Metallothionein mRNA Induction Research Articles

Differential regulation of endoplasmic reticulum stress-induced autophagy and apoptosis in two strains of gibel carp (Carassius gibelio) exposed to acute waterborne cadmium

Previous studies illustrated that gibel carp F strain displays better lipid mobilization and antioxidant ability and compared to the A strain. We therefore hypothesized that the F strain would exhibit superior defense to cadmium exposure. Comparative studies were conducted between A and F strains using plasma stress biomarkers, histological observations, and analysis of hepatic molecular events to examine exposure to waterborne Cd (11.9 mg L−1) for 48 h and 96 h. Waterborne Cd exposure stimulated stress response and hepatic metallothionein mRNA induction in both gibel carp strains confirming exposure. Antioxidant responses were stimulated to counteract Cd toxicity, suggested by the upregulation of mRNA levels of genes associated with nuclear factor erythroid 2-related factor 2 (nrf2) signaling. Cd exposure induced endoplasmic reticulum (ER) stress, meanwhile, branches of genes in unfolded protein response (UPR) were activated. Slight time-dependent effects were implied by greater ER stress, UPR, and apoptosis signals with the duration of Cd exposure. Genotype-specific effects were identified, revealing that the F strain showed greater stress at 96 h exposure and higher antioxidant response compared to the A strain, as indicated by the mRNA levels of genes in nrf2 signaling. ER stress and UPR were also stronger in the F strain after Cd exposure. In contrast, the A strain showed higher autophagy and apoptosis response compared to the F strain. Collectively, combined autophagy and apoptosis were triggered under ER stress, which might serve as defense strategies in both gibel carp strains. The F strain showed greater antioxidant detoxification response and UPR to mitigate Cd toxicity, whereas excessive ER stress contributed to higher autophagy and apoptosis in the A strain. The present study uncovered the differential regulation and defense strategies in fish strains exposed to metal exposure.

Effects of salinity on metal uptake and metallothionein mRNA levels in the organs of tilapia exposed to cadmium, copper, and zinc ions.

This study aimed to determine the effects of salinity on metal uptake and metallothionein (MT) mRNA levels in tilapia exposed to three metal ions. Male Oreochromis niloticus × O. aureus juveniles (hereafter, "tilapia") were exposed to various concentrations (100, 500, and 1 ppm) of metal ions (Cd(2+), Cu(2+) and Zn(2+)) in freshwater and water with two levels of salinity (10 and 20 ppt) for 7 days. Tests were then performed to investigate the effects of salinity on metal concentrations and MT mRNA induction in the test subjects' organs. Saline decreased cadmium (Cd) uptake and MT mRNA fold induction in various internal organs, but it did not enhance MT mRNA induction in the gills. Exposure to Cu(2+) caused greater copper (Cu) levels in the brains, intestines and livers, but Cu uptake in the intestines and kidneys occurred only at 10 ppm. MT mRNA induction caused by Cu(2+) was observed in various internal organs, but it occurred in the gills only at greater levels of salinity. Exposure at greater salinities also decreased zinc (Zn) uptake and MT mRNA induction in all organs except the gills. Although greater salinity decreased Cd and Zn uptake, the metal content in the water correlated with the MT mRNA levels in most of the organs, except for the intestines. In conclusion, metal accumulations in the livers and kidneys of tilapia correlated with MT mRNA levels. The levels of MT mRNA in the livers and kidneys of tilapia might therefore be used as biomarkers of exposure to Cd(2+), Cu(2+) and Zn(2+) in water of various salinities.

Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels

We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification.

