Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity

Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-³²P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10⁻⁶ M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d -[³H]aspartate uptake. Preexposure of astrocytes to CdCl₂, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d -[³H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl₂ treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl₂ pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.     

Curcumin ameliorates diclofenac sodium‐induced nephrotoxicity in male albino rats

Exposure to drugs often results in toxicity in the kidney which represents the major control system maintaining homeostasis of the body and thus is especially susceptible to xenobiotics. Nephrotoxicity is a life‐threatening side‐effect of nonsteroidal anti‐inflammatory drugs (NSAIDs). Diclofenac is one of the most frequently prescribed NSAIDs and have been reported to cause multiple organs damage. Curcumin (CUR) exhibits nephroprotective properties. Therefore, rats were divided into four groups; rats of groups 3 and 4 received diclofenac (100 mg/kg, i.m.), whereas rats of groups 2 and 4 received CUR (100 mg/kg, p.o.) for 3 days. Diclofenac revealed a significant increase in urea and creatinine levels and malondialdehyde concentration and marked reduction in catalase activity and reduced glutathione concentration. Histopathologically, diclofenac produced fatty changes and eosinophilic casts were detected in the renal tubules, those were attenuated by administration of CUR prior diclofenac.     

Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.     

Induction of metallothionein by superantigenic bacterial exotoxin: probable involvement of the immune system

Metallothionein (MT) can be induced in mouse liver by a bacterial exotoxin, toxic shock syndrome toxin-1 (TSST-1). Hepatic MT was induced by TSST-1 in a dose-dependent manner from 100 μg/kg through 3 mg/kg in CF-1 mice, and by 6 h the induction was almost maximal. The increase of hepatic MT occurred at the mRNA level, also, and both MT-I and II mRNAs increased coordinately. Because TSST-1 is a superantigen, it was investigated whether TSST-1 induces MT through cytokines as a consequences of immunostimulation. In low-cytokine-producing mice (C3H/HeJ), up to a dose of 1 mg/kg of TSST-1, there was only 2- to 3-fold increase of hepatic MT. In contrast, in normal-cytokine-producing mice (C3Heb/FeJ), TSST-1 increased MT in a dose-dependent manner, and at a dose of 1 mg/kg, there was a 25-fold increase in hepatic MT. This suggests that activation of the immune system is probably involved in the induction of MT by TSST-1. Studies on the role of specific hepatic cytokines (IL-1, TNF-α, and IL-6) in TSST-mediated hepatic MT induction showed that TSST-1 did not increase hepatic IL-1 or TNF-α significantly over controls in any of the mouse strains studied. In contrast, TSST-1 induced hepatic IL-6 in all three strains of mice. However, in CF-1 and C3Heb/FeJ mice (normal-cytokine-producing) IL-6 induction preceded MT mRNA induction, but in C3H/HeJ mice (low-cytokine-producing), IL-6 induction did not precede MT and mRNA induction.     

Quantitative gait analysis as a method to assess mechanical hyperalgesia modulated by disease-modifying antirheumatoid drugs in the adjuvant-induced arthritic rat

In the present study, azothioprine, chloroquine, D-penicillamine, methotrexate and sodium aurothiomalate (gold salt) were evaluated for possible disease-modifying effects in the adjuvant-induced arthritis model of human rheumatoid arthritis in rats. Gait analysis was used to examine the role of disease-modifying antirheumatic drugs in the development of pain. Body weights were also measured to monitor the progression of disease and the systemic antiarthritic effects of the test compounds used in this study, as well as their systemic toxicity. Our results showed that azothioprine (5 mg/kg/day), chloroquine (12.5 mg/kg/day), sodium aurothiomalate (2.5 mg/kg/day) and methotrexate (1 mg/kg/week) not only inhibited the macroscopic changes such as erythema and swelling of limbs, but also exhibited significant reversal of gait deficits seen in the untreated or saline-treated arthritic rats. No reduction in the body weights were observed in the arthritic rats treated with azothioprine, chloroquine, sodium aurothiomalate and methotrexate. D-Penicillamine (12.5 mg/kg/day), however, showed a significant reduction (P < 0.03) in the body weights of the arthritic rats over a period of 22 days; furthermore, it was unable to show any reduction in arthritic score (P < 0.1). In earlier experiments, chloroquine and methotrexate failed to suppress carageenan-induced edema, suggesting that the mode of antiarthritic action may be different from those of nonsteroidal anti-inflammatory agents. Since these disease-modifying antirheumatic drugs are reported to have an immunomodulatory role, especially the gold salt, which influences the monocyte–macrophage system, it is suggested that the observed antiarthritic effects of disease-modifying antirheumatic drugs may be partly attributed to their immunomodulatory activity.     

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation. © 1996 Wiley-Liss, Inc.     

