One stone two birds: conjugation of graphene oxide with Rg3–doxorubicin reduces its toxicity and downregulates metallothionein expression for improved liver-targeted therapy

Thallium induces metallothionein gene expression in Huh-7 human hepatoma cells.

The involvement of protein kinase C (PKC) in the induction of metallothionein (MT) gene expression by non-metallic compounds has been reported. However, whether PKC participates in the metal-induced MT gene transcription remains unclear. We used PKC inhibitor, H7 and cherelythrine, to treat CHO CdR and GH3 cells, and found that both Cd- and Zn-induced MT gene expressions were blocked. Protein kinase A (PKA) was apparently not involved in the induction since PKA inhibitor, HA1004, did not affect the expression of MT gene. By using constructs with a reporter gene linked to different regions of MT promoter, we observed that transcription factor API was not associated with the induction. The inactivation of MT gene expression by PKC inhibitor was not due to block of Cd transport since cellular Cd content was not affected by the inhibitor. However, the PKC inhibitor dramatically reduced cellular Zn accumulation when stimulated by Zn ions. Further analyses of MT transcriptional factor, MTF-1, by semi-quantitative reverse transcriptase-polymerase chain reaction indicated that MTF-1 gene expression was not changed by the PKC inhibitor. We also found that vanadate, a phosphatase inhibitor, increased both basal and induced level of MT gene expression. These results suggest that MT gene expression induced by metal ions involves a PKC mediated phosphorylation pathway.

Avian Metallothioneins: Structure, Regulation and Evolution

Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations

Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT

Background The aim of this study was to investigate the effects of selenium, zinc, insulin, and metallothionein on oxidative damage and metallothionein (MT) gene expression levels in streptozotocin (STZ)-induced type 1 diabetic rats exposed to Cd. Methods Rats were categorized under eight groups (control, STZ, Cd, STZ + Cd, Group 5, Group 6, Group 7, and STZ + Cd + MT [n:8/group]) were used. After diabetes was induced by STZ (55 mg/kg, i.p.), Cd was administered (1 mg/kg CdCl, orally) for 4 weeks. In cadmium-treated groups selenium (Na2SeO3 1.5 mg/kg, i.p.), zinc (ZnSO4 10 mg/kg via oral gavage), insulin (insulin glargine, 2U/day, s.c.), and MT (1mg/kg, every other 10 days, s.c.) were administered. MT gene expression levels, MDA levels, GPx, SOD, and CAT activity levels were determined in liver and kidney tissues. Results MT gene expression and MDA levels increased (p < 0.05) while GPx and SOD activity levels decreased (p < 0.05) in STZ, Cd, and STZ + Cd groups. In Group 5, Group 6, Group 7, and Group 8 groups MT gene expression and MDA levels were decreased while GPx and SOD activity levels were increased (p < 0.05). CAT activity significantly increased (p < 0.05) in STZ + Cd group while there were no significance in other groups (p > 0.05). Compared to the control, Group 5, Group 6, Group 7, and Group 8 groups provided no difference for alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine levels (p > 0.05). Conclusions Our results suggest that Se, insulin, Zn and MT may have protective effects against hepatotoxicity and nephrotoxicity caused by Cd exposure in diabetic rats by reducing oxidative stress and MT gene expression levels.

Metallothionein (MT) gene expression is increased in cadmium resistant Chinese hamster ovary cells (CHO Cd(R)) upon medium (regular or serum-free) change during culturing. Among the major components of the medium, NaHCO3 was found to be able to induce MT gene expression in a dose- and time-dependent manner. The same effect was observed with other alkaline solutions, such as HEPES and NaOH. Using MT promoter-luciferase reporter gene constructs, we found that the presence of metal response elements (MREs) in the promoter region is necessary for NaHCO3-induced MT gene transcription. This finding is further supported by the observation that the binding activity between the metal-responsive transcription factor 1 (MTF-1) and the MRE were increased after NaHCO3 treatment. Following NaHCO3 treatment, an increase in cell proliferation was observed in CdR cells but not in the parental CHO K1 cells that do not express MT transcripts due to MT gene methylation. Using synchronized cells, an increase in cell proliferation was observed 9 h after NaHCO3 addition. Notably, proliferation of CHO K1 cells was increased when transfected with an MT gene. The effect of MT on cell growth was affirmed by treating CHO K1 cells with 5-azacytidine (Aza) to demethylate the MT gene. Proliferation increased in Aza-treated CHO K1 cells after NaHCO3 treatment. These results demonstrate that NaHCO3 stimulates MT gene expression and causes an enhancement of cell proliferation in CHO cells.

Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney

• Expression and regulation of Arabidopsis metallothionein (MT) genes were investigated to examine the functions of MTs in plants. • To examine the tissue-specific expression of MT genes, GUS reporter gene activity driven by promoters of MT1a, MT2a, MT2b and MT3 was analysed in transgenic plants. • MT1a and MT2b are expressed in the phloem of all organs and are copper (Cu)-inducible; MT2a and MT3, by contrast, are expressed predominantly in mesophyll cells and are also induced by Cu in young leaves and at root tips. Expression of MT genes is highly induced by Cu in trichomes and increases during senescence. Expression of MT4 genes is restricted to seeds. • We propose that plant MTs have distinct functions in heavy metal homeostasis, especially for Cu: MT1a and MT2b are involved in the distribution of Cu via the phloem, while MT2a and MT3 chaperone excess metals in mesophyll cells and root tips. These functional capabilities may allow MTs to play a role in mobilization of metal ions from senescing leaves and the sequestration of excess metal ions in trichomes.

Differential expression of metallothionein (MT) gene by trace metals and endocrine-disrupting chemicals in the hermaphroditic mangrove killifish, Kryptolebias marmoratus

Metal-induced metallothionein gene expression can be inactivated by protein kinase C inhibitor

Cadmium (Cd) is produced mainly as a by-product of zinc mining. In Thailand, the largest zinc mine is located in the Mae Sot district, Tak Province. Samples of Monopterus albus were collected from paddy fields in 4 sites, three downstream and one upstream from the zinc mine. The upstream site was considered to be uncontaminated while the three downstream sites were considered to be contaminated with Cd. Studies on the accumulation level of cadmium were conducted on the liver of the fish using the atomic absorption spectrophotometer technique. The metallothionein (MT) gene expression level in the liver, as a potential biomarker for long-term Cd exposure in their natural habitat, was also assessed. The level of hepatic MT gene expression was performed by quantitative real-time PCR. The result showed that Cd accumulation in the liver was much higher in swamp eels collected from the downstream sites when compared to those collected from the upstream site. The hepatic MT level in the upstream site was 0.75-fold, while the other three downstream sites were 0.36-, 4.44- and 0.94-fold. There is no parallel correlation between hepatic cadmium levels and hepatic MT gene expression. This study then suggests that MT gene expression biomarkers might be not suitable for swamp eels with prolonged exposure to Cd.

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment.

The regulation of metallothionein (MT) gene expression as important part of the detoxification machinery is only scarcely known in invertebrates. In vertebrates, MT gene activation is mediated by the metal-transcription factor 1 (MTF-1) binding to metal response elements (MREs). In invertebrates, the mechanisms of MT gene activation seems to be more diverse. In some invertebrate species, MTF-1 orthologues as well as their ability to activate MT genes via MREs have been uncovered. Although earthworm MTs have been well studied, a MTF-1 orthologue has not yet been described and MT gene activation mechanisms are largely unknown. Analyses of the earthworm wMT2 promoter by reporter gene assays have been performed. We could show that the wMT2 promoter was active in mouse embryonic fibroblasts (NIH/3T3) as well as in mouse MTF-1−/−cells (DKO7). The presence of mouse MTF-1 (mMTF1) led to a significant increase in reporter gene activity. We observed that cadmium as well as zinc had an effect on promoter activity. In the presence of zinc, promoter activity doubled in NIH cells, however, we did not observe a significant effect in the DKO7 cell line. Cadmium decreased promoter activity in DKO7 cells, but this effect could be reversed by providing mMTF1 in a co-transfection experiment. We suggest that MT gene expression in the earthworm is not entirely dependent on a MRE binding protein. Interestingly, the shortest promoter fragment including MRE1 showed the highest promoter activity under control conditions.

Selenium (Se), one of the most widely distributed elements of the earth’s crust, is required in trace amounts for normal growth and development of biological activity but its increasing level in soil poses productivity problems in many crops including sugarcane. In the present investigation, a promising line of sugarcane (CoLk 94184) was used to assess the impact of selenium on growth, physio-biochemical attributes vis-a-vis expression of metallothionein (MT) gene. Single bud setts of sugarcane (Saccharum spp. hybrids) was planted with differential levels of selenium (sodium selenite) viz., 0, 10, 50 and 100 ppm under soil tray culture conditions. At higher concentrations (50 and 100 ppm Se), symptoms of metal toxicity as stunted growth, reduced plant height, vigor, root, shoot weight and leaf chlorosis were observed. Biochemical analysis revealed reduction in content of chlorophylls, carotenoids, proline and induction of lipid peroxidation in terms of malondialdehyde content and higher activity of peroxidase enzyme. qRT-PCR analysis indicated increase in expression of MT gene in leaf tissue with an increase in Se supply and highest expression was observed at 50 ppm Se. At 100 ppm supply, adverse effect of Se was very severe and a minor increase in expression of MT gene was observed. Results suggest that MT gene is related to the Se homeostasis which in turn helps in tolerance to Se toxicity in sugarcane.