Hepatic Metallothionein mRNA Research Articles

A comparison of RT-PCR and Northern blot analysis in quantifying metallothionein mRNA levels in killifish exposed to waterborne cadmium

Reverse transcriptase PCR (RT-PCR) was evaluated as an alternative to Northern blot analysis in quantifying levels of metallothionein (MT) mRNA. Killifish were exposed to water-borne cadmium (Cd) at concentrations ranging from 0 ppb to 46 ppb and their levels of hepatic MT mRNA were quantitated by both Northern blot analysis and RT-PCR. Both methods provided comparable results in that low constitutive levels of MT mRNA were observed in fish exposed to clean water, dose-responsiveness was seen in fish exposed to increasing concentrations of Cd, and sensitivities of both techniques were similar in detecting induced levels of MT mRNA in Cd exposed fish. These results suggest that RT-PCR may provide a sensitive and quantitative method to evaluate gene expression in fish from small, non-invasively sampled tissues.

Effect of glutathione depletion on metallothionein synthesis induced by paraquat in mice.

The effect of Glutathione (GSH) depletion on the induction of metallothionein (MT) synthesis by paraquat (PQ) was examined in ICR mice. An increase in hepatic MT level in mice was observed after a single PQ administration. Pretreatment of mice with L-buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced the induction of hepatic and renal MT synthesis by PQ depending on the decreased tissue GSH level. A similar result was obtained by pretreatment with diethylmaleate, a GSH depleting agent. The ratio of hepatic MT-I to MT-II induced by PQ was not changed by BSO pretreatment. An increase in the hepatic MT level in GSH depleted mice was observed from 3 hr to 24 hr after PQ administration. An increase in the hepatic MT-I mRNA level after treatment with PQ was observed prior to hepatic MT induction in BSO pretreated mice. Pretreatment with actinomycin D, an inhibitor of mRNA synthesis, inhibits the PQ-induced increase in hepatic MT and MT-I mRNA levels in BSO pretreated mice. Pretreatment with BSO did not affect the induction of MT synthesis by zinc, cadmium or dexamethasone. Pretreatment with dexamethasone, an anti-inflammatory agent, enhanced the hepatic MT induction by PQ treatment in GSH depleted mice, while dexamethasone reduced the MT induction by turpentine oil, which is known to induce inflammation and hepatic MT synthesis. These findings suggest that GSH depletion enhances the induction of MT synthesis by PQ because of an increase in the transcription rate, and this enhancement of MT synthesis is not due to an inflammatory response caused by PQ.

The toxic milk mouse does have elevated hepatic metallothionein mRNA

The toxic milk mouse does have elevated hepatic metallothionein mRNA

Effect of chlorpromazine pretreatment on cadmium toxicity in the male wistar (WF/NCr) rat

A recent report has indicated that cadmium-induced testicular damage in CF-1 mice can be prevented by pretreatment with calmodulin inhibitors such as chlorpromazine (CPZ), trifluoperazine, or N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7). However, the basis of this tolerance to cadmium is unclear and has not been demonstrated in any species other than mice. Thus, we examined the effects of the calmodulin inhibitor CPZ on cadmium toxicity in male Wistar (WF/NCr) rats. A single sc injection of 25 mumol CdCl2/kg proved nonlethal over 24 h but caused the typical spectrum of testicular lesions and increases in hemoglobin content (as assessed by hemoglobin absorbance in testicular supernatant). Pretreatment with 40 mumol CPZ/kg had no effect on cadmium-induced testicular lesions but did reduce testicular hemoglobin content, while 120 mumol CPZ/kg moderately reduced the severity of testicular lesions and hemoglobin contents. CPZ pretreatment in some cases increased cadmium content in liver and reduced testicular content but had no effect on renal levels. Cadmium treatment markedly increased hepatic and renal metallothionein (MT), a metal-binding protein often associated with tolerance to cadmium. CPZ alone likewise increased hepatic MT and MT mRNA, but did not modify renal MT, renal MT mRNA, or testicular MT mRNA. In contrast to liver and kidney, testicular cadmium-binding protein (TCBP) decreased in rats exposed only to cadmium or to CPZ, while CPZ pretreatment had no further effect on cadmium-induced reductions in TCBP levels. These results indicate that, like mice, CPZ in rats can reduce the testicular toxicity of cadmium as indicated by CPZ-induced reductions in testicular vascular lesions and hemoglobin contents. However, in rats CPZ has a less dramatic effect on such cadmium-induced lesions than in mice. The CPZ-induced stimulation of hepatic MT gene expression or modification of toxicokinetics may both play roles in this acquired tolerance to cadmium.

Differential stimulation of hepatic and brain metallothioneins by ethanol

Administration of ethanol induces the synthesis of hepatic metallothionein and metallothionein mRNA in the liver but not in the brain. Furthermore, ethyl alcohol, methyl alcohol and isopropyl alcohol enhance the synthesis of metallothionein in Chang cells but not in neuroblastoma IMR-32 cells in culture. The results of this study are interpreted to suggest that the mechanisms of synthesis of metallothionein and the utilization of essential metal nutrients in the brain and peripheral tissues are not identical.

Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro

1. 1. The induction of metallothionein (MT) protein by TPA ( O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [ 35S]cysteine incorporation into MT protein. 2. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6–4.1-fold, as assessed by [ 35S]cysteine incorporation, 1.34–2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5–5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36–5.88-fold (dot blot) and 7.4–22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71–15.26-fold in EC3 cells and 2.23–8.43-fold in 2M cells. MT mRNA was 0.54kb, and α-tubulin mRNA was 1.62kb in size on Northern gels. 4. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.

Abundance of hepatic metallothionein mRNA is increased by protein-synthesis inhibitors. Evidence for transcriptional activation and post-transcriptional regulation

Ongoing protein synthesis is a prerequisite in the expression of some genes. We studied the effect of various protein synthesis inhibitors on the expression of the avian metallothionein (MT) gene. Chicken embryonic hepatocytes in culture were exposed to various concentrations of cycloheximide, puromycin and pactamycin. At concentrations which decreased total protein synthesis by about 90% each inhibitor increased MT mRNA accumulation approx. 5-fold at 9 h of incubation. Incubation with puromycin or zinc for 2 h markedly increased the rate of MT gene transcription. Estimates of the half-life of MT mRNA by using actinomycin D suggested for cycloheximide, but not puromycin, decreased the decay rate of MT mRNA. These data suggest the potential for post-transcriptional regulation of the avian MT gene. We conclude that different antibiotics increase the accumulation of hepatocyte MT mRNA by different mechanisms and that the possibility of multiple mechanisms should be considered in other studies of the role of protein synthesis in gene expression.

Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.

Endotoxin induction of murine metallothionein gene expression.

Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (IL-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha). In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-20-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and MT mRNA. In each organ examined, LPS, IL-1 alpha, or IL-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver. IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

Elevation in metallothionein messenger RNA in rat tissues after exposure to X-irradiation

Metallothionein (MT) mRNA levels in tissues were measured in rats following whole-body X-irradiation (2 and 20 Gy). When compared with control rats, the elevation in MT mRNA levels of liver, kidney, and thymus was observed in irradiated rats at 9 or 72 hr after irradiation. However, the elevation in MT mRNA levels was not observed in brain, spleen, lung, heart, and testis. When compared with other tissues, testicular MT mRNA levels in control rats were extremely high, and treatment with X-irradiation produced a slight decrease of testicular MT mRNA levels. Time-course experiments indicated that hepatic and renal MT mRNA reached a maximum at 6 hr after irradiation. In lowdose (2 Gy) irradiated rat, these values were returned to control values by 4 days after irradiation. However, in high-dose (20 Gy) irradiated rat, the values were not decreased to control values. These data indicate that treatment with X-irradiation produces an elevation in MT mRNA in rat tissues.

Structure and Expression of Chicken Metallothionein

The chicken metallothionein (MT) cDNA and gene were cloned, and their nucleotide sequences determined. The cDNA clones encode a cysteine-rich protein of 63 amino acids which shares extensive structural homology with the mammalian MTs. Southern blot analyses of total genomic DNA, and cloned chicken DNA indicated that the MT gene is a unique gene sequence. The chicken MT gene is structurally homologous with the mammalian MT genes; consisting of three exons separated by two intervening sequences. The placement of the intervening sequences in the chicken gene is nearly identical with that in the mammalian MT genes. Levels of hepatic MT mRNA were rapidly induced by metal ions (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide (LPS). MT mRNA was present in low levels in embryonic liver, but was inducible in ovo by injection of metal ions, glucocorticoids or LPS. Hepatic MT mRNA increased to high levels soon after hatching, before decreasing again to the basal levels found in adult liver. Levels of hepatic MT mRNA in hatched chicks were influenced by dietary metals. The results establish that the structure of the MT protein and gene has been highly conserved between birds and mammals, which suggests a functionally important role(s) for this protein.

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.

Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat.

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.

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.

Levels of Metallothionein Messenger RNA in Foetal, Neonatal and Maternal Rat Liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.

Metallothionein mRNA expression in fetal mouse organs

The regulation of metallothionein biosynthesis in mammalian development was investigated by examining organs of 17-day fetal mice for biologically active metallothionein mRNA. Metallothionein was identified in cell-free translation products by migration in polyacrylamide gels and its characteristic elution on Sephadex G-50 columns. Metallothionein constitutes ∼7.5% of [ 35S]cysteine incorporated into polypeptides directed by mRNA from fetal liver, but it is not detectable in mRNA-directed products of fetal kidney, small bowel, heart, or adult liver. Consistent with a fetal-specific role, hepatic metallothionein mRNA content decreases abruptly in newborn mice, becoming undetectable within 12 days.