Kidney dysfunction and cadmium exposure – Factors influencing dose–response relationships

Our early toxicological studies showed that metallothionein (MT) is a protein that carries cadmium (Cd) to the kidney, explaining why Cd exposures during long time periods may give rise to kidney dysfunction. This dysfunction is usually considered to be the critical effect, i.e. the adverse effect that occurs at the lowest exposure level. MT also provides intracellular protection against cadmium toxicity. In studies of population groups in cadmium contaminated areas in China, we investigated factors that affected the relationship between internal dose of Cd, as indicated by blood Cd (BCd) or urinary Cd (UCd), and the prevalence of kidney dysfunction. We found dose–response relationships between UCd and the prevalence of increased levels of biomarkers of renal tubular dysfunction (urinary beta-2-microglobulin, B2M, or N-acetyl-beta-d-glucosaminidase – NAG) or urinary albumin (UAlb), a biomarker of glomerular kidney dysfunction. Two years after Cd intake from contaminated rice was diminished, renal tubular dysfunction appeared unchanged or aggravated among those with higher UCd; Another 8years later, i.e. 10years after Cd intake was decreased, the prevalence of renal tubular dysfunction was still increased but UAlb had returned to normal. Factors that influenced the dose–response relationships were: (1) time after maximum exposure. (2) Concomitant exposure to other nephrotoxic agents such as inorganic arsenic. (3) Cd induced metallothionein mRNA levels in peripheral blood lymphocytes, used as a biomarker of the ability of each person, to synthesize MT. (4) The occurrence of increased levels in blood plasma of autoantibodies against MT. The two last points further support a role in humans of MT as a protective protein against tissue damage from cadmium and gives support to previous ideas developed partly in experimental systems.

Enhanced metallothionein gene expression induced by mitochondrial oxidative stress is reduced in phospholipid hydroperoxide glutathione peroxidase-overexpressed cells

Mitochondria are major compartments in cells responsible for generating reactive oxygen species, which can cause the development of diabetes, Parkinson's disease and premature aging. Antioxidant systems in mitochondria are important for the prevention of diseases and reduction in the speed of aging. We investigated whether the reactive oxygen species generated in mitochondria induced the expression of metallothionein as an antioxidant. We compared the expression level of metallothionein mRNA in mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx)-overexpressed (PHGPx-ov) cells with that in control cells. These cells were treated with respiratory inhibitors, including rotenone and 2, 4-dinitrophenol; under these conditions, the PHGPx-ov cells were more resistant to cell death than the control cells. In addition, the intracellular reactive oxygen species level that was induced by these inhibitors was lower in PHGPx-ov cells than in control cells. This indicates that PHGPx degrades the membrane phospholipid hydroperoxide that is formed via the reactive oxygen species generated in mitochondria. The enhanced expression of metallothionein-I and metallothionein-II mRNA in rotenone-treated control cells was significantly decreased in rotenone-treated PHGPx-ov cells, suggesting that the hydrogen peroxide that is formed by superoxide anions generated in mitochondria diffuse into the cytosol and induce metallothionein mRNA expression. Conversely, the expression of manganese-superoxide dismutase (Mn-SOD) mRNA, which is localized in mitochondria, was not correlated with the intracellular reactive oxygen species level that was induced by rotenone treatment. These results suggest that metallothionein expression is sensitively and strictly regulated by the oxidative state that is induced by mitochondrial respiration.

Functional Analysis of Avian Metallothionein Isoforms: An Ecotoxicological Approach for Assessing Potential Tolerability to Element Exposure

To assess the inducibility of avian metallothionein (MT) genes and potential tolerability of their protiens to element exposure, we investigated the transcriptional inducibilities of cormorant (Phalacrocorax carbo) and mallard (Anas platyrhynchos) MT genes in CV-1 cells by elements and detoxification potencies of their in vitro synthesized MT proteins. About 1.3 kb of 5'-upstream regions were sequenced for cormorant MT1 and 2 genes, where two metal-responsive elements were identified. Both cormorant MT promoters were dose dependently activated by Cd, Zn, Cu, and CH3Hg, whereas no transactivation was detected by Pb, TI, Ag, inorganic Hg, Cr(VI), Cr(III), As(V), As(III), Ni, Co, Rb, and Bi, suggesting a shared transactivational mechanism of both MTs for specific elements. These findings support our previous results, where Cu and Zn concentrations were shown to be positively correlated with hepatic MT1/2 mRNA levels in wild cormorants. Comparison of EC50 and LOEL (lowest observed effect level) for each element revealed that Cd was the most potent inducer of MT1/2 promoters, followed by CH3Hg, Zn, and Cu. Since LOELs of CH3Hg for both MT promoters were higher than the hepatic levels in wild cormorants, hepatic CH3Hg concentration may not be high enough to induce MT mRNA in the wild population. Although LOELs of Cd were much lower than the hepatic concentrations detected in wild cormorants, no significant correlation was observed between hepatic Cd levels and MT mRNAs. This may be due to the masking effect of multiple elements, probably by Zn and/or Cu that were highly accumulated in wild cormorants. Cotreatment of Cd with Zn supported a possible suppression of Cd-induced MT expression by Zn in wild cormorants. MT1 and 2 proteins of cormorant and mallard endowed Escherichia coli with significantly higher growth rate than control to Cd exposure (500-1000 microM), implying that avian MTs could be involved in the detoxification of intracellular Cd. This study provides the first evidence on the inducibility of avian MT isoforms by specific elements and functional significance of each avian MT isoform in detoxifying intracellular heavy metals. Our in vitro approaches demonstrate their validity in predicting the response of MTs to element exposure in a wild avian population.