Enhanced lipid peroxidation is not necessary for induction of metallothionein-I by oxidative stress

A study has been made of factors which may influence the induction of metallothionein-I (MT-I) synthesis by the superoxide radical generating agent, paraquat (PQ). Hepatic concentrations of zinc (Zn) and MT-I increased in rats injected with PQ (40 mg/kg, s.c.) or fasting, but were greater in the former. Renal concentration of MT-I increased in fasted rats but not in PQ-treated rats. The data suggest that the increase in MT-I concentrations in PQ-treated rats is not caused by reduction in food intake. Administration of PQ increased hepatic concentrations of Zn, MT-I and thiobarbituric acid-reactive substances (TBA-RS), indicating the occurrence of lipid peroxidation. Treatment of rats with vitamin E (400 mg/kg, s.c.) on 4 successive days before injection of PQ prevented only the enhancement of lipid peroxidation. The data indicate that the induction of MT synthesis by PQ is not correlated with enhancement of lipid peroxidation. Similar results were obtained in the liver of rats subjected to the radical-generating conditions, such as fasting and exposure to carbon tetrachloride. Free radicals may induce MT synthesis by direct or indirect mechanisms.     

Different patterns in the induction of metallothionein mRNA synthesis among isoforms after acute ethanol administration

The induction of metallothionein (MT) isoform synthesis was investigated in mouse cerebral cortex 18 h after oral ethanol administration. The expression of MT-I isoform mRNA increased in a dose-dependent manner after ethanol loading at doses between 2 g/kg (ethanol/body weight) and 8 g/kg. Lipid peroxide formation, measured as the amount of malondialdehyde-reactive substances, remained at the control level after all of the administered ethanol doses. The expression of MT-III isoform mRNA remained at the control level up until an ethanol loading dose of 4 g/kg and then finally increased to a significant level at a dose of 8 g/kg, which is almost the LD₅₀ for oral ethanol in mice. The different patterns of MT synthesis induction among MT isoforms suggests that the MT-I isoform, which is ubiquitous in mammalian tissues, plays a significant role as an antioxidant. On the other hand, the MT-III isoform, which has a limited tissue distribution, especially in the central nervous system, seems to be implicated in tissue repair and/or protection against critical tissue injury.     

Pharmacological analysis of the antiinflammatory effects of low-intensity extremely-high-frequency electromagnetic radiation

The antiinflammatory effect of low-intensity extremely-high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.1 mW/cm²) was studied in comparison to the effects of the antiinflammatory drug sodium diclofenac and the antihistamine clemastine in acute inflammatory reaction in mice of NMRI outbred stock. The local inflammatory reaction was induced by intraplantar injection of zymosan to the left hind paw. Intraperitoneal injections of 2, 3, 5, 10, and 20 mg/kg of sodium diclofenac or 0.02, 0.1, 0.2, 0.4, and 0.6 mg/kg of clemastine were made 30 min after the initiation of inflammation. An hour after the initiation of inflammation, animals were whole-body exposed to EHF EMR for 20 min. The inflammatory reaction was assessed 3–8 h after initiation by measuring the footpad edema and hyperthermia of the inflamed paw. Sodium diclofenac (5–20 mg/kg) reduced the exudative edema by ～26% compared to the control. Hyperthermia of the inflamed paw decreased by 60% with an increase in the diclofenac dose to 20 mg/kg. EHF EMR reduced both the footpad edema and hyperthermia by ～20%. This was comparable to the effect of a single therapeutic dose of diclofenac (3–5 mg/kg). The combination of diclofenac and exposure to EHF EMR produced a partial additive effect. Clemastine (0.02–0.4 mg/kg) did not affect the exudative edema, but at a dose of 0.6 mg/kg, edema was reduced by 14–22% five to eight hours after zymosan injection. Clemastine caused a dose-dependent increase in hyperthermia of inflamed paw at doses 0.02–0.2 mg/kg and did not affect the hyperthermia at doses 0.4 and 0.6 mg/kg. A combination of clemastine and EHF EMR exposure resulted in a dose-dependent abolishment of the antiinflammatory effect of EHF EMR. Our results suggest that both arachidonic acid metabolites and histamine are involved in the achievement of the antiinflammatory effects of low-intensity EHF EMR.     

