Chronic exposure to hypoxia occurs in lowland populations, including soldiers, adventurers, tourists, and porters, during their ascent to high altitude. Hypobaric hypoxia induces oxidative stress and impairs the metabolism of micro minerals, including selenium, zinc, iron, and copper. The role of metalloproteins and their associated microminerals in preventing oxidative stress at different altitudes is incompletely understood. In this study, plasma samples were collected from lowlanders at sea level (SL), moderate altitude (A1-1585m, A2-2290m), and high altitude (A3-3500m). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to quantify selenium (Se), zinc (Zn), copper (Cu), and iron (Fe) levels. Antioxidant metalloproteins, including Copper-Zinc Superoxide dismutase (Cu-Zn SOD), Glutathione Peroxidase (GPx), Metallothionein (MT), Selenoprotein 1 (SEPP1), were quantified by enzyme-linked immunosorbent assay. Lipid peroxidation was assessed through malondialdehyde (MDA) and hydroperoxides through ferrous oxidation xylenol orange; FOX1 assay. We found that Se, Zn, Cu, and Fe levels were higher (p < 0.01) at A2 compared to SL and lower (p < 0.01) at A3 compared to A2. MT levels were higher (p < 0.01) at A1 and A3 compared to SL. SEPP1 levels were higher (p < 0.001) at A3 compared to SL, A1, and A2. Cu-Zn SOD levels were higher at A3 (p = 0.006). GPx levels remained consistent across all altitudes. MDA levels were significantly elevated (p = 0.013) at A3 compared to SL, while the FOX assay showed increased hydroperoxide levels (p < 0.001) at A2 and A3 as compared to SL. Our findings demonstrate that hypoxia leads to variations in micromineral levels at different altitudes, accompanied by elevated oxidative stress and dysregulated antioxidant metalloproteins potentially reflecting adaptive responses to oxidative stress at high altitude.

Heavy-metal contamination of wastewater-irrigated farmland in the Kano River Basin notably lead, cadmium, chromium, nickel, copper and zinc has been characterised repeatedly at the soil and tissue level, but local molecular tools for monitoring how crops respond to it are lacking. This paper presents the development, validation and first field application of a multi-gene quantitative real-time PCR (qPCR) panel targeting phytochelatin synthase (PCS1) and glutathione reductase (GR-1) detoxification related genes with actin (ACT) as the reference gene in tomato (Solanum lycopersicum). Primer performance was established by serial dilution standard curves and melt-curve specificity analysis: ACT amplified with an efficiency of 90.6% (slope -3.57, R² = 0.998), PCS1 produced a single specific product (melt temperature 83.5 ± 0.0 °C; R² = 0.971), and GR-1 produced a single product at 81.1 ± 0.2 °C. Metallothionein-2 (MT-2) primers were excluded on specificity grounds. The validated panel was then applied to root and shoot tissue of field-grown tomato sampled in December 2025 from three contaminated sites along a rural-to-industrial gradient Challawa, Wudil and Dambatta and a control at Bayero University, Kano. Exposure was from natural field contamination rather than controlled dosing. GR-1 was preferentially expressed in roots and most strongly at the most contaminated site while PCS1 expression was more evident in shoot tissue patterns consistent with the metal partitioning documented in the same agroecosystem in earlier studies indicating that the panel returns biologically coherent signal. The panel provides a locally validated, low-cost tool for transcript-level monitoring of metal detoxification in field crops.

Histopathological and molecular effects on the gills and kidneys of Oligosarcus argenteus chronically exposed to metal-contaminated tailings from the Fundão dam collapse.

Accurate and accessible screening tools for periodontitis are essential for early detection and disease prevention. This study evaluated a non-invasive diagnostic approach integrating sociodemographic data, self-reported oral health (SROH) questionnaires, and salivary biomarkers, using both conventional statistical and machine learning (ML) predictive models. Seventy-seven adults completed a validated SROH questionnaire and provided saliva samples for quantification of six biomarkers: interleukins (IL-1β, IL-6), tumour necrosis factor (TNF-α), matrix metalloproteinases (MMP-8, MMP-9), and metallothionein (MT). Participants were clinically classified as having (i) periodontal health, (ii) gingivitis, or (iii) periodontitis. Predictive models were developed using Logistic Regression (LR), Random Forest (RF), and Naive Bayes (NB) across three feature sets: (i) SROH, salivary biomarkers and sociodemographic, (ii) SROH and salivary biomarkers, (iii) SROH and sociodemographic and (v) SROH alone. Model performance was assessed using 10-fold cross-validation and standard evaluation metrics. The RF model trained on SROH, and salivary biomarkers achieved the highest accuracy with area under the receiver operating characteristic curve (AUC = 0.856), with superior precision (70.13%), sensitivity (0.701) and lower error rates (RMSE = 0.371) compared with NB (AUC = 0.795) and LR (AUC = 0.724) models in detecting periodontitis. This non-invasive, SROH and biomarker-integrated approach shows potential as a first-line screening and referral tool in primary care and population-based settings where comprehensive periodontal examination is not routinely available. Further validation in larger, more diverse populations is warranted to support clinical translation.

