Gene expression profiles in the liver and kidney of metallothionein-null mice

It has been reported that the expression of certain genes was altered in rodent cells lacking metallothioneins (MTs). To further explore the effects of MT deficiency, we screened genes differentially expressed in the liver and kidney of MT-null mice by cDNA microarray analysis. In the liver, 29 of 8737 genes analyzed were altered in their expression levels: 19 and 10 genes were up-regulated and down-regulated, respectively. Particularly, 14 of the 29 genes were related to energy metabolism, and some of these suggested that loss of MTs might lead to obesity and irregular ATP synthesis. In the kidney, 41 differentially expressed genes were observed: 27 and 14 genes were up-regulated and down-regulated, respectively. Eleven of the 41 genes were also related to energy metabolism. Microarray results were confirmed by Northern blot analysis for five of the energy metabolism-related genes.     

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C₃-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 × 10¹¹ M⁻¹ s⁻¹, demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions.     

The genotoxic effect of carcinogenic PAHs, their artificial and environmental mixtures (EOM) on human diploid lung fibroblasts

Long-term exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. One potential mechanism of estrogen carcinogenesis involves catechol formation and these catechols are further oxidized to electrophilic/redox active o-quinones, which have the potential to both initiate and promote the carcinogenic process. Previously we showed that 4-hydroxyequilenin (4-OHEN) autoxidized to an o-quinone and caused a variety of damage to DNA. Since these deleterious effects could contribute to gene mutations, we investigated the Chinese hamster V79 cells to ascertain the relative ability of estradiol, 4-hydroxyestradiol, 17β-hydroxyequilenin, 4,17β-hydroxyequilenin, estrone, 4-hydroxyestrone, equilenin, and 4-hydroxyequilenin to induce the mutation of the hypoxanthine–guanine phosphoribosyltransferase (hprt) gene. All the 4-hydroxylated catechols induced significantly more colony formations in V79 cells as compared to the parent phenols at 100 nM, suggesting that the catechol estrogen metabolites are more mutagenic towards the hprt gene than estrogens. Since 4-OHEN induced the highest mutation frequency, we examined a biomarker for transformation potential of this compound in MCF-10A cells using an anchorage-independent growth assay. Although 4-OHEN induced anchorage-independent growth of these cells, the isolated clones were not able to grow as tumors in vivo when injected into nude mice. These cells were assayed for genetic changes using cDNA microarrays. Real time RT-PCR confirmation of some of the differentially expressed genes showed down-regulation of metallothionein 2A, p53, BRCA1, and c-myc. Moreover, we showed the involvement of other genes important in cell transformation and oxidative stress, strengthening the hypothesis that this mechanism plays a considerable role in 4-OHEN-induced anchorage-independent growth.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

Variation in metallothionein gene expression is associated with adaptation to copper in the earthworm Dendrobaena octaedra

Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins.     

Different patterns in the induction of metallothionein mRNA synthesis among isoforms after acute ethanol administration

The induction of metallothionein (MT) isoform synthesis was investigated in mouse cerebral cortex 18 h after oral ethanol administration. The expression of MT-I isoform mRNA increased in a dose-dependent manner after ethanol loading at doses between 2 g/kg (ethanol/body weight) and 8 g/kg. Lipid peroxide formation, measured as the amount of malondialdehyde-reactive substances, remained at the control level after all of the administered ethanol doses. The expression of MT-III isoform mRNA remained at the control level up until an ethanol loading dose of 4 g/kg and then finally increased to a significant level at a dose of 8 g/kg, which is almost the LD₅₀ for oral ethanol in mice. The different patterns of MT synthesis induction among MT isoforms suggests that the MT-I isoform, which is ubiquitous in mammalian tissues, plays a significant role as an antioxidant. On the other hand, the MT-III isoform, which has a limited tissue distribution, especially in the central nervous system, seems to be implicated in tissue repair and/or protection against critical tissue injury.     

Bile and liver metallothionein behavior in copper-exposed fish

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2 mg L⁻¹) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC–ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.     

Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd(2+) ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd(3)Cys(9) and Cd(4)Cys(11) based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.