Metallothionein mRNA Expression Research Articles

Upregulation of Metallothionein-I mRNA Expression in a Rodent Model for Amyotrophic Lateral Sclerosis

Metallothionein (MT) mRNA expression was investigated in a rodent model (G93A SOD1 transgenic mouse) for a lethal motor neuron disease, amyotrophic lateral sclerosis (ALS). In 8-wk-old mice that did not yet exhibit motor paralysis, MT-I mRNA expression was already significantly upregulated in the region of the spinal cord responsible for motor paralysis. The expression of another isoform, MT-III, was not changed. In the cerebellum, which is not responsible for motor paralysis in ALS, neither the expression profiles of MT-I nor MT-III were altered. In 16-wk-old mice exhibiting motor paralysis, the expression of MT-I mRNA remained upregulated and the MT-III level tended to be elevated. Although no significant differences were found in the levels of both isoforms in the liver or kidney of 8-wk-old mice, the MT-I mRNA expression level was significantly upregulated in the kidney and liver of 16-wk-old mice. These results indicated that the MT-I isoform, but not the MT-III isoform, is associated with motor neuron death in ALS and suggested that the disease might be a systemic disorder to which the spinal cord is particularly susceptible.

Tributyltin inhibits osteoblastic activity and disrupts calcium metabolism through an increase in plasma calcium and calcitonin levels in teleosts

To examine the direct effects of tributyltin acetate (TBTA) on osteoclasts and osteoblasts, teleost scale, which has both osteoclasts and osteoblasts and is similar to mammalian membrane bone, was used in the present study. The activities of tartrate-resistant acid phosphatase and alkaline-phosphatase, as respective indicators of activity in both cells, were used. In freshwater teleost (goldfish) and marine teleosts (nibbler and wrasse), the osteoclastic activity in the scales did not change as a result of TBTA treatment (10 − 9 to 10 − 5 M). However, the osteoblastic activity decreased in the goldfish, nibbler, and wrasse after 6 h of incubation. In goldfish, even 10 − 10 M of TBTA significantly inhibited the osteoblastic activity. The inhibitory activity in goldfish was stronger than that in nibbler and wrasse. Therefore, details of the mechanism were examined using goldfish. The mRNA expressions of the estrogen receptor and insulin-like growth factor-I, which participate in osteoblastic growth and differentiation, decreased in the TBTA-treated scales. However, the mRNA expression of metallothionein (MT), a metal-binding protein that protects the organism from heavy metal, increased much less than those of cadmium and methyl-mercury. Furthermore, we showed that the plasma calcium and hypocalcemic hormone (calcitonin) level increased in goldfish kept in water containing TBTA (10 − 10 and 10 − 8 M). The current data are the first to demonstrate that, in teleosts, TBTA inhibits osteoblastic activity without affecting osteoclastic activity and disrupts the calcium metabolism, including the calcemic hormone, in goldfish.

Metallothionein mRNA Expression and Cadmium Tolerance in Metal-stressed and Reference Populations of the Springtail Orchesella cincta

Metal contamination in soil ecosystems is a permanent and often strong selection pressure. The present study investigates metal tolerance in 17 Orchesella cincta (Collembola) populations from metal-contaminated and reference sites, and combines analyses at the phenotypic and molecular level. Metal tolerance was phenotypically assayed by measuring survival times of laboratory cultures during exposure to cadmium. Comparisons of survival curves showed that five out of eight metal-stressed populations tested evolved increased cadmium tolerance (Stolberg, Plombieres, Hoboken, Hygum and Gusum). In addition, the role of the metallothionein (MT) gene in cadmium tolerance of O. cincta was studied by means of quantitative RT-PCR. The constitutive and Cd-induced MT mRNA expression of the laboratory cultures was measured. Results show that the mean constitutive MT mRNA expression of populations from polluted sites was significantly higher than of populations from reference sites. However, no correlation between MT mRNA expression levels after laboratory exposure to cadmium and field cadmium concentrations was observed. Furthermore, no relation between survival rate during exposure to cadmium and MT mRNA expression was detected. Our results suggest that constitutive MT mRNA expression plays a role in early protection against cadmium toxicity, and indicate that mechanisms other then MT up-regulation are involved in tolerance to prolonged exposure to cadmium.

