MT-I mRNA Levels Research Articles

Metallothionein (MT) -I and MT-II Expression Are Induced and Cause Zinc Sequestration in the Liver after Brain Injury

Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II−/−) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II−/− mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver. Conclusion: MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.

Chromium (VI) inhibits mouse metallothionein-I gene transcription by modifying the transcription potential of the co-activator p300

The production of the heavy metal-binding proteins, the metallothioneins (MTs), is induced by heavy metals such as Zn, Cd, and Hg. MTs maintain Zn homeostasis and attenuate heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. Previously, we had reported that Zn induced the formation of a co-activator complex containing metal response element-binding transcription factor-1 (MTF-1) and the histone acetyltransferase (HAT), p300, which plays an essential role in the activation of MT-1 transcription. In addition, we had shown that Cr(VI) inhibits Zn-induced MT-1 transcription by preventing the Zn-dependent formation of the MTF-1-p300 complex. In the current study, we have shown that the inhibition by Cr(VI) was partially overcome by the overexpression of p300 or MTF-1 in an MT-I promoter-driven luciferase reporter assay system and have used real-time RT-PCR to determine MT-I mRNA levels. It has been reported that Cr(VI) inhibits CYP1A1 transcription by crosslinking histone deacetylase (HDAC) to the promoter. The crosslink inhibits the recruitment of p300 to the MT-1 promoter and blocks HAT-dependent transactivation by p300. However, our results demonstrate that trichostatin A, an HDAC inhibitor, could not block the inhibitory effects of Cr(VI) on MT-1 transcription and that there were no significant differences in the in vitro inhibitory effects of Cr(VI), Cr(III), and Zn on p300 HAT activity. This suggests that the inhibitory effects of Cr(VI) on MT-I transcription may be due to its effects on the HAT-independent transactivation ability rather than the HAT-dependent, HDAC release-related transactivation ability of p300.

Attenuation of cadmium-induced testicular injury in metallothionein-III null mice

Aims In order to evaluate the role of metallothionein (MT)-III in cadmium (Cd)-induced testicular toxicity, we examined the sensitivity of MT-III null mice to severe testicular injury caused by Cd. Main methods Male MT-III null mice, MT-I/II null mice and wild-type mice were given a subcutaneous injection of CdCl 2 (15 μmol/kg). The testis was collected from each mouse at 6, 12 and 24 h after Cd administration. Key findings Testicular hemorrhages by evaluating morphology, hemoglobin content and histological parameters in the 3 types of mice were elevated by Cd injection in a time-dependent manner. The degree of hemorrhage in Cd-injected MT-I/II null mice was similar to that in the wild-type mice. In contrast, hemorrhage in the MT-III null mice was attenuated compared with that in wild-type mice and MT-I/II null mice. Cd levels, MT-I and MT-II mRNA levels and Cd-binding molecules in the testis were similar between MT-III null mice and wild-type mice. In microarray analysis, high expression of purine-nucleoside phosphorylase 2 (Pnp2), retinal degeneration 3 (Rd3), and cadherin-like 24 (Cdh24) was revealed in the testis of MT-III null mice under normal or Cd-treated conditions. Significance MT-III null mice were found to show attenuation of Cd-induced severe testicular toxicity. These results suggest the lack of MT-III contributes to protection of testis from Cd. In addition, regulation of Pnp2, Rd3, and Cdh24 mRNA levels may involve the sensitivity of MT-III null mice to Cd.

Metallothionein-I and -III expression in animal models of Alzheimer disease

Previous studies have described altered expression of metallothioneins (MTs) in neurodegenerative diseases like multiple sclerosis (MS), Down syndrome, and Alzheimer’s disease (AD). In order to gain insight into the possible role of MTs in neurodegenerative processes and especially in human diseases, the use of animal models is a valuable tool. Several transgenic mouse models of AD amyloid deposits are currently available. These models express human β-amyloid precursor protein (AβPP) carrying different mutations that subsequently result in a varied pattern of β-amyloid (Aβ) deposition within the brain. We have evaluated the expression of MT-I and MT-III mRNA by in situ hybridization in three different transgenic mice models of AD: Tg2576 (carrying AβPP harboring the Swedish K670N/M671L mutations), TgCRND8 (Swedish and the Indiana V717F mutations), and Tg-SwDI (Swedish and Dutch/Iowa E693Q/D694N mutations). MT-I mRNA levels were induced in all transgenic lines studied, although the pattern of induction differed between the models. In the Tg2576 mice MT-I was weakly upregulated in cells surrounding Congo Red-positive plaques in the cortex and hippocampus. A more potent induction of MT-I was observed in the cortex and hippocampus of the TgCRND8 mice, likely reflecting their higher amyloid plaques content. MT-I upregulation was also more significant in Tg-SwDI mice, especially in the subiculum and hippocampus CA1 area. Immunofluorescence stainings demonstrate that astrocytes and microglia/macrophages surrounding the plaques express MT-I&II. In general, MT-I regulation follows a similar but less potent response than glial fibrillary acidic protein (GFAP) expression. In contrast to MT-I, MT-III mRNA expression was not significantly altered in any of the models examined suggesting that the various MT isoforms may have different roles in these experimental systems, and perhaps also in human AD.

