cytosol concentration of cdmium and the percentage of this metal associated with metallothionein was likewise increased. [³H]Amino acid incorporation studies indicated that the accumulation of cadmium resulted in de novo synthesis of the 1 and 2 forms of metallothionein.     

Failure of cis-platinum to alter metal concentrations in the liver and kidney of the rat

The anticancer drug, cis-diamminedichloroplatinum, was administered to male rats at a dose of 0.5, 1.0, 2.0 or 4.0 mg platinum/kg body weight. After 48 hrs, the animals were killed and platinum, zinc, copper and metallothionein were measured in renal and hepatic tissue and platinum, zinc and copper were measured in the plasma. Administration of this platinum-containing compound did not induce hepatic or renal metallothionein and did not significantly alter the concentrations of copper or zinc in hepatic or renal tissue or in the plasma.     

The identification of soluble adult antigen on the tegumental surface of juvenile Fasciola hepatica.

An antiserum was raised in rabbits against a soluble extract of fresh homogenized adult Fasciola hepatica of rat origin and was then absorbed with rat and mouse tissue antigens. This antiserum reacted specifically with the surface coat of adult flukes, of both rat and mouse origin, by indirect immunofluorescence to show the detail of surface spines. When tested against juvenile stages recovered from mice the reaction was positive with all but the earliest hepatic parenchyma stages. No reaction was present on the tegumental surface of newly excysted juveniles or stages 1 or 2 days post-infection (p.i.) whether recovered from the peritoneal cavity or the hepatic parenchyma.     

Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of cis-dichlorodiammine platinum II

After the administration of the anticancer drug cis-dichlorodiammine platinum II (cisplatin) to male rats, the Pt in the soluble fraction of the kidney is isolated, by gel filtration, in association with a high molecular weight component and a low molecular weight fraction. At 24 h, Pt is also recovered in a metallothionein-like fraction which elutes from Sephadex G-50 with a lower apparent molecular weight than endogenous (Cu, Zn)-thionein or Cd-thionein isolated from the kidneys of Cd2+-treated rats. None of these low molecular weight metal-binding fractions binds to Octyl Sepharose CL-4B. On DE-52 ion exchange chromatography, Cd-thionein is resolved into two isometallothioneins whereas the low molecular weight Pt-binding fraction is only partially purified and contains at least six components which elute at higher gradient concentrations than metallothionein. Pretreatment with Cd2+ which stimulates the synthesis of renal and hepatic metallothionein has no effect on the uptake and subcellular distribution of Pt in the liver and kidneys. Cisplatin treatment reduces the concentration of Cu and Zn in the renal metallothionein and other soluble protein fractions in the kidney. When administered to Cd2+-pretreated rats, cisplatin promotes the loss of Zn from the soluble protein fractions but causes the redistribution of Cd from the metallothionein to the high molecular weight fraction and fails to inhibit the Cd2+-induced accumulation of Cu in the kidneys and the binding of Cu to the soluble protein fractions. It is suggested that metallothionein probably does not have a significant role in the renal metabolism of Pt following the administration of cisplatin to rats.     

The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium

The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 μmoles of ¹⁰⁹CdCl₂/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for about 4 days. The second phase of ¹⁰⁹Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75–80% of the ¹⁰⁹Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein took place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of ³⁵S-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.     

The apparent lack of effect of supplementary dietary zinc on zinc metabolism and metallothionein concentrations in the turbot, Scophthalmus maximus (Linnaeus)

Turbot were fed either a basal diet containing approximately 100 mg zinc kg⁻¹ or a similar diet supplemented with 1000 mg kg⁻¹; the fish were sampled over a period of 220 days. At final sampling there was no significant difference between control and test animals in hepatic and renal metallothionein levels, hepatic and renal zinc levels and white muscle, skin and bone zinc concentrations.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE^−/−) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1β at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes—and not epithelial cells—and was dependent on NLRP3 inflammasome activation and IL1β-signalling. In females, CFA-induced TSLP was independent of IL1β and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR^−/− mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE^−/− and ApoE^−/−/TSLPR^−/− mice with either CFA/IFA or PBS. ApoE^−/− mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE^−/−/TSLPR^−/− mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Summary Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAF-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.Abbreviations MT Metallothionein - PBS Phosphate Buffered Saline - SDS Sodium Dodecyl Sulfat - PAGE Polyacrylamide Gel Electrophoresis Part of the work was reported in Proceedings of 54th Annual General Meeting of the Society of Biological Chemists, India, 1985.     

Developmental changes in hepatic metallothionein, zinc, and copper levels in genetically altered mice.

Using mice that either overexpress metallothionein 1 (MT-1*) or do not express metallothionein 1 and 2 (MT-null) and a control strain (C57BL/6), the essential metal storage function of hepatic metallothionein and its subcellular localization were investigated during development. Hepatic metallothionein, zinc, and copper levels were measured in all groups from gestational day 20 to 60 days of age. Hepatic metallothionein levels were maximal during the perinatal period in both MT-1* and C57BL/6 mice with levels approximately three times higher in MT-1* mice. MT-null mice had no detectable hepatic metallothionein throughout development. Hepatic zinc levels were highest in the neonatal period of MT-1* and C57BL/6 mice and declined to adult levels by 30 days of age, while hepatic zinc levels in MT-null mice did not vary markedly throughout development. Hepatic copper profiles were very similar in MT-1* and MT-null mice as compared with the C57BL/6 mice. Correlation analysis showed a strong positive correlation between hepatic metallothionein and zinc levels in MT-1* mice, moderate correlation between hepatic metallothionein and metals in C57BL/6 mice, but only a very weak correlation between hepatic metallothionein and copper levels in MT-1* mice. Immunohistochemical localization showed specific nuclear staining in both MT-1* and C57BL/6 mice during the neonatal period with a gradual shift to the cytoplasm. The results show that hepatic metallothionein is a major determinant of zinc but not copper levels during murine development. Additionally, hepatic metallothionein levels and localization are regulated in a similar manner in MT-1* and C57BL/6 mice. The MT-null mice maintain a basel level of zinc sufficient for development, which was found to be 15.9 micrograms/g. This value was similar to the levels of hepatic zinc that was not bound to metallothionein in MT-1* and C57BL/6 mice during development.