Effects of copper supplementation on copper absorption, tissue distribution, and copper transporter expression in an infant rat model

Infants are exposed to variable copper (Cu) intake; Cu in breast milk is low, whereas infant formulas vary in Cu content as well as the water used for their preparation. Little is known about the regulation of Cu absorption during infancy. The objectives of this study were to determine effects of Cu supplementation on Cu absorption and tissue distribution and the expression of Cu transporters in an infant rat model. Suckling rat pups were orally dosed with 0, 10, or 25 microg Cu/day. Intestine and liver were collected at days 10 and 20, and Cu concentration, Cu transporter-1 (Ctr1), Atp7A, Atp7B, and metallothionein (MT) mRNA and protein levels were measured. 67Cu absorption was measured at days 10 and 20. Total 67Cu absorption decreased, and intestinal 67Cu retention increased with increased Cu intake. At day 10, intestine Cu concentration, MT mRNA, and Ctr1 protein levels increased with supplementation, but no changes in Atp7A or Atp7B levels were observed. At day 20, intestine Cu concentration was unaffected by Cu supplementation, but Ctr1 protein and Atp7A mRNA and protein levels were higher than in controls. In liver, Cu level reflected Cu intake at days 10 and 20. There was a significant increase in Ctr1, Atp7B, and MT mRNA expression in liver at both ages with Cu supplementation. In conclusion, the ability of suckling rat pups to tolerate varying amounts of dietary Cu may be due to changes in Cu transporters, facilitated by transcriptional and posttranslational mechanisms. Despite these adaptive changes, Cu supplementation resulted in elevated alanine aminotransferase levels, suggesting a risk of Cu toxicity with supplementation during infancy.

The effect of timing of pneumoperitoneum on the inflammatory response

We examined the effects of an identical period of pneumoperitoneum applied at three different time points after lipopolysaccharide (LPS) challenge. Two different insufflation gases were also compared. Male rats (n = 70) were injected intravenously with 1 mg/kg of LPS (time 0). The time relationship between a 1.5-h period of insufflation and initial LPS stimulation was the experimental variable. All rats were killed 6 h after injection. CO2 and helium insufflation were investigated. Ten control rats received LPS only. Serum interleukin-6 (IL-6) levels were determined by enzyme-linked immunosorbent assay (ELISA). Hepatic expression of alpha2-macroglobulin, beta-fibrinogen, and metallothionein were measured by Northern blot analysis. Statistical analysis was performed using one-way analysis of variance (ANOVA). Expression of alpha2-macroglobulin mRNA was lower in CO2 groups compared to the control group (p < 0.05 at time 120 and 270). beta-Fibrinogen message was diminished in CO2 0 and 120 groups compared to control. Serum levels of IL-6 and expression of metallothionein mRNA did not show significant differences between groups. These findings suggest that CO2 pneumoperitoneum downregulates the inflammatory response to LPS challenge. Start time of CO2 insufflation does not appear to alter hepatic expression of acute phase genes. The mechanism of alpha2-macroglobulin downregulation does not appear to be due to IL-6.

