Induced synthesis of metallothionein by ascorbic acid in mouse liver

The concentration of metallothionein in mouse tissues after administration of ascorbic acid was determined by the Cd-hem method. The concentration of metallothionein in the liver was increased significantly after an intraperitoneal injection of L-ascorbic acid at the dose of 1000 mg/kg. A simultaneous injection of cycloheximide inhibited the increase of the metallothionein concentration. Mortality of mice injected with a lethal dose of cadmium was decreased significantly by the pre-injection of L-ascorbic acid. All of these results indicate that metallothionein is induced in the liver after the administration of ascorbic acid.     

Induction of metallothionein in mouse liver by valproic acid

Intraperitoneal injection of valproic acid (VPA) induces metallothionein (MT) in mouse liver. There was a clear-cut dose-dependency and the maximal amount of MT induced by VPA was about 6 times the basal level. The hepatic MT level reached the peak value at 24 h after VPA injection. Neither Cu nor Zn concentrations showed any significant changes after VPA administration in either the whole liver, or in the mitochondrial or supernatant fractions of the liver homogenate. Gel filtration profiles for the supernatant of the liver homogenate of the VPA-treated mice, however, clearly showed that induced MT was chiefly Zn-thionein. Therefore, Zn molecules necessary for the elevated MT levels seem to originate mainly from cytosolic Zn-containing proteins except for MT.     

Cadmium-induced metallothionein synthesis in the rat liver slice system

In vivo exposure of a rat to cadmium results in elevation of the hepatic metal-binding protein, metallothionein (MT). The present work describes the induction of MT in the in vitro liver slice system. Incubation of rat liver slices with CdCl₂ resulted in a dose-dependent elevation of tissue MT. The cadmium-binding protein was increased by 140 and 220% in the presence of 10 and 20 μ CdCl₂, respectively. A lower level (5 μ ) of the inducer had only a slight effect. A time-course study showed a gradual increase in the MT level following incubation of the liver slices for 4 and 6 hr with 10 μ -cadmium. Characterization of the metal-binding protein by Sephadex G-75 gel filtration revealed that it is composed of MT and also of a high-molecular-weight fraction that might be either a polymerized or aggregated form of MT, or another type of cadmium-binding protein. These findings indicate that the response of the liver slice system to toxic agents is similar to that of the intact animal. These are encouraging results which need to be extended before the system can be introduced into the routine screening of hepatotoxic agents.     

Increased metallothionein in mouse liver, kidneys, and duodenum during lactation.

Lactation-induced increases in cadmium absorption and retention have been demonstrated in mid-lactating mice, but no systematic measurements of endogenous metal-binding protein concentrations during lactation have been reported. Using Cd/hemoglobin radioassay, this study detected significant increases in metallothionein (MT) concentrations in liver (4-fold), kidneys (2-fold), and duodenum (2-fold), but not jejunum, of mouse dams on days 13 and 20 of lactation. These increases occurred in the absence of cadmium exposure and were specific to the lactation period; dams 5 days after weaning showed MT levels that were similar to those of nonpregnant (NP) mice. Similarly, Northern blot analyses of livers from lactating mice demonstrated that MT mRNA concentrations in maternal liver during mid-lactation were 6-fold higher than those observed 5 days after pups were weaned. Gel filtration of final supernatants from the Cd/hemoglobin assay confirmed that the Cd-binding molecule induced during lactation was indeed metallothionein. In addition, chromatographic analyses of cytosols from tissues taken from dams administered small amounts of Cd (66 ng/mouse) showed that the trace amounts of Cd absorbed through the maternal gastrointestinal tract during mid-lactation were also bound to the MT. These results indicate MT induction in mouse dams occurs as a physiological consequence of lactation, requiring no external stimulus. This induced MT participates in binding low levels of dietary cadmium consumed by the dam. During lactation, elevated maternal MT may affect pathways for essential trace metals as well as sequester toxic metals harmful to the neonate. Multiparous humans may have increased risk of accumulating environmental Cd.     

Dose-dependent increases in metallothionein synthesis in the lung and liver of paraquat-treated rats.