Cadmium-induced elevation of hepatic isometallothionein concentrations in inbred strains of mice

Susceptibility to Cd toxicity differs among inbred strains of mice. For example, C3H/He mice are sensitive to Cd-induced hepatotoxicity while DBA/2 mice are resistant. Metallothionein (MT), which in rodents exists predominantly as two isoproteins (MT-I and MT-II), is an important endogenous protein in the detoxication of Cd. The present investigation examines the possibility that strain-dependent susceptibility to Cd-induced liver injury is mediated by an inherited inability to accumulate a specific isoform of MT in response to Cd exposure. Hepatic concentrations of MT-I and MT-II were measured in C3H/He (Cd-sensitive) and DBA/2 (Cd-resistant) mice at various times after the administration of non-toxic (2.5 mumol Cd/kg) to hepatototoxic (80 mumol Cd/kg) dosages of Cd. The concentration of MT-I and MT-II in these strains was similar 24 h after injection of non-hepatotoxic dosages of Cd (10 mumol Cd/kg or less) as well as 6-12 h after a mildly hepatotoxic dose of Cd (20 mumol Cd/kg). The concentration of total MT in liver of Cd-sensitive mice was greater than that present in resistant mice 24-72 h after 20 mumol Cd/kg injection. The data indicates that susceptibility to Cd-induced hepatotoxicity observed in C3H/He mice is not due to a deficit in the induction of a particular isoform of MT.     

Effect of nitric oxide synthesis inhibition on mouse liver and brain metallothionein expression

The role of nitric oxide (NO) production on metallothionein (MT) regulation in the liver and the brain has been studied in mice by means of the administration of nitric oxide synthase (NOS) inhibitors. Mice injected with either the arginine analog N^G-monomethyl-L-arginine (L-NMMA) or the heme binding compound 7-nitro indazole (7-NI) showed consistently increased liver MT-I mRNA and MT-I+II total protein levels, suggesting that NO is involved in the hepatic MT regulation. In agreement with the liver results, in situ hybridization analysis demonstrated a significant upregulation of the brain MT-I isoform in areas such as the cerebrum cortex, neuronal CA1-CA3 layers and dentate gyrus of the hippocampus, and Purkinje cell layer of the cerebellum, in 7-NI treated mice. The same trend was observed for the brain specific isoform, MT-III, but to a much lower extent. The effect of NOS inhibition was also evaluated in a MT-inducing condition, namely during immobilization stress. In both the liver and the brain, stress upregulated the MT-I isoform, and 7-NI significantly reduced or even blunted the MT-I response to stress, suggesting a mediating role of NO on MT-I regulation during stress. Stress also increased the MT-III mRNA levels in some brain areas, an effect blunted by the concomitant administration of 7-NI, which in some areas even decreased MT-III mRNA levels below the saline injected mice. Results in primary culture of neurons and astrocytes demonstrate significant effects of the NOS inhibitors in some experimental conditions. The present results suggest that NO may have some role on MT regulation in both the liver and the brain.     

Secretoneurin in the human aqueous humor and the absence of an effect of frequently used eye drops on the levels

Secretoneurin (SN) was detected in the aqueous humor (AH) of patients treated topically with tobramycine eye drops alone or tobramycine and cyclosporine A, tobramycine and diclofenac or tobramycine and rimexolone. The levels of the peptide were found to be higher in the uninflamed human than in the rabbit aqueous humor which may be the result of species differences and/or age-related circumstances. Furthermore, they are approximately one hundred times higher than those of classical neuropeptides indicating release from nerve fibers and/or secretion from non-pigmented ciliary epithelium cells. Despite a slight tendency by rimexolone to decrease the levels, there was no significant effect seen for either of the drops. It must be considered that aminoglycosides are known to have toxic side effects and that they can influence the levels of SN which may be not diminished by low topical doses of corticosteroids or nonsteroidal antiinflammatory drugs. The high levels of the peptide are of relevance and may indicate a significant role of secretoneurin in the anterior segment of the eye. This should encourage performing functional studies.     

Glucocorticoid regulation of metallothionein during murine development

During the second half of gestation in the mouse there is a rise in both fetal (4-fold) and maternal (10-fold) metallothionein-I (MT-I) mRNA in the liver (but not in the kidney). There is a large increase in plasma corticosterone (the predominant murine glucocorticoid hormone), as well as an increase in hepatic zinc, which is coincident with the induction of MT-I mRNA. Considering that both of these compounds are known to be effective inducers of MT-I mRNA, we set out to determine whether either one or both were involved in the developmental regulation of MT-I genes. Several lines of evidence suggest that corticosterone is the principal inducer of fetal MT-I mRNA: The induction of MT-I mRNA in the liver, but not in the kidney, mimics glucocorticoid regulation but not metal regulation. Reduction of maternal corticosterone levels by treating mice with metyrapone lowered MT-I mRNA levels but had no effect on zinc levels. A line of transgenic mice carrying a metallothionein-growth hormone fusion gene that is responsive to metals but unresponsive to glucocorticoids was not developmentally regulated. Based on these observations, we propose that corticosterone is responsible for the induction of MT-I mRNA and that the resulting MT sequesters zinc and copper which may be used later in development.