Ultrastructural Demonstration of Mercury Granules in the Placenta of Metallothionein-Null Pregnant Mice after Exposure to Mercury Vapor

The placenta plays an important role in the regulation of maternal to fetal transfer of toxic substances, including nonessential metals. Metallothioneins (MTs), which are known to have protective effects against heavy metal toxicity, exist in the placenta, but the exact localization of placental MTs (both MT-I and MT-III) and their physiological role in the placenta exposed to mercury are unclear. The present study was performed to examine the localization of MTs and mercury granules in the placenta of mice exposed to mercury vapor. On gestational day 16, MT-I & II-null and wild-type mice were exposed to mercury vapor at 4.9 to 5.9 mg/m3 for 2 hours. At 24 and 48 hours after exposure, the placentas were examined for mercury distribution (autometallography), MT immunoreactivity, and MT mRNA expression (in situ hybridization). No histological changes were observed in the placentas of either MT-null or wild-type mice. Mercury deposition was demonstrated along the boundary between the junctional zone and the labyrinth zone, as well as in the yolk sac, maternal decidual cells, and labyrinth trophoblasts of both MT-null and wild-type mice. MT-I & -II immunoreactivity, which was confined to wild-type mice, was demonstrated in the yolk sac and decidual cells; mercury was also shown in both structures, suggesting that mercury granules were bound to MTs. MT-III mRNA expression was observed in the yolk sac, decidual cells, and spongiotrophoblasts in both MT-null and wild-type mice. There was, however, no evidence of MT at the boundary between the junctional and labyrinth zones, where substantial mercury deposits were demonstrated. These results suggest that placental MTs and the other unknown molecules may be related to the barrier to the placental transfer of mercury.

Two 14-epi analogues of 1,25-dihydroxyvitamin D3 protect human keratinocytes against the effects of UVB.

In search of photoprotective agents, we recently demonstrated a protective effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] against different events mediated by ultraviolet B (UVB) in human keratinocytes. Pharmacological doses of 1,25(OH)(2)D(3) were required to obtain significant UVB protection; however, these doses cannot be used in vivo due to the calcemic properties of 1,25(OH)(2)D(3). Therefore, we evaluated the photoprotective capacities of two low-calcemic 14-epi analogues of 1,25(OH)(2)D(3), 19-nor-14-epi-23-yne-1,25(OH)(2)D(3) (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3) (TX 527). Using cultured human keratinocytes, we investigated the influence of TX 522 and TX 527 on two hallmark events in UVB-irradiated keratinocytes: the induction of apoptosis and the production of interleukin-6 (IL-6). Treatment of the keratinocytes with TX 522 or TX 527, 24 h before irradiation, resulted in a significant and dose-dependent reduction of both UVB-induced apoptosis and IL-6 production. Both analogues were equally efficient in their anti-UVB effects and at least 100 times more potent than 1,25(OH)(2)D(3). We further demonstrated that metallothionein (MT) mRNA expression was clearly induced by 1,25(OH)(2)D(3) and both analogues. MT acts as a radical scavenger in oxygen-mediated UVB injury and its induction may therefore be relevant for the anti-UVB effects of 1,25(OH)(2)D(3) and both analogues. Taken together, these findings create new perspectives for the use of active vitamin D analogues as photoprotective agents.

Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release.

Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

Field-selected cadmium tolerance in the springtail Orchesella cincta is correlated with increased metallothionein mRNA expression

Populations of the springtail Orchesella cincta that live in metal contaminated soils have developed tolerance to cadmium by increased metal retention in the midgut epithelium and excretion at every moult. Regulation of the MT gene was studied in a tolerant population (Plombières, Belgium) and a laboratory culture. Animals were exposed to a range of concentrations of cadmium in the food (0–1.5 μmol Cd/g food). RNA was extracted after 5–14 days of cadmium exposure and used for Northern blot analysis to quantify MT mRNA. MT expression levels were significantly higher ( p<0.01) in individuals from laboratory-raised strains originating from the soils of the metal contaminated forest Plombières (2.4- to 7.8-fold expression) compared to the reference population (1.5- to 2.4-fold expression). No variable sites were found in the complete MT coding sequence. Southern blot analysis suggests that in both populations the gene is not tandemly repeated. This is the first evidence of evolution of metal tolerance via gene regulation of MT in a natural population. These data indicate a higher fitness of the tolerant population in the polluted environment due to selection of high MT expression phenotypes.

