Metallothionein Gene Expression Research Articles

P0173 The impact of dietary composition on metallothionein gene expression in the intestinal epithelium by exclusive enteral nutrition

Abstract Background The mechanism by which exclusive enteral nutrition (EEN) is effective in the treatment of Crohn’s disease remains elusive. Epidemiologic studies have shown that a substantial proportion of patients with Crohn’s disease are deficient in micronutrients such as zinc, which has been shown to play an important role in the integrity of intestinal epithelial barrier function. Zinc plays a critical role in the transcriptional regulation of metallothioneins (MT), cysteine rich binding proteins that regulate both epithelial and immune function where they are generally believed to play a beneficial role in reducing disease. Methods Transcriptomic analyses were performed by both bulk RNAseq as well as GeoMx spatial transcriptomics. Enzymatic food digests using both bile acids and pancreatic enzymes, analyzed by Proton NMR metabolomics, were used to generate cell culture media for studies in intestinal epithelial cells. Results RNAseq of rectal biopsies obtained from healthy human subjects consuming EEN (Modulen-IBD, Nestlé Health Science) vs. an omnivore diet revealed a very specific induction of genes involved in mineral transport, namely MTs such as MT1G and MT2A. Spatial transcriptomics revealed that the induction of MTs was specific to the colonic epithelium. To develop a cellular model to characterize the mechanism underlying this response, we investigated the impact of animal protein, vegetable, and EEN based foods on the transcriptome of intestinal epithelial cells in culture by both RNAseq and targeted quantitative RT-PCR. This model involved the development of a physiologically relevant modality to digest whole food to generate serum-free cell culture media that was characterized using NMR-based metabolomics. Animal protein and EEN diets induced the expression of specific MTs whereas vegetable diets reduced their expression. We also show that zinc induced MT gene expression in intestinal epithelial cells whereas phytic acid, a phytochemical with known anti-nutrient properties that chelates zinc, reduces the ability of protein-based foods and EEN to induce MT gene expression. Conclusion The consumption of EEN leads to a very specific transcriptomic signature of MT gene expression in the rectal epithelium of humans that can be reproduced in cell culture by exposure to both animal protein-based foods and EEN. By contrast, vegetable-based foods inhibit MT gene expression possibly through the chelation of zinc. These results are consistent with the notion that the exclusion of plant-based foods through the consumption of EEN may have beneficial effects in the treatment of Crohn’s disease by reducing exposure to anti-nutrient phytochemicals that reduce the absorption of beneficial micronutrients such as zinc.

Water pollution caused by ash from grassland fires alters the molecular, biochemical, and morphological biomarkers of non-biting midge larvae.

The frequency and intensity of wildfires have been increasing in many parts of the world, which may result in biodiversity loss. Wildfires can devastate plant communities, generating toxic ash that pollutes watercourses through runoff. However, our understanding of the effects of ash exposure on aquatic biodiversity is still limited. Here, Chironomus sancticaroli larvae were exposed to various concentrations of grassland ash (ranging from 0.01 to 100 g/L) in acute and subchronic assays. The tested ash concentrations are environmentally relevant, as similar levels have been observed in nature after moderate runoff in post-fire conditions. We experimentally tested whether ash-contaminated waters increase mortality, induce molecular and biochemical biomarker changes, and reduce body size in chironomid larvae. Results showed that grassland ash contains micro and nanoparticles with high solubility and is rich in metals and polycyclic aromatic hydrocarbons. Adding ash to the water increased pH, conductivity, and total solids while decreasing dissolved oxygen levels. The lethal concentration (LC50) of grassland ash for C. sancticaroli was estimated at 31.43 g/L after acute exposure. However, exposure to sublethal ash concentrations led to higher metallothionein and hemoglobin gene expression related to metal sequestration and adaptation to lower oxygen levels. At the biochemical level, ash exposure increased the activity of enzymes associated with detoxification capacity, such as alpha and beta esterases. It also led to oxidative stress and damage to cell membranes, as indicated by the increased enzymatic activity of superoxide dismutase, glutathione S-transferase, and lipid peroxidation. Finally, larvae exposed to ash reduced body size by up to 55 % at exposure to higher concentrations. As wildland fires produce ashes that are later moved to the aquatic ecosystem when lixiviated, impacting the resident aquatic biota, environmental managers should adopt measures to reduce the likelihood of wildfires and the ash load to watercourses.

Effects of ECMF Isolated from Mining Areas on Water Status, Photosynthesis Capacity, and Lead Ion Transport of Populus alba Under Pb Stress.

Native ectomycorrhizal fungi (ECMF) are generally more effective than non-native ECMF in facilitating the phytoremediation of heavy metal (HM) ions from contaminated soils. This study aimed to investigate the contributions of four ECMF species-Suillus luteus, Suillus flavidus, Suillus variegatus, and Gomphidius glutinosus-that were isolated from mining areas to the growth, water status, photosynthesis, and metallothionein gene expression of Populus alba exposed to varying concentrations of lead (Pb). The experiment lasted two months and involved P. alba cuttings subjected to Pb concentrations of 0, 200, and 400 mg kg-1, representing no Pb stress, moderate Pb stress, and severe Pb stress, respectively. Results indicated that S. flavidus significantly enhanced the growth, water status, photosynthesis parameters, and metallothionein gene expression of P. alba under Pb stress, whereas S. luteus only exhibited positive effects under severe Pb stress. S. variegatus negatively affected the growth, water status, photosynthesis, and metallothionein gene expression of P. alba under Pb stress, while no significant difference was observed between the control treatment and G. glutinosus symbiosis. Therefore, S. flavidus and S. luteus are promising ECMF species for ecological restoration in mining areas, especially in P. alba woodlands.