Nitric oxide induces metallothionein-I gene expression in mesangial cells

In various forms of injury involving the renal glomerulus, mesangial cells are exposed to potentially toxic concentrations of nitric oxide (NO) caused by activation of the inducible isoform of nitric oxide synthase (NOS). Whether mesangial cells possess systems that can defend against NO mediated oxidative injury is unknown. One putative system is Metallothionein (MT). Metallothioneins constitute a family of cysteine proteins and play a significant role as anti-oxidants. The authors assessed whether NO upregulates MT-I expression in cultured glomerular mesangial cells. Northern blot analysis revealed that steady state MT-I mRNA levels were increased by three different NO donors: sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), and Spermine-NONOate (Sper/NO). The increase in MT-I mRNA levels induced by SNAP-derived NO was attenuated by the antioxidant N-acetylcysteine (NAC), a glutathione (GSH) precursor, which indicates that the mechanism of NO-mediated MT-I expression may involve an oxidative stress response. These observations identify MT-I as a putative antioxidant system in NO-mediated mesangial cell injury.

The Role of Metallothionein in Hard Tissue

We studied the metallothionein (MT) induction by cadmium (Cd) in the dental pulp and bone in rats. In order to clarify the cell response to Cd in the hard tissue, the isoform-specific expression of MT mRNAs (MT-I and MT-II) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method, and the localization of MT was confirmed by immunohistochemical studies. Both MT-I and MT-II mRNA levels were increased within 3 hours by Cd administration in both the dental pulp and bone. In the dental pulp, MT protein was localized in the specific cell type of odontoblasts (secretory odontoblasts and resting odontoblasts). In the bone, MT-positive cells were time-dependently increased, and the positive cells were mainly localized in osteocytes. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced the hard tissue injury in vivo.

Ribozyme‐mediated downregulation of human metallothionein IIa induces apoptosis in human prostate and ovarian cancer cell lines

Human metallothioneins (MTs) are low-molecular-weight, cysteine-rich, metal ion-binding proteins that constitute the majority of intracellular protein thiols. They are overexpressed in prostate and ovarian cancers and are believed to confer resistance to radiation and cytotoxic anticancer drugs. The aim of this study was to investigate the roles of MTs in prostate and ovarian cancer cells and their possible relationship with other cancer development and progression factors. The main problem in investigating the role of MT, however, is the absence of any known specific inhibitor. To this end, in a previous study, we had developed sequence-specific ribozymes (Rzs) targeting MT and had shown their in cellulo efficacy. Here we show that transient transfection of a vector carrying a hammerhead Rz (Rz4-9), designed to cleave class II MT, in the human prostate cancer cell line PC-3 and the ovarian cancer cell line SKOV-3 resulted in a dose-dependent attenuation of MT-II(a) transcripts and dramatic cell loss. Transient transfection with 2 microg of Rz4-9 vector DNA completely abolished MT-II(a) mRNA levels and induced a 94% and a 67% reduction in cell number in PC-3 cells and SKOV-3 cells, respectively. Fluorescence-activated cell sorting (FACS) showed that the Rz-induced cell loss probably was due to apoptosis, because it was associated with marked increases in the hypodiploid cell population, reaching maximums of 52% and 64% in cultures of PC-3 and SKOV-3, respectively. Additionally, annexin V-propidium iodide double-staining, followed by FACS, confirmed that Rz4-9-induced cell death was due to apoptosis and showed a vector DNA-dependent increase in late apoptotic cell numbers that reached maximums of 80% and 42%, respectively, in PC-3 and SKOV-3 cell cultures transfected with the highest concentration of vector DNA. In parallel experiments, transfection with a vector containing the enzymatically inactive mutant Rz-3-3 or the empty vector was not effective in inducing similar responses. The Rz-induced loss of MT-II(a) mRNA-associated cell death in these cancer cell lines was attended by dose-dependent downregulation of the proto-oncogene c-myc and the apoptosis inhibitory mediator bcl-2, suggesting that these signaling pathways are involved in the process. In conclusion, our data indicate that MT-II(a) is an important cell-survival or anti-apoptotic factor for prostate and ovarian cancer cells and that downregulation of its expression via transgene expression of a sequence-specific Rz is a feasible target for cancer therapy.