In situ hybridisation for flow cytometry: a molecular method for monitoring stress-gene expression in hemolymph cells of oysters

In the present study, we developed a molecular method for flow cytometry to detect the effects of environmental factors on the stress-response in immunocompetent cells of the European flat oyster, Ostrea edulis. Stress-generating conditions were applied to individuals acclimated in the laboratory to environmental salinity and temperature. Oligonucleotidic probes were applied to quantify the expression of HSP/C70 or metallothionein genes. After a heat-stress, a response was reflected in hemocytes by an increased amount of HSP/C70 mRNA and quantitative changes in HSP/C70 protein expression. The technique of in situ hybridisation described here also allowed to quantify the expression of metallothionein mRNA in oysters exposed to a heavy metal-exposure. The detection of stress protein markers by using such quantitative methods could be applied in contamination studies in oysters and other bivalves where monitoring the status of hemolymph cells is required.

Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters ( Crassostrea rhizophorae)

A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters ( Crassostrea rhizophorae), using β-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

Hepatic metallothionein in patients with chronic hepatitis C: relationship with severity of liver disease and response to treatment

Objectives Reactive oxygen species may be involved in the pathogenesis of chronic hepatitis C virus infection. Metallothionein (MT) is an essential protein for the protection of cells against reactive oxygen species. The aim of this prospective study was to assess the influence of the hepatic level and cellular distribution of MT in hepatitis C virus (HCV) infection and in the liver disease outcome. Methods In liver biopsy samples of 32 patients with chronic HCV infection and of 12 control subjects, quantification of MT was performed by radioimmunoassay, MT, interleukin (IL)-1 and -6, and tumor necrosis factor (INF)–α mRNA by reverse transcription–polymerase chain reaction (PCR) and cellular distribution by immunohistochemistry. Results In HCV-infected patients, MT liver protein level was 3-fold lower than in control specimens. A significant inverse linear regression between MT protein or mRNA expression and the Histological Activity Index, the necroinflammatory grade, and the stage of fibrosis was observed. MT immunostaining was located in the nucleus and cytoplasm in hepatocytes of control subjects, whereas it was mainly cytoplasmic in HCV-infected patients. Before interferon (IFN) therapy, the hepatic MT level in patients that were nonsustained responders was half that of sustained responders. Intrahepatic IL-6 and MT were simultaneously down-regulated, but no correlation was found between MT and intrahepatic cytokine mRNA expression in patients with chronic HCV infection. Conclusions This study shows that hepatic MT expression could reflect the severity of chronic HCV infection and could be one of the factors associated with a favorable clinical outcome in the response to interferon therapy.

Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague–Dawley rats received a s.c. injection of 4.0 μmol Cd/kg and 1, 3, 6, or 24 h later and untreated animals (0 h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 μmol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3 h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6 h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0–3 h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0–3 h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that 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.

Changes of serum sex hormone levels and MT mRNA expression in rats orally exposed to cadmium

It has been demonstrated that cadmium (Cd) is carcinogenic to rodent prostate. However, the mechanism of its toxicity is far from fully understood. In the present study, the effects of oral Cd exposure (0, 50, 100, 200 ppm in drinking water) on serum sex hormone levels, the expression of MT-I and MT-II mRNA, and the zinc content of rat prostate were assessed. With Cd administration, serum testosterone (T) levels significantly increased in all Cd groups after 3 months and in the 200 ppm Cd group after 6 months. A significant depression in the serum luteinizing hormone (LH) level was seen in the Cd group (200 ppm) after 6 months. It was noted that Cd administration resulted in a significant down-regulation in the expression of MT-I and MT-II mRNA in the rat ventral prostate. However, no Cd-induced changes in the mRNA expression of Metallothioneins (MTs) were detected in the dorsolateral prostate. After Cd administration, the content of Cd in both the ventral and dorsolateral lobes of the prostate significantly increased with increasing dose and duration of Cd administration. In contrast, the Zn content decreased with Cd administration in both the ventral and dorsolateral lobes of the rat prostate. Taken together, these results suggest that oral Cd exposure may disrupt endocrine homeostasis, changing the distribution of Zn and the mRNA expression of MTs in rat prostate, and that such Cd-induced changes may contribute to the susceptibility of prostate to the carcinogenicity of this heavy metal.