Effect of interleukin-6 neutralization on CYP3A11 and metallothionein-1/2 expressions in arthritic mouse liver

Rheumatoid arthritis is characterized by chronic inflammation of the synovial tissue. We examined the effect of interleukin (IL)-6 neutralization on the expression of cytochrome P450 or metallothionein-1/2 (metallothionein) during chronic phase inflammatory disease using rheumatoid arthritis model mice, human T-cell leukemia virus type I (HTLV-I) transgenic mice. Serum IL-6 concentrations of arthritis-developed HTLV-I transgenic mice were 129.9 ± 26.1 pg/ml. Moreover, signal transducer and activator of transcription (STAT) 1/3 phosphorylations was observed in arthritic HTLV-I transgenic mouse livers. CYP3A11 mRNA was more strongly reduced by the development of arthritis in HTLV-I transgenic mouse livers as compared with CYP2C29 or CYP2E1 mRNAs. CYP3A protein and testosterone 6β-hydroxylation activity also changed in a similar manner to the corresponding CYP3A11 mRNA level. On the other hand, metallothionein mRNA was significantly induced as compared with that of wild-type or non-arthritic mice. CYP3A suppression and metallothionein mRNA overexpression activity seen in the developed arthritic mice returned to the gene conditions of the non-arthritic HTLV-I transgenic mice by IL-6 antibody at 48 h after treatment. The present study has revealed that CYP3A11 and metallothionein expressions are affected by the release of IL-6 by arthritis and its systemic circulation, and neutralization of IL-6 recovered from the down-regulation of CYP3A11 mRNA and the induction of metallothionein mRNA in arthritic HTLV-I transgenic mice.

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1–1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.

Human Metallothionein Gene Expression Is Upregulated by .BETA.-Thujaplicin: Possible Involvement of Protein Kinase C and Reactive Oxygen Species

Recently, we discovered that beta-thujaplicin (BT) induces metallothionein (MT) expression in mouse keratinocytes, both in vivo and in vitro. However, the molecular mechanisms by which BT exerts its biological effects have not been elucidated. The purpose of this study is to explore the signal transduction pathway involved in the MT mRNA induction by BT. Using a HaCaT keratinocyte cell line, Northern blotting was performed for analyzing the human MT-IIA mRNA expression levels in combination with BT and a number of protein kinase (PK) inhibitors including H7, HA1004 and a PKC-specific inhibitor chelerythrin. CAT assays with the MT-IIA gene promorter-CAT construct were conducted for examining the transcriptional regulation by BT of MT. A free radical scavenger N-acetylcysteine (NAC) was used for analyzing a role of oxidative stress for the MT gene induction by BT. BT increased MT-IIA gene transcript levels and CAT activity in a dose-dependent fashion in HaCaT cells. The increase in MT-IIA mRNA levels and CAT activity were completely suppressed by H7 but not by HA1004. In addition, chelerythrin prevented BT-inducible MT-IIA promoter activation. Furthermore, NAC suppressed BT-inducible MT-IIA promoter activation. These results demonstrate that BT is a potent activator of the MT-IIA gene promoter and that PKC activation and reactive oxygen species are implicated in BT-inducible MT-IIA gene expression. BT may be a useful tool for dissecting the signal transduction pathway mediating MT-IIA promoter activation.