Spermidine alleviates sepsis-induced cardiomyopathy by improving mitochondrial quality and quantity via a Metallothionein 1-dependent antioxidant pathway.

Urban coastal lagoons are heavily impacted by untreated sewage, industrial effluents and diffuse pollution, heightening ecotoxicological risks. Herein, Nile tilapia (Oreochromis niloticus) was employed as a sentinel species concerning metal exposure at the Jacarepaguá Lagoon Complex (JLC), a critical case of ecological degradation under intense anthropogenic pressure in Rio de Janeiro, Southeastern Brazil. Samplings were conducted between 2022 and 2023 during the dry (autumn-winter) and rainy (spring-summer) seasons. Organosomatic indices and total (muscle) and subcellular (liver and muscle) metal levels were determined. Antioxidant system alterations were evaluated through superoxide dismutase (SOD), glutathione-S-transferase (GST), reduced glutathione (GSH), total antioxidant capacity (TAC) and metallothionein (MT) assessments. Lipid peroxidation (LPO) and protein carbonylation (PTC) levels were also determined as oxidative effect markers, while vitellogenin (VTG) was used as an estrogenic effect indicator. Arsenic (0.018-0.033mg L⁻¹), Mn (0.041-0.050mg L⁻¹), Rb (0.019-0.023mg L⁻¹), and V (0.008-0.010mg L⁻¹) were detected in water samples in both sampling seasons; while Fe was detected only in the dry season (0.023 ± 0.03mg L⁻¹). Subcellular As, Co, Cu, Fe, Mn, Ni, Pb, Rb, Ti, V, and Zn were quantified in both liver and muscle, albeit with no significant differences between seasons or sexes. Seasonal variations were noted for male VTG, although not statistically significant, with attenuation of sexual VTG differences from the dry (M/F = 0.67) to the rainy season (M/F = 1.20). The multibiomarker approach applied to wild tilapia under diffuse urban pollution indicates mild sublethal effects while highlighting the need for expanded, integrated monitoring of additional endocrine and oxidative biomarkers. Although centered on a specific lagoon complex system, these mechanisms recur in urban lagoons worldwide, indicating the need for consumption-risk evaluations and actionable remediation/management frameworks.

Zinc flux in skin: Identity and function of metallothionein 1\u2013positive cells in skin homeostasis

Industrial wastewater is a major source of heavy metal contamination, posing serious environmental and public health risks. In this study, indigenous heavy metal-resistant bacteria were isolated from industrial wastewater and evaluated for their bioremediation potential with a focus on metallothionein (MT) production. A total of 38 bacterial isolates were screened for resistance to lead, cadmium, and nickel, among which Pseudomonas aeruginosa and Bacillus spp. exhibited the highest tolerance levels. Three superior isolates (6M, 8M, and 21F) were selected based on minimum inhibitory and bactericidal concentrations, growth stability, and reproducibility. Molecular analysis confirmed the presence of MT-related genes, and protein profiling by SDS-PAGE revealed MT-associated bands with molecular weights of approximately 6-7kDa. Metal removal assays demonstrated removal efficiencies of up to 83.4% for Cadmium, 76.9% for Cobalt, and 69.9% for nickel by Bacillus thuringiensis under laboratory conditions. This study provides an integrated evaluation of indigenous industrial wastewater bacteria by correlating metallothionein gene detection, proteomic evidence of low-molecular-weight metal-binding proteins, and quantitative multi-metal removal efficiency within the same isolates. The findings highlight the potential application of these strains in heavy metal bioremediation and provide a foundation for future scale-up and wastewater treatment strategies.