Renal expression of metallothionein in rats treated with cadmium

Background. The intrarenal localization of metallothionein (MT) expression has not been studied in detail. Therefore, we investigated the effects of acute exposure to cadmium on the renal expression of MT. Methods. MT mRNA expression was estimated quantitatively at 2, 6, 24, and 72 h after the injection of cadmium (1 mg/kg) to the intraperitoneum of male Sprague-Dawley rats. The site of cadmium-induced MT mRNA expression was determined, by the reverse transcription polymerase chain reaction (RT-PCR), using microdissected tubules. Immunoblotting and immunohistochemistry were also performed, to determine the localization of MT protein. Results. The mRNAs of MT-I and MT-II were significantly upregulated at 2 h and peaked at 24 and 6 h, respectively. RT-PCR revealed the expression of cadmium-induced MT mRNA mainly in the proximal tubules. Immunoblotting detected a clear 14-kDa band corresponding to MT protein, and immunohistochemistry showed abundant MT protein in the renal cortices of cadmium-treated rats. Conclusions. Our findings suggest that the proximal tubules are responsible for the cadmium-induced renal expression of MT mRNA and protein.

In vitro bioavailability of heavy metals in pressure-treated wood dust.

Pressure treatment with chromium, copper, and arsenic (CCA) is the most prevalent method for protecting wood used in outdoor construction projects. Although these metals are tightly bound to the wood fibers and are not released under most conditions of use, we examined the bioavailability of metals in CCA pressure-treated wood dust in vitro. Cytotoxicity and metallothionein (MT) mRNA expression were examined in V79 Chinese hamster lung fibroblast cells incubated with respirable-size wood dust generated by sanding CCA-treated and untreated (control) Southern yellow pine. In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/- 77 microg/ml, mean +/- SE) compared with control wood dust (883 +/- 91 microg/ml). Increased cytotoxicity with CCA wood dust also occurred in an arsenic resistant subline of V79 cells, thus suggesting that arsenic was not responsible for the increased cytotoxicity. Metallothionein mRNA was significantly increased after 48 h of treatment with CCA wood dust compared with control wood dust. Incubation of CCA wood dust in cell culture media resulted in the transfer of copper, but not chromium or arsenic, into the media. Moreover, the treatment of cells with this filtered extract resulted in significantly increased metallothionein mRNA, suggesting that bioavailable copper is responsible for inducing metallothionein mRNA in V79 cells. Thus, these bioassays suggest that metals become bioavailable during in vitro culture of phagocytic V79 cells with CCA wood dust.

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout ( Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 μg Cd l −1), Zn (150 or 1000 μg Zn l −1) or Cd/Zn mixtures (1.5 μg Cd l −1 with 200 μg Zn l −1 or 10 μg Cd l −1 with 1000 μg Zn l −1). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT–PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.

Rottlerin Stimulates Metallothionein Gene Expression but Inhibits Metal Transport in Chinese Hamster Ovary Cells