Metallothionein expression and localization in rat bone tissue after cadmium injection

We investigated the induction of metallothionein (MT) by cadmium (Cd) in the bone tissue of rats. To clarify the cell response to Cd in bone, the isoform-specific expression of MT mRNAs (MT-I and MT-II) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Both MT-I and MT-II mRNA levels were increased within 3 h by Cd administration. MT (MT-I/MT-II) localization after single Cd injection were also confirmed by immunohistochemical studies. Notably, MT-positive cells were time-dependently increased, and the positive cells were mainly localized in osteocytes. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, we also found that MT was consecutively expressed in some osteoclasts of control rats. This finding suggested a new role of osteoclasts in bone metabolism.

Localization of Metallothionein (MT) and Expression of MT Isoforms Induced by Cadmium in Rat Dental Pulp

We investigated the induction of metallothionein (MT) by cadmium (Cd) in the dental pulp of rat incisors. Time-course studies of MT mRNA expression after single Cd injection were observed by Northern-blot analysis. The isoform-specific expressions of MT mRNAs (MT-I, MT-II and MT-III) were observed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Both MT-I and MT-II mRNA levels increased within 3 h, peaked at 3 h and then decreased. These findings demonstrated that MT-I and MT-II mRNA were rapidly induced by Cd in dental pulp. MT-III mRNA was constitutively expressed in rat dental pulp, but the expression level did not change by Cd treatment. The localization of MT protein in Cd-treated rat dental pulp was determined by immunohistochemical staining using anti-MT antibody against MT-I and MT-II. MT protein was localized in the specific cell type of odontoblasts (secretory odontoblasts and resting odontoblasts). In conclusion, it is likely that stained MT in the immunohistochemical study should be MT-I and/or MT-II. Furthermore, MT-I and/or MT-II in Cd-treated rat dental pulp was localized in odontoblasts, in which accumulation of Cd were reported. The cell-specific synthesis of MT may be associated with its metal storage and detoxification role in dental tissues.

Metallothionein induction in human CNS in vitro : neuroprotection from ionizing radiation

Purpose : There have been extensive studies on the regulation of metallothionein (MT) synthesis, and its biological role in liver and kidney. Although there are few reports on brain MT, there is a growing interest in the role of MT in brain. There have been no publications to date on MT synthesis in the human central nervous system (CNS) following exposure to ionizing radiation. In the present study, primary human CNS cultures were used to examine the effect of ionizing radiation on MT mRNA and protein synthesis. In the same cultures, the neuroprotective effects of zinc (Zn) and cadmium (Cd)-induced MT synthesis from high-dose radiation were also examined. Materials and Methods : Primary, serum-free, human CNS cultures were exposed to 30 or 60 Gy γ-rays. The total MT protein was then measured by a Cd-heme assay, and mRNA for MT-II and MT-III was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytotoxicity was measured by LDH release and apoptotic cell death by DNA fragmentation analysis. Sublethal neuroglial injury was assessed morphologically using specific astrocytic (glial fibrillary acidic protein-GFAP) and neuronal (microtubule-associated protein 2-MAP2) immuno-histochemical markers. Results : The total MT protein content was increased 12 h after exposure to 30 Gy. The increase in MT content in response to 60 Gy was not statistically significant. MT-II mRNA levels increased at 3 and 6 h after exposure to 30 Gy γ-rays, with a maximum expression at 12-24 h. MT-III mRNA was not significantly affected. Exposure to 60 Gy, but not 30 Gy, caused a marked increase in LDH release. Cells exposed to 30 Gy or less showed some apoptotic cell death by DNA fragmentation analysis, while exposure to 60 Gy resulted in a DNA smear confirmed by LDH assays. Preinduction of MT by 5 μ m Cd or 100 μ m Zn resulted in a significant reduction in radiation-induced LDH release. Morphological evaluations revealed that Cd or Zn preincubation led to relative preservation of MAP2 staining and GFAP. Conclusion : Both MT protein and MT-II mRNA can be induced in human CNS cells by ionizing radiation. Furthermore, induction of MT synthesis with Zn and Cd can protect human CNS cells from radiation-induced cytocidal and sublethal injuries. Both findings have implications in the development of strategies to protect human CNS tissue from damage during radiotherapy.