Dose-dependent changes in the concentration of metallothionein-1 (MT-1) in rat tissues were determined following subcutaneous administration of paraquat (PQ), a superoxide radical-generating agent. At 24 hr after injection. MT-1 concentrations in the lung increased linearly with PQ dose. Concentrations in the liver increased with dose until a plateau was reached at a dose of 30 mg/kg body wt. In the kidneys, MT-1 concentrations did not increase even at high doses of PQ. Zn was the principal metal bound to MT in the liver. PQ-induced MT-1 synthesis may reflect de novo protein synthesis, since the increase in MT-1 in the liver was reduced by pretreatment of the rats with actinomycin D. Although MT-1 synthesis in the liver was well correlated with lipid peroxidation, as evaluated by measurement of thiobarbituric acid-reactive substances, there was no such correlation in the kidneys. PQ increased the lipid peroxidation in kidneys without any increase in MT-I content. The data suggest that enhanced lipid peroxidation is not necessarily related to the increased MT-1 synthesis caused by radical-generating agents.     

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

Rates of hepatic and renal metallothionein synthesis were estimated by measuring the incorporation of [³H]cysteine into metallothionein prepared from mice at various times following a single intraperitioneal injection of cadmium acetate (2 mg of Cd/kg). Tissue metallothionein concentrations were measured indirectly as a function of the total cadmium-binding capacity of the isolated metallothionein. Maximal incorporation of [³H]cysteine into hepatic metallothionein occurred 6–12 hr following cadmium exposure, while renal metallothionein synthesis was maximal after 3 hr. Incorporation of [³H]cysteine into metallothionein as well as metallothionein concentrations was greater in the liver than in the kidney. It is concluded that the liver is the primary site of cadmium-induced metallothionein synthesis.     

Induction of hepatic metallothionein in mouse liver following administration of chelating agents

Chelating agents commonly used in therapy of heavy metal intoxication alter the levels of essential metals in liver, kidneys, and serum. Induction of metallothionein synthesis in liver occurs following exposure to a variety of chemical and environmental insults and, in some cases, has been attributed to enhanced hepatic uptake of zinc. Therefore, the effect of acute exposure to seven common metal chelators on the concentration of metallothionein in liver was investigated. Adult male Swiss Webster mice were injected intraperitoneally with the chelators and hepatic metallothionein was quantified by the cadmium radioassay. Ethylenediaminetetraacetic acid (EDTA) produced a 5- to 6-fold increase in hepatic metallothionein 24 hr after injection of 0.75 to 3.0 g/kg. No significant increase in hepatic MT was observed until 12 hr following injection of EDTA (1.5 g/kg, ip). Maximal levels were reached between 12 and 48 hr following EDTA injection. Cadmium, a known inducer of hepatic metallothionein, produced a 15-fold increase in the concentration of MT in liver 24 hr following injection. By comparison, 2,3-dimercaptopropanol and diethyldithiocarbamate produced a 9-fold and 13-fold increase in hepatic metallothionein levels, respectively, 24 hr following injection. A 4- to 6-fold increase in metallothionein was observed 24 hr following injection of 2,3-dimercaptosuccinic acid, D,L-penicillamine, diethylenetriaminepentaacetic acid, and EDTA, while nitrilotriacetic acid elevated hepatic metallothionein levels by 2-fold. Alterations in the concentration of hepatic metallothionein by chelators may have implications for their efficacy in the treatment of cadmium intoxication.     

Protein synthesis inhibition in rat liver by the mycotoxin patulin.

Patulin, a carcinogenic mycotoxin, inhibits in vivo and in vitro protein synthesis in rat liver. The in vivo inhibition culminates 5 h after toxin administration and reaches a maximum of 65% with regard to control values; a breakdown of polysomes is associated with the translational blockage. However in in vitro systems, the cellular fractions obtained from patulin-treated rats appear equally as active in protein synthesizing ability and as sensitive to the toxin action as those prepared from control animals. The in vitro inhibition by patulin is dose related. The postmitochondrial system is less sensitive than that functioning with isolated polysomes and pH 5 enzyme. This difference might be due to the presence of soluble factor(s) that counteract patulin action. We propose that the inhibition of protein synthesis by patulin may result from an interaction of the drug with active SH groups at the membrane level (amino acid transport, ion equilibrium) and/or at the cytoplasmic level (enzymes and factors involved in the translational process).     

The inhibition of CYP enzymes in mouse and human liver by pilocarpine.