Metallothionein (MT) is a cysteine-rich protein that displays a high redox activity, acts like an acute phase protein, and forms metal-thiolate clusters. However, little is known about how the presence of metals in MT or their absence in Thionein (T) modify the structure of the protein and if such differential conformations alter the protein-protein interactions that MT/T establishes with other proteins. Ribonucleases (RNases) are a group of enzymes that hydrolyze different classes of RNA and due to the relevance of their function, they are highly regulated by specific inhibitors. Earlier results showed a decrease in RNase activity during an inflammatory process that appears to be a consequence of a de novo synthesis of an unidentified RNase inhibitor (RI) (Brambila et al in Exp Mol Pathol 71:125–131, https://doi.org/10.1006/exmp.2001.2383 , 2001). Therefore, we performed experimental studies to evaluate the ability of MT/T to inhibit RNase A activity in vitro. Computational methods were utilized to characterize the tertiary structure of MT/T in the presence and absence of zinc ions and to evaluate the stability of a complex with RNase A. Our results show that only in the absence of metals, T, the homodimeric protein has an inhibitory effect on RNase A activity via a mechanism that involves the formation of a complex between RNase A and T by specific interactions. Also, the involvement of residues, including cysteines that coordinate the cations, that participate in intermolecular interactions in the metal-bound form, aids in explaining the metal dependent inhibitory properties in the metal-free protein. This work suggests an additional role of T in the regulation of enzyme activity and protein synthesis.

This study for the first time investigated the effects of poultry litter containing cypermethrin (740μg kg-1 d.w.) and metals on Folsomia candida, assessing lethality, behavior, reproduction, and oxidative stress biomarkers, which were combined into an Integrated Biomarker Response (IBR) assessment. The sublethal effects covered application rates equivalent to those reported for different cropping systems (2.0-25.0tha-1). Compared to control soil, poultry litter showed elevated concentrations in mg kg-1 of Cu (27.5), Zn (36.6), Mn (37.7), and Cr (0.93). The median lethal concentrations (LC50) were 10.82% at 7 days and decreased to 3.59% at 14 days. Significant avoidance and reproductive effects were detected at poultry litter concentrations of 0.5% (10tha-1) and 0.1% (2tha-1), respectively. The corresponding median effective concentrations (EC50) were 1.38% for avoidance and 0.188% for reproduction. Antioxidant biomarkers, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), reduced glutathione (GSH), and metallothionein (MT), together with oxidative damage indicators, malondialdehyde (MDA) and protein carbonyls (CARB), revealed a response pattern: at intermediate concentrations (0.75-1.0%), an increase in enzyme activities and antioxidant defenses levels was observed; however, at 0.75% and 1.25%, oxidative damage occurred. Principal Component Analysis and correlation analyses indicate that higher litter concentrations impair antioxidant defenses, increase oxidative damage, and trigger compensatory detoxification in F. candida, while the IBR revealed progressive changes in the antioxidant system in response to increasing poultry litter concentrations. Notably, to the best of our knowledge, this study provides the first evidence of pyrethroid occurrence in poultry litter, revealing a previously unrecognized contamination pathway and underscoring potential risks to soil ecosystems.

Cadmium (Cd), being a heavy metal, causes myriad clinical problems if exposed to the environment through food or drinking water or through inhalation. The river Churni of West Bengal is highly contaminated with heavy metals, particularly Cd, due to the disposal of waste materials from paint, sugar, and textile industries. People residing in the catchment area of the river are using the river water for drinking, cooking, and household work, and, therefore, are exposed to high levels of Cd throughout the years. Evaluation of biomarkers in chronic Cd-induced health hazards. A total of 105 study participants were recruited to evaluate chronic Cd-exposure-induced biomarkers. Among them, 50 were from the Cd-exposed population, and 55 were from the unexposed control. Cadmium concentration was measured by Graphite Furnace AAS in drinking and cooking water samples, and the biological samples (blood, urine) collected from every participant. The extent of oxidative DNA damage, protein carbonylation, plasma nitrate, and malondialdehyde (MDA) concentration was measured in people and found to be significantly higher in the Cd-exposed population in comparison to the unexposed control. Expression of the metallothionein (MT) gene and protein has also been studied in both populations, with significantly higher MT gene expression observed in the Cd-exposed population. Evaluation of cell death using the comet assay and acridine orange/propidium iodide staining revealed significantly higher comet tail moment and apoptosis in isolated lymphocytes from exposed participants. Thus, it can be concluded that Cd exposure is associated with oxidative damage and health risk. Cadmium-induced oxidative damage and MT gene expression in the exposed population can serve as a biomarker. This cross-sectional study demonstrates that Cd-induced oxidative damage and MT gene expression can serve as biomarkers in a chronically exposed population. In the future, protein carbonylation, oxidative DNA damage, and MT gene expression may serve as biomarkers of Cd exposure-induced health hazards for diagnostic purposes. Graphical abstract showing the effects of Cd on the human body by food chain contamination.

