Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS.

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell - inductively coupled plasma - mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.

METALLOTHIONEIN EXPRESSION IN A PARASITIC CRUSTACEAN, LAMPROGLENA CLARIAE (CRUSTACEA: COPEPODA), ON CLARIAS GARIEPINUS (TELOESTEI: CLARIIDAE) CORRESPONDS TO WATER QUALITY.

Globally, parasites are sensitive toward environmental changes, and, in some cases, they are even more sensitive than their hosts. However, there is limited knowledge on the physiological responses of parasites and their effects on their hosts in relation to environmental degradation. In this study, metallothioneins (MTs) were isolated and compared between the ectoparasite Lamproglena clariae and its host fish Clarias gariepinus. Differences in the levels of MTs in the parasite and host were compared to physicochemical water quality variables and metals to determine if MT expression was linked with changes in water quality. Clarias gariepinus individuals were sampled from 2 sites of differing water quality along the Vaal River using gill nets and assessed for L. clariae. Gill, muscle, and liver tissue of the host and L. clariae were collected and stored in liquid nitrogen for analysis of MT. Water and sediment samples were collected for metal analysis by inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-mass spectrometry. Nutrient levels and water hardness in water samples were assessed using spectrophotometry. MTs were quantified using spectrophotometry and size exclusion chromatography in the host and parasite, respectively. Infections by L. clariae differed between sites, with higher parasite intensity at the unpolluted Vaal Dam site. Concentrations of MT in host tissues and L. clariae were significantly higher at the polluted site, below the Vaal River Barrage, compared to the Vaal Dam site. Parasite MT concentrations were significantly lower compared to concentrations in the liver and gill tissue of C. gariepinus individuals. In conclusion, differences in the concentrations of MT and infection biology of L. clariae reflected the state of the environment and support the usefulness of this parasite and other Lamproglena spp. as bioindicators.

Metallothioneins alter macrophage phenotype and represent novel therapeutic targets for acetaminophen-induced liver injury.

Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

ZnO nanoparticle-based seed priming modulates early growth and enhances physio-biochemical and metabolic profiles of fragrant rice against cadmium toxicity.

BACKGROUND: Cadmium (Cd) is amongst the most toxic heavy metals that severely affects crop growth, whereas application of nanoparticles (NPs) to negate the toxic effects of heavy metals could be an effective management approach. In the present study, the seeds of two fragrant rice varieties i.e., Yuxiangyouzhan and Xiangyaxiangzhan under normal and Cd stress conditions i.e., 0 and 100 mg L- 1 applied with four levels of ZnO NPs i.e., 0, 25, 50, and 100 mg L- 1. RESULTS: Seed priming with ZnO NPs had no significant effect on the seed germination (p > 0.05) however, it substantially improved the seedling growth and other related physiological attributes under the Cd stress. The mean fresh weight of the shoot, and whole seedling was increased by 16.92-27.88% and by 16.92-27.88% after ZnO NPs application. The root fresh weight, root-shoot length was also substantially improved under ZnO NPs treatment. Moreover, application of ZnO NPs induced modulations in physiological and biochemical attributes e.g., the superoxide dismutase (SOD) activity in root and shoot, the peroxidase (POD) activity and metallothionein contents in root were increased under low levels of ZnO NPs. The α-amylase and total amylase activity were improved by ZnO NPs application under Cd Stress. Besides, modulation in Zn concentration and ZnO NPs uptake in the seedling were detected. The metabolomic analysis indicated that various pathways such as alanine, aspartate and glutamate metabolism, phenylpropanoid biosynthesis, and taurine and hypotaurine metabolism were possibly important for rice response to ZnO NPs and Cd. CONCLUSION: Overall, application of ZnO NPs substantially improved the early growth and related physio-biochemical attributes in rice. Our findings provide new insights regarding the effects of ZnO NPs on seed germination, and early growth of rice, and its potential applications in developing crop resilience against Cd contaminated soils.

Spatial expression of metallothionein, matrix metalloproteinase-1 and Ki-67 in human epidermal wounds treated with zinc and determined by quantitative immunohistochemistry: A randomised double-blind trial.