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.

Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6(-/-)) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6(-/-) mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn(2+) and (65)Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.

Effects of short-term copper exposure on gill structure, metallothionein and hypoxia-inducible factor-1α (HIF-1α) levels in rainbow trout ( Oncorhynchus mykiss)

Rainbow trout ( Oncorhynchus mykiss) were exposed to 1.65 μM of waterborne copper for 24 h. Fish were then transferred to metal-free water. Metallothionein mRNA induction in rainbow trout liver and gill tissue, hypoxia-inducible factor-1 (HIF-1α) accumulation in gill tissue and arithmetic mean thickness of gill epithelium ( H ar) were determined at 4 and 24 h of exposure as well as 48 h after transfer to metal-free water. The arithmetic mean distance from water to blood was significantly elevated after both 4 and 24 h of exposure ( H ar was 4.67 and 4.66 μm, respectively in exposed fish, compared to 3.81 and 3.62 μm for the corresponding control fish). During the 48 h recovery H ar returned towards the control values; the recovery value of 4.21 μm was significantly lower than values during exposures. There was also a significant increase in gill metallothionein mRNA levels after the 4 h exposure with MT/GAPDH ratio of 1.288 versus the control value of 0.988. In liver, metallothionein induction was not observed. HIF-1α protein showed an increased accumulation in gills after 4 h, with the HIF-1α/α-tubulin ratio of 0.562 being significantly higher than the 24 h exposure value of 0.232. These results suggest that exposure to copper for four hours causes hypoxia in the gill epithelium, which is adequate for the activation of HIF-1α.

Induction of Metallothionein mRNA in Mouse Lens Epithelial Cell by UV Irradiation

Induction of Metallothionein mRNA in Mouse Lens Epithelial Cell by UV Irradiation

Potential role of p53 on metallothionein induction in human epithelial breast cancer cells.

The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 μM CdCl2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79±1.36-fold. Other breast cancer cell lines which are p53-negative (p53−) and oestrogen-receptor-negative or weakly oestrogen-receptor-positive showed a small induction ranging from 1.40±0.10 to 3.65±0.30-fold. RT–PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61±0.08-fold, while in HCC1806 cells (p53−, oestrogen-receptor-negative) by 1.11±0.13-fold, and in MDA-MB-231 (p53−, oestrogen-receptor-negative) by 1.25±0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 μM CdCl2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10–40 μM CdCl2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl2, it peaked at 6–8 h and appeared again at 48 h for all concentrations of CdCl2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10–40 μM CdCl2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 μM CdCl2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p53− and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.British Journal of Cancer (2002) 87, 1019–1026. doi:10.1038/sj.bjc.6600549 www.bjcancer.com© 2002 Cancer Research UK

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.

Metallothionein protects against severe oxidative stress-induced apoptosis of human trophoblastic cells.

Oxidative stress induces cellular apoptosis. Many agents producing intracellular oxidative stress, including H(2)O(2) and steroid hormones, have also been found to induce metallothionein (MT) expression. Recently, MT has been recognized as potentially having antioxidant activity. This action may be essential for survival of terminally differentiated cells subject to oxidative stress, such as syncytiotrophoblasts, placental cells producing pregnancy hormones and forming the maternal-fetal barrier. We previously demonstrated an inverse relationship between basal MT expression and apoptotic incidence in the trophoblastic cell line, JEG-3. Using JEG-3 cells transfected with MT in sense or antisense orientation, we have examined here the effect of altered basal MT levels on trophoblastic function and apoptosis following treatment with H(2)O(2) or diethylstilbestrol (DES). Induction of MT mRNA was observed in control and transfected JEG-3 cells following exposure to severe oxidative stress. Changes in the localization of MT protein, however, were apparent after a low oxidative stress challenge. Exposure to H(2)O(2) resulted in a dose-dependent decrease in human chorionic gonadotropin secretion in all JEG-3 cultures regardless of basal MT expression, whereas no change was detected following DES treatment. With respect to apoptosis, a significant protective effect was observed proportional to the basal MT level. These results suggest that although MT does not ameliorate oxidative stress-induced perturbation of some trophoblastic functions, its expression is critical for protection of these cells from severe oxidative stress-induced apoptosis. MT thus appears to act as an anti-apoptotic antioxidant in trophoblastic cells.