Effects of Cadmium on Metallothionein-I and Metallothionein-II mRNA Expression in Rat Ventral, Lateral, and Dorsal Prostatic Lobes: Quantification by Competitive RT–PCR

Highly sensitive, sequence-specific competitive reverse transcriptase–polymerase chain reaction (RT–PCR) protocols were established for the detection and quantification of metallothionein (MT)-I and MT-II messages, in absolute values, in rat tissues. Detection limits for these protocols were in the range of 5 to 10 amol per μg total RNA. Levels of MT-I and MT-II transcripts in the three major prostatic lobes, kidney, and testis were measured in untreated and cadmium (Cd)-treated rats. The dorsal prostate (DP), lateral prostate (LP), kidney, and testis expressed substantial levels of MT-I and MT-II mRNA while the ventral prostate (VP) had extremely low levels of the transcripts. Cd treatment induced higher levels of MT-I and/or MT-II mRNA expression in all tissues studied with the exception of LP. In the LP, Cd treatment caused reductions of MT-I and MT-II mRNA levels. The Cd-induced levels attained in the VP following Cd exposure were still markedly lower than those found in the kidney, testis, LP, and DP of untreated animals. These findings contradict previous claims that the MT genes in rat VP are unresponsive to Cd activation. The susceptibility of VP to Cd toxicity/carcinogenicity may therefore be explained by low levels of Cd-induced expression rather than lack of induction of MTs.

Molecular Analysis of Mammalian Timeless

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER–mTIM interactions. The data suggest that PER–PER interactions have replaced the function of PER–TIM dimers in the molecular workings of the mammalian circadian clock.

Metallothionein Induction in Response to Restraint Stress: TRANSCRIPTIONAL CONTROL, ADAPTATION TO STRESS, AND ROLE OF GLUCOCORTICOID

Metallothioneins (MT) have been implicated in the protection of cells from oxidative stress. We studied the molecular mechanism of induction of MT-I and MT-II in response to restraint stress using a mouse model system in which the animals were restrained in well ventilated polypropylene tubes for 12 h each day (one cycle). Here, we show that MT-I and MT-II mRNA levels were elevated as much as 10-20-fold after just one cycle of this simple stress. Stress-mediated MT induction occurred at the transcriptional level. The level of MT mRNA correlated with the stress-induced increase, and not with the diurnal variation, in the level of serum glucocorticoid. Treatment of the mice with RU 486, a glucocorticoid receptor antagonist, prior to restraint stress inhibited MT induction by at least 50%. Furthermore, the glucocorticoid responsive element-binding activity in the liver nuclear extracts from the stressed mice was significantly higher than that in the control mice. The complex formations between the transcription factor Sp1, MTF1, or MLTF/ARE and the respective specific oligonucleotides were not altered in the liver from the stressed mouse. The MT mRNA levels returned to the basal level at the end of nine cycles of stress, indicating habituation of the animals to restraint stress. At this stage, exposure of the animals to another type of stress, treatment with heavy metals, resulted in further induction of MT. These data indicate that glucocorticoid is the primary physiological factor responsible for MT induction following restraint stress, and the glucocorticoid receptor is the major transcription factor involved in this process.

Dimethyl sulfoxide, but not acidosis-induced metallothionein mRNA expression in neonatal rat primary astrocyte cultures is inhibited by the bioflavonoid, quercetin

Metallothionein (MT) mRNA levels were analyzed following exposure of neonatal rat primary astrocyte cultures to physiologic pH (7.4), acidosis (pH 6.5 and 6.0), and dimethyl sulfoxide (DMSO). Treatments were carried out both in the presence and absence of the bioflavonoid, quercetin. Total RNA was probed on northern blots with [ α 32P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. MT-I and MT-II mRNA levels in astrocytes exposed to pH 6.5 or pH 6.0 were increased compared to controls (pH 7.4). Treatment with DMSO in the presence and absence of acidosis, also increased MT-I and MT-II mRNA levels compared to controls (pH 7.4). The DMSO-induced increase in MT mRNA expression was reversed by treatment of astrocytes with quercetin, such that MT-I and MT-II mRNA levels in DMSO plus quercetin-treated astrocytes were indistinguishable from mRNA levels in their respective controls at pH 7.4, pH 6.5, and pH 6.0. These findings suggest that both acidosis and DMSO exposure are associated with increased astrocytic MT synthesis at the mRNA level, and that quercetin, effectively blocks MT mRNA induction by DMSO.