Metallothionein (MT) is a multifunctional metal-binding protein with broad applications in medicine, healthcare, and food industries, but its large-scale use is limited by inefficient industrial synthesis. To address this and obtain optimal fermentation parameters for large-scale MT production, this study used the recombinant marine-derived MT-producing Pichia pastoris strain SMD1168-MT. We first optimized the strain’s growth and induced fermentation conditions, then constructed a Back Propagation (BP) neural network model for in-depth parameter optimization and accurate MT expression prediction. Results showed the optimal growth conditions for SMD1168-MT were: 30 °C, initial pH 8.0, shaking speed 220 r/min, and 4% inoculum size. The BP model exhibited high accuracy (training set: R2 = 0.8430, MAE = 0.0129, RMSE = 0.0175; validation set: R2 = 0.8337, MAE = 0.0144, RMSE = 0.0174). Combined with Particle Swarm Optimization (PSO), the optimal fermentation conditions were: 7.7% methanol, initial OD600 8.2, 240 r/min, 50 h induction, and 125 μmol/L Zn2+. Validation confirmed MT expression reached 0.2141 mg/mL (2.93-fold). This study demonstrates that the BP neural network effectively optimizes recombinant P. pastoris-based marine-derived MT fermentation, improving yield and providing a basis for industrial scale-up.

Accumulation of heavy metal in fish not only causes morphological, biochemical, haematological and genetic changes but also poses serious health risks to their consumers. The present study investigated the effects of chromium (Cr) exposure on the alteration of physiological parameters and the expression pattern of three candidate genes: metallothionein 2 (MT-2), interleukin 1 beta (IL-1β) and tumour necrosis family 1 (TNF) in a climbing perch (Koi fish). The potassium dichromate (KCr2O7) was used as a source of Cr for fish feed and fed at three experimental doses (1, 2 and 5 ppm of Cr/kg feed) along with a control (0 ppm) for 90 days under aquarium conditions. Cr concentration found in the organs followed the order: muscle > gill > liver. Cr deposition was above the recommended level (0.05 ppm) and was significantly higher in muscle than in gill and liver. The growth in fish length, weight, specific growth rate (SGR) and food conversion ratio (FCR) did not vary significantly with different Cr doses but showed a negative correlation of overall weight gain, SGR and FCR with Cr deposition. Blood haemoglobin, glucose levels and protease activity decreased with increasing chromium concentrations, showing a strong negative correlation between total chromium accumulation and protease activity (r = 0.64, p = 0.022). The overall expression of the MT-2 gene was downregulated in the Cr-exposed fish with time and dose but did not significantly affect its expression in the gills and muscles. However, genes encoding TNF and IL-1β showed an upregulation in the muscle, gill and liver tissues. The sensitivity of these candidate genes to heavy metals was also evident by its positive correlation with Cr concentration and few growth parameters. Therefore, the findings of the present study will help conduct routine health assessments of the fish populations in areas prone to Cr contamination.

Biochar application decreases heavy metal accumulation in ants (Formica japonica).

Acute mercury exposure affects the redox status system, fatty acid profile and the ovarian morphology of the solitary ascidian Ciona intestinalis (Chordata, Tunicata).

Zinc, redox regulation and brain development.

Redox-active iron (Fe) overload and antioxidant zinc (Zn) deficiency are hallmarks of chronic liver fibrosis, yet their upstream regulatory mechanisms remain elusive. Here, we identify the redox-sensitive metallothionein (MT) as a critical regulator in the injured liver that integrates trace-metal homeostasis with Hippo-Yap signalling. MT expression was transiently induced in early-stage murine and human liver fibrosis but declined sharply in advanced disease. Genetic deletion of MT in mice exacerbated CCl4-induced liver injury, characterized by severe Zn depletion, pathological Fe accumulation, and aggravated fibrosis. Mechanistically, MT loss impaired the activity of Zn-dependent matrix metalloproteinases and unleashed pathological Fe deposition, which amplified lipid peroxidation and oxidative damage. We revealed that MT deficiency suppressed the upstream Hippo kinase Mst1, promoting the nuclear translocation and activation of the transcriptional coactivator Yap. Crucially, pharmacological inhibition of Yap with verteporfin rescued the Fe overload and attenuated fibrosis in MT-deficient livers but did not correct the Zn depletion, establishing Yap as a key downstream driver of Fe dysregulation. The analysis of human fibrotic and cirrhotic livers confirmed a conserved inverse correlation between MT levels and nuclear Yap accumulation, accompanied by severe Fe deposition. These findings reveal MT as a key hepatocyte integrator of trace-metal homeostasis and Yap signalling and suggest that therapeutic strategies enhancing MT activity or combining Zn supplementation with Yap inhibition may offer new avenues to prevent or reverse chronic liver fibrosis.