Metallothionein (MT) can be induced by various metals. We have shown previously that H7, a protein kinase C (PKC) inhibitor, inactivates metal-induced MT gene expression. To investigate whether a specific PKC isoform is involved in the induction process, inhibitors for various PKC isoforms were administered to cadmium-resistant Chinese hamster ovary (CdR) cells. None of the inhibitors used can reduce metal-induced MT gene expression. However, a PKCδ inhibitor, rottlerin, induced MT mRNA expression in CdR cells in the presence or absence of Cd. Notably, the induction occurs through the activation of the MT transcriptional factor (MTF-1) and is not related to an increase of metal influx. Furthermore, metal accumulation is reduced in the presence of rottlerin. Pulse-labeling analysis indicated that MT protein synthesis increased in CdR cells upon rottlerin treatment. These results suggest that rottlerin blocks metal transport but stimulates MT synthesis in CdR cells. Since rottlerin is capable of reducing the cellular accumulation of Cd, it was expected that the cytotoxic effect of Cd would decrease in the presence of rottlerin. Treating the parental cell of CdR with Cd and rottlerin together indeed showed a decline of cytotoxicity compared to cells treated with Cd alone. We further examined how MTF-1 was activated by rottlerin. Rottlerin-induced MTF-1 activity was not affected in CdR cells by the addition of EDTA. It was, however, diminished by administering an intracellular Zn chelator, TPEN. The result implies a mobilization of intracellular Zn ions after rottlerin treatment in CdR cells. To investigate whether the described results occur in all types of cells, another cell line (GH3) was used to study the effect of rottlerin on MT gene expression. The result revealed that rottlerin did not increase the amount of MT mRNA in GH3 cells. This differential effect between cell lines may be useful for investigating the regulatory mechanism of MT gene expression.

ACUTE EXPOSURE TO ARSENITE INDUCES METALLOTHIONEIN ISOFORM-SPECIFIC GENE EXPRESSION IN HUMAN PROXIMAL TUBULE CELLS

The expression of metallothionein (MT) mRNA and protein was determined in human proximal tubule cells (HPT) following acute exposure to the classic stimulators of the stress response, heat and sodium arsenite (As3+). Treatment of the cells with 100 µ M As3+ for 4 h resulted in a significant increase in the MT-1 and MT-2 proteins immediately preceding and following removal of the stress. The level of the MT-3 isoform protein was unchanged as a result of As3+ treatment. An analysis of the MT isoform-specific mRNA demonstrated that control cells express the MT-1E, MT-1F, MT-1X, MT-2A, and MT-3 genes, but not the MT-1A, MT-1B, MT-1G, MT-1H, and MT-4 genes. Treatment with As3+ resulted in a significant increase in the expression of the MT-1X gene and appearance of mRNA for the MT-1A gene. Expression of the other MT genes was unaffected by As3+ exposure, except one isolate expressed a low level of MT-1G mRNA at several time points. It is likely that the increase in MT protein seen in As3+-treated cells is due to the increased expression of the MT-1X gene because its expression is much greater than the MT-1A isoform. Treatment of the HPT cells with heat shock had no marked effect on the levels of MT protein or mRNA. This study demonstrates that acute exposure to As3+ increases the levels of MT protein and that this elevation most likely arises from increased expression of the MT-1X isoform.

Dexamethasone Prevents Acute Cadmium-Induced Hepatic Injury but Exacerbates Kidney Dysfunction in Rabbits

Cadmium is a potent hepatotoxicant for which neither effective preventive methods nor the mechanism of toxicity has been established. We investigated the preventive effect of dexamethasone against cadmium toxicity on cadmium-induced liver injury in rabbits. Pretreatment with dexamethasone at 1 mg/kg increased the rate of survival in rabbits administered 2.5 mg/kg iv cadmium. Cadmium induced acute severe liver injury characterized by hepatocellular necrosis, infiltration by inflammatory cells, and increases of plasma GOT, GPT, LDH, and LDH5. Dexamethasone mitigated the acute hepatotoxic effect of cadmium, but exacerbated cadmium-induced kidney dysfunction, with destruction of renal tubular cells and increases in excretion of protein, glucose, and amino acids into urine. The cadmium concentration in liver and kidney of rabbits administered cadmium was not changed by dexamethasone pretreatment. Although metallothionein mRNA expression induced by cadmium was not affected by dexamethasone in liver or kidney, cadmium-induced metallothionein protein production was augmented at the early phase in liver and decreased at the later phase in kidney. Neutrophilia observed after cadmium administration was enhanced initially by dexamethasone pretreatment. These results indicate that dexamethasone pretreatment potently prevented cadmium-induced liver injury, but exacerbated renal tubular dysfunction.