Reepithelialisation is fundamental to wound healing, but our current understanding largely relies on cellular and animal studies. The aim of the present randomised double-blind three-arm controlled trial was to correlate genuine epidermal wound healing with key proteins and topical zinc treatment in humans. Sixty wounds were produced using deroofed suction blisters in 30 healthy volunteers and randomised to topical zinc sulphate (n = 20), placebo (n = 20), or control (n = 20) treatment for 4 days. All wounds with perilesional skin were processed for automatic immunostaining of paraffin tissue sections with monoclonal antibodies against Ki-67, metallothionein (MT) and matrix metalloproteinase (MMP)-1. Protein expression was quantified by automated digital image analysis. Epidermal Ki-67 and MT labelling indices were increased in keratinocytes in the neoepidermis (∼1.1 mm) and at the wound edge (0.5 mm) compared to normal skin. Increased MMP-1 immunostaining was restricted to the neoepidermis. MT was robustly upregulated in the upper dermis of the wounds. Zinc treatment enhanced MMP-1 expression beneath the neoepidermis via paracrine mechanisms and MT under the neoepidermis and in the nonepithelialised wound bed via direct actions of zinc as indicated by the induction of MT2A mRNA but not MMP-1 mRNA in cultured normal human dermal fibroblasts by zinc sulphate. The present human study demonstrates that quantitative immunohistochemistry can identify proteins involved in reepithelialisation and actions of external compounds. Increased dermal MT expression may contribute to the anti-inflammatory activities of zinc and increased MMP-1 levels to promote keratinocyte migration.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

Metallothionein determination can be applied to learn about aquatic metal pollution and oxidative stress detoxification mechanisms through Problem-based Learning.

A simple, repeatable and inexpensive laboratory practice applied to teach and discuss aquatic metal pollution and oxidative stress detoxification mechanisms through biomarker analyses, as well as important ecotoxicology concepts, is presented herein. It has been implemented in a university in Brazil to both undergraduate and Master's and PhD students, indicating usefulness to all these levels. Students learned to detect metallothionein and reduced glutathione concentrations in biological samples and investigate several variables of interest in biomonitoring assessments. In addition, statistical correlations were used to indicate the potential dual role played by MT in aquatic organisms, allowing for biological inferences regarding both aquatic metal pollution and oxidative stress detoxification mechanisms and maturing of ecotoxicological and biomonitoring concepts discussed and presented both theoretically and integrated to the laboratory findings.

Protein Corona-Triggered Catalytic Inhibition of Insufficient POSS Polymer-Caged Gold Nanoparticles for Sensitive Colorimetric Detection of Metallothioneins.

Metallothioneins (MTs) are important biomarkers for the early diagnosis of heavy metal poisoning and malignancies. Convenient and cost-effective approaches for the rapid detection of MTs are therefore highly desirable for clinical monitoring. Herein, by taking advantage of the enzyme-mimetic activity of nanoparticles and protein corona-based recognition, insufficient polyhedral oligomeric silsesquioxane (POSS) polymer-caged gold nanoparticles (denoted as PP-AuNPs) are developed for the sensitive colorimetric analysis of MTs. In the presence of MTs, the catalytic reduction of yellow 4-nitrophenol to colorless 4-aminophenol is inhibited due to masking of the exposed PP-AuNPs catalytic surface with MTs corona. MTs are quantified by the presented color contrast with a superior sensitivity up to a 1.5 nM detection limit. Most importantly, the heavy metal ion- and aptamer-free PP-AuNPs platform exhibits excellent selectivity toward MTs over various ions, neutral biomolecules, and protein species, and successful applications are demonstrated by the detection of MTs in complex biological samples.

Morphostructural and immunohistochemical study for evaluation of nano-TiO2 toxicity in Armadillo officinalis Duméril, 1816 (Crustacea, Isopoda, Oniscidea).

Invertebrates are precious organisms in order to study environmental pollution. In particular, they appear to be suitable as a bioindicator species for pioneer ecotoxicity studies on new xenobiotics such as nanoparticles. In fact, they are able to absorb nanomaterials scattered in the environment in different ways and it's known the compartmentalization of nano-sized contaminants in selected tissues and intracellular organelles. Titanium dioxide represents the most used nanoparticulate, destined to become probably ubiquitous in the environment. Recently, some research has been published on the toxic potential of nano-TiO2 in several animal species. Among all invertebrates, Oniscidean Isopods are the only taxon of Crustaceans that has become completely terrestrial, known as excellent bioindicators and bioaccumulators. They have a digestive gland, the hepatopancreas, which is the location of election for the accumulation of pollutants. For this reason, they are considered efficient animal models to ecological studies. For this study, we collected Armadillo officinalis from Natural Oriented Reserve of "Vendicari" (Sicily, Italy), to evaluate the toxicity of titanium dioxide (TiO2 ) on their hepatopancreas, after a short period of exposure. We conducted morphostructural and immunohistochemistry assays. The results suggested a great capacity of the species of bioaccumulation of nanoparticles in the hepatopancreas, where a strong positivity to the metallothioneins was highlighted. Our study confirms that Oniscidean Isopods, in particular Armadillo officinalis, proved to be an appropriate indicator of pollution in terrestrial ecosystems from nanoparticles.