Quantitative reverse transcription polymerase chain reaction of a molluscan metallothionein mRNA

A quantitative assay based on competitive reverse transcription polymerase chain reaction (RT-PCR) was developed for metallothionein (MT) mRNA of the mollusc Crassostrea virginica and applied to analysis of MT mRNA of hemocytes. The assay was based on titration of a competitive external standard cRNA derived from the coding region of the oyster MT mRNA. Serial dilutions of the cRNA standard were coamplified with a constant amount of total RNA using biotinylated primers common to both target and standard sequences. Amplified products were bound to streptavidin-coated plates and hybridized to sequence-specific fluorescein-labeled probes. Detection was based on single photon counting of chemiluminescence generated by an alkaline phosphatase-conjugated antifluorescein antibody. For quantification, the target chemilumincescence was normalized to that of the standard, and the amount of target MT mRNA in the sample was derived from the titration. Cadmium-induced MT mRNA equivalent to that in 180 hemocytes was easily detected, and, for routine quantitative analysis, was sufficiently sensitive to quantify basal and induced MT mRNA. Basal hemocyte MT mRNA of 133±8 (1 S.E.) amol per microgram total RNA was induced 5-fold to 573±14 amol per microgram total RNA by in vitro exposure to 15 μM CdCl 2 for 20 h.

Effect of ultraviolet A on ornithine decarboxylase and metallothionein gene expression in mouse skin.

Ultraviolet A (UVA) is known to induce the expression of many stress responsive genes due to the generation of reactive oxygen species (ROS). However, UVA's role in inducing metallothionein (MT) gene expression has not been studied. Furthermore, our group demonstrated that UVA enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of ornithine decarboxylase (ODC) activity in mouse skin (1). We examined the interaction of UVA, TPA and antioxidants on the induction of MT and ODC mRNA in mouse skin. Female CD-1 mice were exposed to UVA (19 J/cm2) and total RNA was isolated from the skin. Northern blot analysis for MT and ODC mRNAs was performed. ODC activity in mouse epidermis was also determined in some experiments. UVA induced MT mRNA in mouse skin; however, it did not increase ODC mRNA. 1,4-Diazabicylo-[2,2,2]-octane (DABCO), a singlet oxygen scavenger, reduced UVA-mediated induction of MT mRNA by 40%. The data suggest that ROS produced by UVA exposure may contribute to its ability to induce MT mRNA. UVA slightly enhanced TPA-mediated ODC mRNA induction, while it enhanced ODC enzyme activity 70%. UVA additively intensified TPA-mediated MT mRNA induction. alpha-Tocopherol pretreatment inhibited the induction of ODC enzyme activity by TPA treatment combined with UVA exposure (TPA + UVA); however, alpha-tocopherol had less of an inhibitory effect on ODC mRNA induction by TPA + UVA. Curcumin, a plant pigment, dramatically inhibited both TPA- and TPA + UVA-induced expression of ODC and MT genes. These results demonstrate that UVA can induce MT gene expression and enhance TPA-induced ODC and MT gene expression. The data further suggest that these effects are partially mediated by ROS.