Drought is a major abiotic stress factor for tomato plants, which require frequent and regular irrigation. Tomatoes are cultivated worldwide and are widely used as a model organism in scientific research. For sustainable production, identifying varieties resistant to biotic and abiotic stresses is crucial. This study is the first report on investigating the morphological and physiological responses of two tomato varieties, Naim and Şölen, to drought stress. The mRNA expression of three genes was also analyzed: Ethylene Response Factor 1 (ERF1), Catalase 1 (CAT1), and Metallothionein 1 (MT1). Quantitative Real-Time PCR (qRT-PCR) analysis showed that ERF1 expression decreased in both varieties under protracted drought stress conditions compared to the control. CAT1 expression significantly increased in all drought treatments of the Naim variety and only in the 5-day drought treatment of the Şölen variety. MT1 expression increased significantly in the Naim variety in all groups except the 5-day drought treatment, while it decreased in the Şölen variety. Morphological and physiological parameters also showed statistically significant differences between the two varieties across different drought stress periods (p&amp;lt;0.0001), indicating that Naim appeared more sensitive to drought than Şölen. Ultimately, these results provide a comprehensive scientific foundation for selecting varieties tolerant to protracted drought stress and developing protective pre-treatments in tomato cultivation.

Heavy metal toxicity is a severe global threat, adversely affecting environmental health, food safety, and human well-being. Microbial biosorbents are a renowned effective solution for environmental pollution; however, their success depends on the adsorption capacity of the microbes. Metallothionein (MT) proteins are high in cysteine content and have a strong affinity to bind divalent metal ions. Thus, deliberately increasing cysteine content in bacterial MTs might hold promise in enhancing their metal-binding capacity. However, expressing these MTs in bacterial hosts is challenging due to many genetic constraints. In this study, we applied sequence-based protein design to generate three novel synthetic MT proteins (M_MT01, M_MT02, and M_MT03) with 32-38% cysteine content and enhanced metal ion binding potential. When expressed in Escherichia coli (E. coli) cytosol and on the cell surface, these MTs exhibited enhanced Cd/Cu tolerance and bioaccumulation capabilities. Notably, cytosolic expression of M_MT02 removed 153.6% more Cu and grew faster than the control, while M_MT03 enhanced Cd removal by 214.5%. However, cytotoxicity affected the growth rate. Moreover, the MT recombinant cells demonstrated antioxidant activity up to 81.7% higher than the control. To overcome intracellular metal toxicity, we further anchored M_MT02 and M_MT03 to the bacterial surface using the Lpp-OmpA (lipoprotein-outer membrane protein A) system to develop bacterial cell surface expression proteins that we called Lp-M_MT02 and Lp-M_MT03, accordingly. The Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDS) assay verified the expected cell surface expression and metal binding. Recombinant E. coli expressed Lp-M_MT02 and Lp-M_MT03 on their cell surface exhibited faster growth in Cd- and Cu-containing media than cells with only cytosolic expression. In fact, Lp-M_MT03 expression removed 46.4% more Cd from the media compared to M_MT03 expression. This study demonstrates the potential of de novo MT design technology and opens the door to utilizing the developed MTs for strain engineering to advance bio-absorption technologies.

ABSTRACTChronic obstructive pulmonary disease (COPD) is a highly heterogeneous disease with complex pathogenesis. Identifying high‐risk populations and implementing timely prevention strategies are critical to reducing the disease burden. Single‐cell RNA sequencing of lung tissue from control never‐smokers, patients with pre‐COPD, and COPD patients revealed a novel T cell subset characterized by high expression of metallothionein (MT) genes, designated MT‐high T cells. These cells were progressively depleted in the lungs with disease progression. A similar decline was observed in the peripheral blood using flow cytometry, highlighting the potential of these cells to serve as an accessible biomarker of disease progression. Functional analysis indicated that MT‐high T cells suppress CD8+ T cell cytotoxic activity, suggesting a key immunoregulatory role in disease pathogenesis. Receiver operating characteristic curve analysis demonstrated the excellent potential of MT‐high T cell frequency to predict susceptibility to COPD. These findings establish MT‐high T cells as promising biomarkers for identifying individuals at risk for COPD and as novel targets for future therapeutic and prophylactic strategies.