1. Pilocarpine is a cholinomimetic natural alkaloid. Its interactions with testosterone hydroxylations, coumarin 7-hydroxylase (COH), dimethylnitrosamine N-demethylase (DMNA), pentoxyresorufin O-dealkylase (PROD) and 7-ethoxyresorufin O-deethylase (EROD), which are indicative of the activities of cytochrome P4502A5 (CYP2A5) or 6, 2E1, 2B, 1A, were examined in mouse and human liver microsomes. 2. In mouse liver microsomes the IC50 values of pilocarpine were 6 microM for COH and testosterone 15 alpha-hydroxylase (T15 alpha OH) activities, 4 microM for PROD, approximately 100 microM for DMNA and testosterone 6 beta-hydroxylase (T6 beta OH) activities and > 1 mM for EROD activity. 3. In human liver microsomes, the IC50 value for COH was 6 microM and for DMNA 10 microM; T15 alpha OH and PROD activities were not detectable but T6 beta OH and testosterone 16 beta/2 beta-hydroxylase activities were moderately inhibited (IC50 70 microM). 4. These results suggest that pilocarpine has (i) a high affinity towards phenobarbitone-inducible CYP2A4/5 and CYP2B activities in mouse liver, (ii) a high affinity towards CYP2A6 in human liver microsomes and (iii) a moderate affinity towards CYP3A enzyme(s) in both microsomal preparations. 5. The low IC50 concentrations in vitro indicate potential metabolic interactions between pilocarpine and several P450 enzymes.     

Dietary calcium restriction enhances cadmium-induced metallothionein synthesis in rats.

Experiments were conducted with adult male rats to investigate the effects of dietary calcium (Ca) restriction upon intake and tissue distribution of cadmium (Cd), and Cd-metallothionein (Mt) synthesis. Four groups of animals were fed either a low-Ca, semisynthetic diet (0.1% Ca) or the same diet supplemented with 0.8% Ca (normal diet). The caloric intake was similar in all groups. Two groups (low-Ca and normal diet) were used as controls, and two groups (low-Ca and normal diet) received 100 mg/l Cd (as CdCl2) in drinking water. Cd levels in liver, kidney, spleen and red cells were measured in all animals after 8 weeks of treatment. Concomitantly, Mt levels in plasma, liver and kidney were evaluated by radioimmunoassay. Ca deficiency entailed marked and significant increases in accumulation of Cd and synthesis of Mt in all assayed tissues. It is concluded that dietary Ca restriction, independent of caloric intake, enhances Cd intestinal absorption and tissue accumulation, which is followed by increased tissue Mt synthesis.     

Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein.

Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 gamma-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus.     

Influence of cadmium on zinc distribution in the mouse liver and kidney: role of metallothionein

Male mice of the ICR strain were given 2.5 mg of Cd/kg by a sc injection of CdCl₂ and were sacrificed by bleeding at 0.5, 4, and 10 hr and 1, 6, and 20 days. Cadmium was detected in the liver and kidney at 0.5 hr after the cadmium injection and the metal concentration increased progressively. In the kidney, zinc concentration at 20 days increased significantly in comparison with that of the control group. In the liver, the zinc concentration was not different from that in control mice up to 10 hr, but after 1 day, the concentration increased significantly. The proportion of cadmium in the liver and kidney supernatant increased with time up to 6 days after cadmium injection. The fact that cadmium content in the supernatant fraction increased with time to be more than 90% of the cadmium in the liver and kidney may suggest that the capacity of this fraction for cadmium accumulation increased with time. In the control group, about 78 and 68% of the total zinc content in the liver and kidney were present in the supernatant. After the injection, these values increased with time up to 6 days. At 20 days after the injection, the decrease of the proportion of zinc was synchronous with the decrease of cadmium. The increased zinc in the liver supernatant may be due mainly to the appearance of metallothionein which was induced de novo by cadmium. At 4 hr after the injection, about 63 and 55% of the cadmium in the supernatant of the kidney and liver, respectively, were detected at the region corresponding to the metallothionein fraction as separated on Sephadex G-75. However, at 4 hr, only about 4.6 and 9.3% of the soluble zinc was detected in this fraction of the kidney and liver, respectively. At later stages, the uptake of cadmium into the metallothionein fraction increased to more than 90%. However, this uptake of zinc was about 47 and 31% at 6 days in the liver and kidney. By further purification of the metallothionein using a DEAE cellulose column, two distinct proteins (Peak II and Peak III), metallothioneins, were obtained. These proteins contained Cd and Zn and were homogeneous as judged by the appearance of a single stainable band on polyacrylamide gel disc electrophoresis. However, the ratio of Cd/n was dependent on the length of time after the cadmium injection. At all times, the protein in Peak II occupied much more cadmium and zinc than that in Peak III.     