Detection of Metallothionein Proteins by Enzyme-Linked Immunosorbent Assay (ELISA).

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind to heavy metals. MTs play a key role in the homeostasis of metal ions, maintaining intracellular redox equilibria and free radical scavenging. In several studies, under different conditions such as cancer development, drug therapy and heavy metal stress, the unique structural changes and functional effects of MT were studied. Although several assays are available to monitor the content and type of Metallothionein (MT) from environmental samples or in biomedical assays, Enzyme-Linked Immunosorbent Assays (ELISA) became the preferred method of MT detection. ELISA is low in cost, specific, simple, and efficient. This review evaluates the advantages and disadvantages of using different types of ELISA in the detection of metallothioneins from environmental or clinical samples as well as ways of its validation and cross-validation.

Metallothionein protein and minichromosome maintenance protein-2 expression in adrenocortical tumors.

AIM: Some resected adrenal-confined adrenocortical carcinomas metastasize and others not. The present study was designed to evaluate the expression of metallothionein protein (MT) and minichromosome maintenance protein-2 (MCM2) in adrenocortical carcinomas and adrenocortical adenomas, and to test the correlation between this and adrenocortical carcinoma aggressiveness. MATERIALS AND METHODS: The study comprised 14 patients operated on for adrenocortical carcinoma, 15 operated on for adrenocortical adenoma and 2 with normal adrenals. Hematoxylin-eosin staining was used for histological evaluation under light microscopy, and sequential sections were used for MCM2 and MT staining. RESULTS: In normal adrenals, positive staining was weak for MT and zero for MCM2. Rates of positive staining for MT and MCM2 were significantly higher in adrenocortical carcinomas than in adrenocortical adenomas (P=0.008 and P<0.001, respectively). In adrenocortical carcinomas, a significant positive correlation was found between MCM2 staining and Weiss revisited score (P=0.022) but not for Weiss score, and a significant positive correlation was found between MCM2 and mitotic rate on histology (P=0.033). MCM2 but not MT staining was also shown to correlate significantly with stage IV carcinoma (P=0.008 and P=0.165, respectively). CONCLUSION: MCM2 and MT are overexpressed in adrenocortical carcinoma, and MCM2 expression correlates significantly with metastatic disease.

Immune response of juvenile common carp (Cyprinus carpio L.) exposed to a mixture of sewage chemicals.

Pharmaceuticals and household chemicals are important components of municipal sewage. Many of them are biologically active, disrupting not only hormonal regulation of aquatic animals but also, indirectly, disturbing their immunological protection. In the environment, chemicals rarely act as individual substances, but as elements of mixtures. Therefore, the aim of this study was to check whether the acute laboratory exposure of common carp juveniles to a mixture of ibuprofen, sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and 17 α-ethynylestradiol in increasing concentrations, modifies the levels of innate immunity (lysozyme, C-reactive protein) as well as general stress (metallothioneins, heat shock proteins HSP70) markers in brain, liver, gills, spleen and mucus. The levels of the markers were measured by an immunodetection technique. Not only do the pharmaceuticals and household chemicals impair immunological reactions of young carp in various tissues but also do that in a concentration-dependent manner in the liver, gills, spleen and mucus. This has a very important implication, since it may result in higher sensitivity of young fish to pathogens due to energy allocation to defence processes. The comparisons of the pattern of stress reactions in the studied organ samples indicated that mucus appeared to be a good, non-invasive material for monitoring of environmental state and fish conditions.

Effect of dietary zinc pectin oligosaccharides chelate on growth performance, enzyme activities, Zn accumulation, metallothionein concentration, and gene expression of Zn transporters in broiler chickens1.