Kinetics and magnitude of metallothionein induction by Cd, Cu, Hg and Zn in European flounder — calibration for environmental monitoring

The inductive response of metallothionein (MT) synthesis to heavy-metal exposure in many Phyla is well documented; however, before this response can be used and interpreted as an environmental biomarker of exposure, the kinetics and dose responses must be known. We have carried out a series of experiments where we have studied these parameters of MT mRNA and MT protein induction in different tissues (liver, kidney, gills) of European flounder after intraperitoneal (ip) injection of Cd (1–320 μg/kg), Cu (25–200 μg/kg), Hg (235–100 μg/kg) and Zn (1–20 mg/kg). Basal hepatic levels of 320 μg MT/g wet tissue for the wild caught flounder used were five times higher than in fish from a pristine area and 10–20 times higher than in plaice or turbot obtained from pristine areas. Transient increases in renal MT mRNA levels were only observed 4–10 days post injection (p.i.) of Cu or Hg and there were no significant increases in MT protein levels in any tissue. Doses of >5 mg/kg Zn induced MT mRNA levels three- to four-fold in all tissues 2–4 days p.i. but only produced a transient elevation in MT levels at 4 days p.i. There was no measurable response to low doses of Cd, doses of >80 μg/kg Cd had no significant effect on hepatic MT levels but caused a significant elevation in gill and kidney levels 10 and 21 days p.i. MT mRNA levels in gill and kidney were induced four- to five-fold 24 h p.i. of Cd (and remained elevated for >10 days) whilst a response was only observed in liver (two-fold) 4 and 10 days p.i. These laboratory results, obtained with a wild population exhibiting moderately elevated basal MT levels indicate that the response to metal exposure is too small and too highly dependent upon the metal and exposure time to provide a robust biomarker of contaminant effect. This supports our previous observation of analyses of flounders from the southern North Sea and Dutch estuaries and would indicate that caging studies with ‘ultra-clean’ animals would be required for any monitoring studies.

Metallothionein (MT) gene expression and cadmium-induced immunotoxicity in hemocytes of the eastern oyster Crassostrea virginica

Metal exposure is known to result in immunotoxicity in bivalve molluscs. To understand the relationship between metallothionein (MT) gene expression and Cd toxicity in oyster hemocytes, methods were developed for quantitative reverse transcription polymerase chain reaction of MT mRNA and selective inhibition of MT expression with antisense phosphorothioate deoxyoligonucleotides (S-ODN). In vitro Cd exposure of hemocytes at sub-cytolethal concentrations up to 15 μM CdCl 2 resulted in concentration-dependent increases in MT induction. This upper Cd concentration also corresponded with the first detection of inhibited reactive oxygen intermediate (ROI) generation and lysosomal neutral red retention. Further Cd increases caused a decline in induced MT mRNA, ROI generation, and neutral red retention. Cell viability was not affected until Cd concentrations exceeded 50 μM. Incubation of hemocytes with antisense S-ODN resulted in significant reductions in basal and Cd-induced MT mRNA as well as significant inhibition of ROI production. These findings suggest a protective role for MT at low concentrations of Cd and a functional role for MT in ROI production.

Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue‐specific inhibition of cadmium‐mediated metallothionein induction by 17β‐estradiol, 4‐OH‐PCB 30, and PCB 104

AbstractIn this study, we haveexamined the basallevel expression and tissue‐specificexpressionpatternsof metallothionein (MT) in Arctic char following metal and E2 (17β‐estradiol) treatment. To study the gene regulation in Arctic char, the two MT isoforms were isolated from a lambda‐ZAP hepatic cDNA library and characterized. Determination of basal MT mRNA and MT expression for 10 different tissues revealed a lack of correlation between MT mRNA and MT levels. The inducibility of MT mRNA and the correlation to resulting MT levels were then determined for liver and kidney. We found a more rapid and stronger induction of MT mRNA in liver than in kidney at day 1 and 3 postinjection, whereas the MT protein quantification showed higher MT levels in kidney than in liver at days 3 and 7 postinjection. These discrepancies indicate that differences in metal handling or posttran‐scriptional regulation of MT exists between tissues. Whereas metals induce MT synthesis, E2 inhibit the hepatic MT expression. To examine the tissue specificity of this inhibition, we determined the effect of 17pj‐estradiol (E2) and two estrogenic PCBs (4′‐OH‐PCB 30 and PCB 104) on Cd‐mediated MT induction in liver and kidney. Although E2 and the estrogenic PCBs inhibited cadmium‐mediated hepatic MT induction, these compounds did not interfere with renal MT induction.