Interleukin-6 and tumor necrosis factor- α type 1 receptor deficient mice reveal a role of IL-6 and TNF- α on brain metallothionein-I and -III regulation

Metallothioneins (MTs) are a family of low molecular weight proteins which in rodents is comprised of several isoforms (MT-I to MT-IV). MT-I and MT-II are widely expressed isoforms, whereas MT-III is mainly expressed in the central nervous system and is the only isoform that inhibits survival and neurite formation of rat cortical neurons in vitro. However, the physiological roles and regulation of these proteins in the brain are poorly characterized. In this report we have studied the putative role of IL-6 and TNF- α on the regulation of brain MT-I and MT-III, by using mice carrying a null mutation in the IL-6 or the TNF- α type 1 receptor genes or both. In situ hybridization analysis revealed that brain MT-I induction by bacterial lipopolysaccharide (LPS) was significantly lower in IL-6- and TNFR1-deficient mice, and to a greater extent in the double mutant mice, in most brain areas studied. These results suggest that the MT-I isoform could be considered an acute-phase protein in the brain, which is consistent with previous studies in transgenic mice overexpressing IL-6 in astrocytes. In contrast to LPS, brain MT-I induction by restraint stress was not affected significantly by IL-6 or TNFR1 deficiencies, suggesting that these cytokines are not important during the stress response in the brain. In basal conditions, it was also observed that the double mutant mice had diminished MT-I mRNA levels in several brain areas. In contrast to MT-I, MT-III mRNA levels were minimally affected by either LPS or stress. Yet, significant decreasing effects of IL-6 and TNFR1 deficiencies were observed in the Purkinje neuronal layer of the cerebellum (after LPS) and ependymal cells (after LPS and stress). In contrast, significant increasing effects, especially of TNFR1 deficiency, were observed in CA1 hippocampal area, retrosplenial and parietal cortex, and in thalamic nuclei (after LPS). These results demonstrate that IL-6 and TNF- α are involved in brain MTs regulation during LPS-elicited inflammatory response but not during the stress response.

Concentration-dependent differential effects of N-acetyl- l-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells

Cadmium induces the expression of the 70 kDa heat shock protein (HSP70) and metallothionein (MT), both of which are considered to be associated with intracellular glutathione (GSH) metabolism in the cellular protection mechanism against cadmium-induced cellular injury. We determined the effects of N-acetyl- l-cysteine (NAC), which increases the intracellular GSH levels, on the induction of HSP70 and MT gene expression in a cultured cell line of human amniotic cells (WISH) exposed to CdCl 2. The mRNA level of MT-II, a major isoform of MT genes, was more prominently increased than that of HSP70 when WISH cells were exposed to CdCl 2 (5–15 μM, for 6 h). The treatment of WISH cells with 1.5 and 30 mM NAC for 2 h increased the intracellular GSH levels by 1.4- and 3.1-fold, respectively. Pretreatment of cells with 30 mM NAC significantly reduced both HSP70 and MT-II mRNA levels in the cells exposed to 50 μM CdCl 2. This concentration of NAC also efficiently suppressed the cadmium-induced lethality. On the contrary, pretreatment with 1.5 mM NAC suppressed only the induction of HSP70 gene expression in the 50 μM CdCl 2-treated cells, and did not inhibit the metal toxicity. However, this low concentration of NAC efficiently suppressed lipid peroxidation which was increased by 50 μM CdCl 2. Furthermore, this low concentration of NAC also decreased the CdCl 2-induced gene expression of HSP32 which represents a general response to oxidative stress. Taken together, NAC seems to have at least two concentration-dependent functions in WISH cells exposed to CdCl 2; the low concentration of NAC can suppress the induction of HSP70 gene expression as well as the increase of lipid peroxidation via an antioxidant pathway, while the high concentration of NAC can suppress the induction of MT-II mRNA as well as cadmium-induced cell death. Our present data suggest that changes in intracellular redox status, as reflected by GSH concentration, have more important effects on the induction of HSP70 mRNA rather than that of MT-II mRNA in human amniotic cells exposed to cadmium.