This study was to investigate the effect of zinc pectin oligosaccharides chelate (Zn-POS) on growth performance, serum enzyme activities, tissue zinc accumulation, metallothionein (MT) concentrations, and gene expression of zinc transporters (ZnT) in broilers. Five hundred forty 1-d-old Arbor Acres broiler chicks were randomly assigned to 5 dietary groups with 6 replicates of 18 birds per replicate. The diets were formulated with the same supplemental Zn level (80 mg/kg diet) but different amount of the Zn-POS: 0, 200, 400, 600, and 800 mg Zn-POS/kg diet. ZnSO4 was used to adjust to the desired amount of the Zn (80 mg/kg) in the Zn-POS diets. Broilers were fed with the experimental diets for 42 d including the starter (days 1 to 21) and grower (days 22 to 42) phases. Our results showed that dietary supplementation of Zn-POS linearly and quadratically increased (P < 0.05) the average daily gain and gain-to-feed ratio during 22 to 42 d and 1 to 42 d as well as body weight on day 42, whereas reduced (P < 0.05) the sum of mortality and lag abnormalities in broilers on day 42. Besides, serum alkaline phosphatase and copper-zinc superoxide dismutase activities increased (P < 0.05) linearly and quadratically in response to dietary Zn-POS supplemental level on day 42. Dietary Zn-POS supplementation increased Zn accumulation in serum (linear, P < 0.05), liver (linear, P < 0.05), and pancreas (linear and quadratic, P < 0.05). In addition, Zn-POS supplementation linearly and quadratically increased (P < 0.01, P < 0.05, respectively) MT concentrations in liver and pancreas of broilers. Pancreatic mRNA levels of MT, ZnT-1, and ZnT-2 increased (P < 0.05) linearly and quadratically, and the mRNA expression of metal response element-binding transcription factor-1 increased linearly (P < 0.05), in response to dietary Zn-POS supplementation. In conclusion, supplementation of Zn-POS in the diet increases Zn enrichment in the metabolic organs such as liver and pancreas and promotes productive performance in broilers.

Seasonal variation of heavy metals and metallothionein contents in Asian swamp eels, Monopterus albus (Zuiew, 1793) from Tumpat, Kelantan, Malaysia.

BACKGROUND: Levels of toxic metal exposure in indigenous inhabitants are key bioindicators of the severity of environmental contamination. This study measured the seasonal variation of heavy metals and metallothionein (MT) contents in Asian swamp eels (Monopterus albus) from a paddy field situated in Tumpat, Kelantan, Malaysia, to identify prevalence, patterns and associations and togain insight on the suitability of MT as a biomarker for metal exposure. METHODS: Gill, muscle and liver tissues of M. albus (n = 50) sampled during the ploughing, seedling, growing and harvesting phases of rice growing were collected. The concentrations of copper (Cu), zinc (Zn), lead (Pb), nickel (Ni), and cadmium (Cd) in these tissues were determined by flame atomic absorption spectrometry. MT from each sample was isolated and purified, and subsequently quantitated using UV spectrophotometry. Associations between metal and MT concentrations, season and tissue type were evaluated using Pearson correlation and ANOVA with post-hoc Tukey HSD analysis. RESULTS: Zn was present in higher quantities in gill and liver tissues, while Cu levels were elevated solely in liver. Patterns of non-essential metal accumulation were varied: Cd was detected in low concentrations in all tissues, while Pb and Ni were abundant in gill tissues across all seasons. MT concentration in liver tissue was consistently higher than that found in muscle or gill tissue, except during the growing phase. Moreover, significant correlations (P < 0.05) were observed for Cd, Ni, and Zn when MT was employed as metal exposure biomarker. However, no significant association was found between high Pb and Ni levels and MT concentration in gill tissue.Variation of bioaccumulation rates of heavy metals among the different tissues was observed. Some of these metal concentration differences were found to be associated with MT concentration and, by extension, to its high metal-binding capacity. CONCLUSIONS: Significant liver MT-Zn, MT-Cd, and MT-Ni correlations found in this study emphasised the role of metallothionein as a biomarker for exposure of zinc, cadmium and nickel metals in M. albus.

Protection of dietary selenium-enriched seaweed Gracilaria lemaneiformis against cadmium toxicity to abalone Haliotis discus hannai.