The effect of captopril on inflammation-induced liver injury in male rats

Abstract

Activation of macrophages-induced by lipopolysaccharide (LPS) is accompanied by the production of reactive oxygen species and inflammatory cytokines to induce hepatic injury. Renin–angiotensin system may play a role in liver damage. In the current study, the effect of captopril on the injury of inflammation-induced liver was investigated. The rats were divided into (1) Control, (2) LPS, and (3–5) LPS as well as 10, 50, or 100?mg/kg captopril for 2 weeks. Captopril decreased NO metabolites, IL-6, and MDA, while it increased CAT, SOD, and total thiol in the liver. Captopril also attenuated AST, ALT, and ALK-P, whereas it increased albumin and total protein in serum. As an ACE inhibitor, captopril improved liver function and attenuated inflammation and oxidative stress induced by LPS injection.     

Prolyl hydroxylase inhibition corrects functional iron deficiency and inflammation-induced anaemia in rats

Background and Purpose

Small-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated.

Experimental Approach

JNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model].

Key Results

Oral administration of JNJ-42905343 to healthy rats increased the gene expression of cytochrome b (DcytB) and divalent metal-ion transporter 1 (DMT1) in the duodenum, and increased plasma EPO. Repeated administration of JNJ-42905343 for 28 days increased blood haemoglobin, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV). The serum iron concentration was increased with low doses (0.3 mg·kg⁻¹) but reduced at high doses (6 mg·kg⁻¹). In PGPS-treated rats, administration of JNJ-42905343 for 28 days corrected FID and anaemia, as reflected by increased blood haemoglobin, MCH and MCV. Increased expression of DcytB and DMT1 genes in the duodenum resulting in increased iron availability was defined as the mechanism for these effects. rhEPO did not affect DcytB and DMT1 and was not effective in PGPS-treated rats.

Conclusions and Implications

PHD inhibition has a beneficial effect on iron metabolism in addition to stimulating the release of EPO. Small-molecule inhibitors of PHD such as JNJ-42905343 represent a mechanism distinct from rhEPO to treat anaemia and FID.     

Ceramide synthase inhibition by fumonisin B1 treatment activates sphingolipid-metabolizing systems in mouse liver.

Sphingolipids are important components of cell structure and cell signaling. Both external and internal stimuli can alter levels of cellular sphingolipids by regulating enzyme activities associated with sphingolipid metabolism. Fumonisin B1, mycotoxin produced by Fusarium verticillioides, is a reportedly specific inhibitor of ceramide synthase. In order to test our hypothesis whether ceramide synthase inhibition by fumonisin B1 alters other sphingolipid-metabolizing enzymes, we investigated the changes in free sphingoid bases and sphingomyelin (SM) and activities of key enzymes for their metabolism, sphingomyelinase (SMase), serine palmitoyltransferase (SPT), and sphingosine kinase (SPHK) in mouse liver. The hepatic free sphingoid bases increased significantly following five daily treatments with fumonisin B1 in mice. The activity of acidic SMase was enhanced by fumonisin B1, accompanied with a decrease in liver SM content. The expression and activities of SPT and SPHK1 in liver increased significantly following fumonisin B1 treatment. Another hepatotoxicant acetaminophen caused liver regeneration similar to fumonisin B1 but did not produce similar effects on liver sphingolipid-metabolizing enzymes, suggesting that activation of sphingolipid metabolism was not a consequence of hepatocyte regeneration. Data suggest that ceramide synthase inhibition by fumonisin B1 treatment stimulates sphingolipid-metabolizing systems to maintain a balance of cellular sphingolipids.     

Nitrofurantoin inhibition of mouse liver mitochondrial respiration involving NAD-linked substrates

In our study, nitrofurantoin (NF) and nitrofurazone (NZ) inhibited respiration of isolated mouse (C57B/6J, adult, male) liver mitochondria. Other aromatic nitro compounds, nitroimidazole, metronidazole, and p-nitrobenzoic acid, did not have any significant effect. The primary site of activity for NF was complex I NADH-ubiquinone oxidoreductase mediated respiration, since only complex I substrates, glutamate, beta-hydroxybutyrate, and alpha-ketoglutarate-mediated respiration were decreased. Respiration supported by succinate, a complex II substrate, was not affected by any of the compounds. NF at a concentration of 50 microM decreased state 3 and dinitrophenol-uncoupled respiration to 28 +/- 1 and 25 +/- 5% of control, respectively, of mitochondria oxidizing glutamate. Studies with mitoplasts oxidizing glutamate showed that NF inhibited both state 3 and 4 respiration. The inhibition of state 3 was prevented by the simultaneous addition of superoxide dismutase (240 micrograms/ml) and catalase (200 micrograms/ml). These results suggest that the mitochondrion, in particular complex I of the electron transport system, is a target for NF toxicity. The effect on respiration may be mediated by NF redox cycling and the generation of reactive oxygen intermediates resulting in the interference of electron flow.     