Transgenic expression of interleukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice.

The metallothionein (MT) gene family consists of several members (MT-I-IV) that are tightly regulated during development. MT-I and MT-II are expressed in many tissues, including the brain, whereas MT-III is expressed mainly in the central nervous system. However, the physiological roles of these isoforms in the brain and their regulation are poorly characterized. In this report, we have studied the putative role of IL-6 in the regulation of brain MT. The present results demonstrated that transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), and which develop chronic progressive neurodegenerative disease, show significantly increased MT-I + II protein levels in specific brain areas. Thus, the MT-I + II levels of 1- and 3-month-old GFAP-IL6 mice (G16 and/or G36 lines) were not altered in hippocampus but they were elevated in the cerebellum (highest induction), medulla plus pons, hypothalamus and remaining brain (lowest induction). The effect of the transgenic expression of IL-6 was more dramatic for MT-I + II protein than for MT-I mRNA levels, with the latter only marginally elevated in the G16 line at 3 months but not at 6 months of age where there was a tendency to decreased levels. Brain MT-I mRNA levels also tended to decrease in the higher expressor G36 line in 3-month-old mice despite the strongly elevated MT-I + II protein levels at this age. Therefore, in addition to increasing MT gene transcription, these results suggest a post-transcriptional effect of IL-6 or of a IL-6-dependent factor, in this chronic situation. The up-regulated brain MT-I + II protein levels in the GFAP-IL6 mice was comparable to the expression of the acute-phase response gene EB22/5, suggesting that these MT isoforms could be considered acute-phase response proteins in the brain. Brain MT-III mRNA levels followed a somewhat similar pattern that those of MT-I mRNA but the decreasing effect of IL-6 transgene production with age was more dramatic for the former, suggesting differential regulation of these MT isoforms by IL-6. The results indicate that these transgenic mice might be a valuable tool for further examining the role of the MT isoforms in brain physiology and pathobiology.

Metallothionein induction by bismuth in neonatal rat primary astrocyte cultures

Metallothionein (MT) proteins and mRNA levels were determined following exposure of neonatal rat primary astrocyte cultures to bismuth. MT mRNA were probed on Northern blots with [α- 32P]dCTP labeled synthetic cDNA probes specific for rat MT-I and MT-II mRNA. The probes for MT-I and MT-II mRNA hybridize to a single mRNA with a size appropriate for MT, approximately 550 and 450 base pairs for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 and 10 μM bismuth for 6 h was increased in a dose-dependent manner over MT-I and MT-II mRNA levels in control cells. Western-blot analysis revealed a time-dependent increase in MT protein synthesis through 72 h of exposure to bismuth (2–10 μM). Consistent with the bismuth-induced increase in MTs at the mRNA and protein levels, a time- (24–72 h) and dose-dependent (2–10 μM) increase in astrocytic MT-immunoreactivity was noted.

Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.

Metallothionein gene expression in testicular interstitial cells and liver of rats treated with cadmium

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. Cd induces predominantly testicular interstitial cell (TIC) tumors. In order to clarify the molecular mechanism underlying tissue differences in Cd sensitivity, we compared Cd-induced metallothionein (MT) gene expression, MT protein accumulation, and Cd retention in freshly isolated TICs and liver. Adult male Fischer rats received a s.c. injection of 4.0 μmol Cd/kg or vehicle and 24 h later tissues were sampled and TICs isolated. MT-I and MT-II mRNA levels were determined by slot-blot analysis followed by densitometry scanning, and MT was estimated by the Cd-heme method. Testicular lesions were not grossly or histologically observed in rats treated with 4 μmol Cd/kg. Both MT mRNA and MT (as determined by Cd-binding capacity) were constitutively present in TICs as well as the liver. TICs isolated from Cd-treated rats accumulated more Cd (4-fold), and had higher levels of MT-I ( 1.9-fold) and MT-II ( 1.4-fold) mRNAs over control, but contained less MT (30% decrease) than TICs isolated from control animals. Cd exposure substantially increased hepatic Cd content (6000-fold), MT (58-fold), and MT-I mRNA (5.3-fold), but did not increase MT-II mRNA. Thus, our findings indicate that, although low-dose Cd exposure results in increases of MT mRNA in TICs it does not enhance MT synthesis within these cells. The inability to induce the metal-detoxicating MT-protein, in response to Cd, might account for higher susceptibility of testes to Cd toxicity and Carcinogenesis relative to liver.