Seaweed Gracilaria lemaneiformis is the main dietary source of the abalone mariculture industry in China. In this study, we examined the protection of selenium (Se)-enriched G. lemaneiformis against cadmium (Cd) toxicity in the abalone, Haliotis discus hannai, using various indices including metal concentration (Se and Cd), growth rate, GPx enzymatic antioxidants, and metallothionein (MT) concentration over a period of 28 days of exposure. The growth rates and Se contents increased significantly in abalones fed with Se-enriched G. lemaneiformis, while the toxicity of Cd was reduced. Seven to 12 days Cd exposure to the Se-enriched G. lemaneiformis not only affected GPx activity but the MT levels fluctuated irregularly. MT concentrations increased after 3 days exposure and then gradually decreased to the control level after Day 7. There were statistically significant positive correlations between MT levels, GPx activity and Se concentrations, and negative relationships between MT levels, GPx activity and Cd levels in abalones. These findings suggest that Se-enriched Gracilaria protects abalone against Cd toxicity. The possible mechanism is the induction of MT with a concomitant increased capacity of GPx enzymatic antioxidants.

Metallothionein Induced Time Dependent Histomorphometric Analysis of Clarias gariepinus Exposed to Cadmium.

Cadmium (Cd), an extremely toxic heavy metal is extensively used in modern era because of its constructive chemical and physical properties. Recently Cd contamination was estimated in India's major cities fresh water ecosystem, which may have firm impact on human health. Hence, this study was aimed to detect the time dependent effect of cadmium in fresh water fish C. gariepinus, a bioindicator species of water pollution. In a controlled environment, fishes were exposed to cadmium for different duration and analyzed for Cd accumulation. Cd induced toxicity was assessed by estimating metallothionein biomarker protein of heavy metal toxicity and histomorphometric changes in liver and kidney. Our results revealed that fish exposed to Cd induced apoptosis in fish tissues via induction of caspases and in contrast the metallothionein was also increased consistently with different doses of Cd exposure. Hence we conclude Cd induced structural damages to fishes are attributed to induction of caspases and estimating MT level in tissues can be effective biomarker to analyze the effect of acute environmental exposure to Cd.

Metallothioneins may be a potential prognostic biomarker for tumors: A Prisma-compliant meta-analysis.

BACKGROUND: Metallothioneins (MTs) were reported to be associated with many kinds of tumors' prognosis, although MTs expression varied greatly among tumors. To assess the prognostic value of Metallothioneins (MTs) in different kinds of tumors, comprehensive literature search was conducted to perform a meta-analysis. METHODS: Eligible studies were identified by PubMed, MEDLINE, Web of Science (WOS), the Cochrane Library of Systematic Reviews, EMBASE, China National Knowledge Infrastructure (CNKI), WANFANG database and SinoMed database up to December 2017, which was designed to assess the prognostic value of MTs in different kinds of tumors. The main endpoint events were overall survival (OS) and disease-free survival (DFS). Hazard ratios (HRs) and its variance were retrieved from the original studies directly or calculated using Engauge Digitizer version 4.1. Random or fixed effects model meta-analysis was employed depending on the heterogeneity. Publication bias was evaluated by funnel plots, Begg and Egger tests. RESULTS: A total of 22 studies were enrolled in this meta-analysis, including 2843 tumor tissues (1517 were MTs negative/low, and 1326 were MTs high). Results showed that there was significant association between MTs expression and tumors' OS (HR = 1.60; 95%CI 1.34∼1.92, P < .00001). Subgroup analysis showed that high level of MTs expression was associated with prolonged OS in liver cancer (HR = 0.65, 95%CI 0.48∼0.89, P = .007), but it was on the contrary in the tumor of ovary (HR = 1.47, 95%CI 1.01∼2.14, P = .04), bladder (HR = 1.71, 95%CI 1.21∼2.42, P = .002), intestine (HR = 3.13, 95%CI 1.97∼4.97, P < .00001), kidney (HR = 3.31, 95%CI 1.61∼6.79, P = .001). However, there was no significant association between MTs expression and OS in breast (HR = 1.02, 95%CI 0.69∼1.51, P = .93). CONCLUSIONS: MTs could be taken as a potential prognostic biomarker for tumors, and uniqueness of MTs prognostic value in liver cancer deserved further study.

The Zinc-Metallothionein Redox System Reduces Oxidative Stress in Retinal Pigment Epithelial Cells.