Cadmium-induced inhibition of hepatic drug oxidation in the rat: Time dependency of tolerance development and metallothionein synthesis

Experiments were undertaken to determine if a correlation existed between the time-dependent synthesis of hepatic metallothionein and the development of tolerance to the inhibition of hepatic drug oxidation elicited by cadmium in the male rat. Maximal rates of metallothionein synthesis were achieved within 2 hr after administering cadmium in a 0.21 mg Cd/kg (ip) dose. The total hepatic concentration of metallothionein, as well as the cadmium binding capacity of this protein, also increased rapidly with maximal values being observed from 8 to 67 hr after cadmium administration. Despite this rapid increase in hepatic metallothionein levels, the tolerance to the inhibition of in vivo drug oxidation induced by a challenge cadmium (0.84 mg Cd/kg) dose did not develop until 16 hr after treatment with the 0.21 mg/kg cadmium dose. Furthermore, hepatic metallothionein levels decreased from a maximum at 67 hr to approximately control levels at 336 hr. Although the tolerance to the inhibition of drug oxidation also decreased from a maximum at 72 hr a modest degree of protection was maintained even at 336 hr. Pretreatment with the 0.21 mg Cd/kg dose increased the hepatic uptake of a ¹⁰⁹Cd challenge dose (0.84 mg Cd/kg). This increase was associated with an increased ¹⁰⁹Cd binding to metallothionein in the cadmium-pretreated animals. While these data are suggestive of a role for metallothionein in tolerance development, the lack of correlation of the time course of metallothionein synthesis with the development of the tolerance would suggest that factors in addition to metallothionein may also participate.     

Cadmium hypersusceptibility in the C3H mouse liver: cell specificity and possible role of metallothionein

The possible involvement of metallothionein (MT) gene expression dysfunction was examined in a strain of mouse which is unusually sensitive to cadmium toxicity, the C3H. C3H mice, and the relatively cadmium-insensitive Swiss mice, were injected sc with 20 microM CdCl2/kg body wt. This dose caused liver damage, visible at the light microscopic level, in the C3H but not the Swiss mice. These studies showed that MT-I mRNA and MT protein accumulation, as well as binding of cadmium by MT, were very similar in the two strains. These data suggested that altered expression of MT in the hepatic parenchyma was not a factor in the C3H hypersusceptibility. An electron microscopic examination of the early effects of cadmium injection indicated that the primary targets for toxicity in the C3H liver may be the endothelial cells. It is hypothesized that the widespread damage seen at later times resulted, secondarily, from ischemia produced in response to endothelial cell damage.     

Pancreatic metallothionein: protection against cadmium-induced inhibition of insulin secretory activity.

Mouse liver and pancreatic concentrations of metallothionein were determined by gel filtration at several intervals after the ip administration of cadmium acetate. Hepatic concentrations of the protein reached peak values 48 hr after the injection, whereas the pancreatic concentrations were highest at 6 hr. The exposure of mouse isolated pancreatic islets to a concentration of 1 × 10^?6m cadmium significantly reduced glucose-stimulated insulin secretion. When mice were administered cadmium 6 hr prior to islet isolation, the time required for peak concentrations of metallothionein to develop in the pancreas, subsequent exposure to the metal did not reduce glucose-stimulated insulin secretion. The results suggest that the pancreas synthesizes metallothionein and that the protein serves a protective role against the acute effects of cadmium.     

Glucocorticoid inhibition of fibroblast contraction of collagen gels

When skin fibroblasts are grown in culture on collagen gels, the collagen gels contract. We have studied the effects of various steroids on the contraction process. Cortisol, beta-estradiol and dexamethasone inhibited fibroblast-mediated gel contraction at low (10(-8) to 10(-9) M) concentrations whereas dihydrotestosterone was without effect. These effects were time and concentration dependent and could be reversed if the steroids were removed. This system may be useful for assaying the activities of various steroids in terms of their activities and modulating effects on connective tissue.