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques. The Zn-MT system was induced in an in vitro model of RPE cells and determined by elemental mass spectrometry with enriched isotopes and mathematical calculations. Induced-oxidative stress was quantified using fluorescent probes. We observed that 25, 50 or 100 µM of zinc induced Zn-MT synthesis (1.6-, 3.6- and 11.9-fold, respectively), while pre-treated cells with zinc (25, 50, and 100 µM) and subsequent 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) treatment increased Zn-MT levels in a lesser extent (0.8-, 2.1-, 6.1-fold, respectively), exerting a stoichiometric transition in the Zn-MT complex. Moreover, AAPH treatment decreased MT levels (0.4-fold), while the stoichiometry remained constant or slightly higher when compared to non-treated cells. Convincingly, induction of Zn-MT significantly attenuated oxidative stress produced by free radicals' generators. We conclude that the stoichiometry of Zn-MT plays an important role in oxidative stress response, related with cellular metal homeostasis.

Increased Expression Levels of Metalloprotease, Tissue Inhibitor of Metalloprotease, Metallothionein, and p63 in Ectopic Endometrium: An Animal Experimental Study.

OBJECTIVE: To characterize the patterns of cell differentiation, proliferation, and tissue invasion in eutopic and ectopic endometrium of rabbits with induced endometriotic lesions via a well- known experimental model, 4 and 8 weeks after the endometrial implantation procedure. METHODS: Twenty-nine female New Zealand rabbits underwent laparotomy for endometriosis induction through the resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of animals (one with 14 animals, and the other with15) were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised along with the opposite uterine horn for endometrial gland and stroma determination. Immunohistochemical reactions were performed in eutopic and ectopic endometrial tissues for analysis of the following markers: metalloprotease (MMP-9) and tissue inhibitor of metalloprotease (TIMP-2), which are involved in the invasive capacity of the endometrial tissue; and metallothionein (MT) and p63, which are involved in cell differentiation and proliferation. RESULTS: The intensity of the immunostaining for MMP9, TIMP-2, MT, and p63 was higher in ectopic endometria than in eutopic endometria. However, when the ectopic lesions were compared at 4 and 8 weeks, no significant difference was observed, with the exception of the marker p63, which was more evident after 8 weeks of evolution of the ectopic endometrial tissue. CONCLUSION: Ectopic endometrial lesions seem to express greater power for cell differentiation and tissue invasion, compared with eutopic endometria, demonstrating a potentially invasive, progressive, and heterogeneous presentation of endometriosis.

OBJETIVO: Caracterizar o padrão de diferenciação celular, proliferação e invasão tecidual em endométrio eutópico e ectópico de coelhas com lesões de endometriose induzidas por um modelo experimental 4 e 8 semanas após o procedimento de implantação endometrial. MéTODOS: Vinte e nove coelhas fêmeas Nova Zelândia foram submetidas a laparotomia para indução de endometriose através da ressecção de um dos cornos uterinos, isolamento do endométrio e fixação do tecido no peritônio pélvico. Dois grupos de animais (14 animais em um grupo e 15 animais no outro) foram sacrificados 4 e 8 semanas após a indução da endometriose. A lesão foi excisada junto com o corno uterino contralateral para determinação da presença de glândulas e de estroma endometrial. Reações de imunohistoquímica foram realizadas no tecido endometrial eutópico e ectópico para análise dos seguintes marcadores: metaloprotease (MMP9) e inibidor tecidual da metaloprotease 2 (TIMP-2), os quais estão envolvidos na capacidade de invasão do tecido endometrial; e metalotioneina (MT) e p63, os quais estão envolvidos na diferenciação e proliferação celular. RESULTADOS: A intensidade da imunomarcação para MMP9, TIMP-2, MT e p63 foi mais alta nos endométrios ectópicos do que nos endométrios eutópicos. Contudo, quando as lesões foram comparadas entre 4 e 8 semanas, nenhuma diferença foi observada, com exceção do marcador p63, o qual foi mais evidente depois de 8 semanas de evolução do tecido endometrial ectópico. CONCLUSãO: Lesões endometriais ectópicas parecem expressar maior poder de diferenciação celular e de invasão tecidual comparadas com endométrios eutópicos, demonstrando o potencial de invasão, de progressão e de apresentação heterogênea da endometriose.

The application of capillary electrophoresis, mass spectrometry and Brdicka reaction in human and rabbit metallothioneins analysis.

BACKGROUND: Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. OBJECTIVES: The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. MATERIAL AND METHODS: Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. RESULTS: In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. CONCLUSIONS: The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.