ZIP10 (SLC39A10) Is an Important Transport Protein in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) represents a challenging hematologic malignancy characterized by a high rate of relapse after initial remission and significant mortality. Despite improvements in understanding genetic and epigenetic drivers of AML progression, the impact of micronutrients such as zinc on disease pathogenesis remains poorly understood. Transport proteins, including ZIP10 (SLC39A10), play a crucial role in cellular zinc homeostasis and are implicated in various cellular processes critical for cancer biology, including proliferation and apoptosis resistance. A recent study highlighted zinc's potential role in enhancing immune reconstitution after allogeneic stem cell transplantation (Iovino et al., Blood, 2022). Hence, we sought to further investigate the relevance of zinc in AML pathogenesis. Methods: In this study, we employed a comprehensive approach utilizing AML cell lines (MV4-11, THP-1, NB-4) and samples obtained from AML patients at diagnosis or relapse (n=43). All patients provided written informed consent. The protocol was approved by the Ethics Committee at RWTH Aachen University Hospital (Germany) (EK 206/09; project #11-2022). All experiments were conducted in accordance with the Declaration of Helsinki. Cellular zinc levels were quantified using flame atomic absorption spectrometry. Gene expression of zinc transporters (ZIP1-ZIP14 and ZnT1-ZnT10) and metallothioneins (MT1/2) were analyzed by qRT-PCR. Additionally, cellular responses to zinc deficiency were evaluated by using the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) or zinc-deficient medium (ZDM) generated by Chelex 100-treated RPMI 1640. The efficacy of zinc depletion was verified by comparing zinc concentrations in ZDM vs. untreated medium (0.066mg/l vs. 0.199mg/l, n=5; p=0.0001). Results: Patients were between 29 and 80 years old (19 female and 24 male). Our study revealed elevated protein-adapted zinc levels in peripheral blood (AML: n=6, Control: n=3; p=0.0069) and bone marrow (AML: n=8, Control: n=6; p=0.0215) from AML patients. RNA analysis of bone marrow cells demonstrated a significant upregulation of ZIP6 (SLC39A6) (p=0.0429), ZIP9 (SLC39A9) (p=0.0070), and ZIP10 (SLC39A10) (p=0.0171) in AML (AML: n=7, Control: n=6), whereas expression of MT1/2 was notably reduced (AML: n=10, Control: n=9; p=0.0253). This was accompanied by increased staining of free intracellular zinc in CD34+ AML blasts via the zinc-specific dye Zinpyr-1 (n=6; p=0.0488). An increased amount of the ZIP10 surface protein was observed by Western Blot analyses (AML: n=10, Control: n=9; p=0.0002) and immunohistochemical staining (n=3). In addition, overexpression of ZIP10 (SLC39A10) in AML was confirmed by data from the BeatAML2.0 (n=805) and Leukemia Mile (n=542) study. Functional studies showed impaired colony formation after treatment of AML cells for 72h under zinc deficient conditions via TPEN (n=5; p=0.0016) or ZDM (n=5; p=0.0443). Experiments with MV4-11, THP-1, and NB-4 cells demonstrated that zinc deficiency caused decreased cell proliferation and increased apoptosis in myeloid cell lines. Furthermore, ZDM impaired myeloid signaling after stimulation with G-CSF and improved differentiation of NB-4 cells. ZDM induced an upregulation of ZIP10 (SLC39A10) in MV4-11 (n=7; p=0.0438) and THP-1 (n=11; p=0.0207). Blocking the ZIP10 transporter with an anti-ZIP10 antibody led to decreased intracellular zinc levels in MV4-11 cells (n=11; p=0.0081) accompanied by a dose-dependent decrease in cell growth (n=13, p<0.0001) and cell viability (n=11, p<0.0001). Conclusions: Our findings underscore a critical role of zinc homeostasis, particularly as mediated by ZIP10, in AML pathogenesis. Dysregulated zinc metabolism, characterized by elevated zinc transporter expression and altered zinc storage mechanisms, appears to be essential to AML biology. Targeting the ZIP10 transporter, thereby reducing zinc uptake, offers further evidence that zinc modulation disrupts myeloid cell proliferation and increases apoptosis. These results suggest that blocking zinc transporters deserves being further investigated as a supplementary therapeutic approach in AML management and advocates further exploration of the role of zinc and other micronutrients in the progression of myeloid malignancies.

Reprogramming human urine cells into intestinal organoids with long-term expansion ability and barrier function

Generation of intestinal organoids from human somatic cells by reprogramming would enable intestinal regeneration, disease modeling, and drug screening in a personalized pattern. Here, we report a direct reprogramming protocol for the generation of human urine cells induced intestinal organoids (U-iIOs) under a defined medium. U-iIOs expressed multiple intestinal specific genes and showed resembling gene expression profiles to primary small intestines. U-iIOs can be stably long-term expanded and further differentiated into more mature intestinal lineage cells with high expression of metallothionein and cytochrome P450 (CYP450) genes. These specific molecular features of U-iIOs differ from human pluripotent stem cells derived intestinal organoids (P-iIOs) and intestinal immortalized cell lines. Furthermore, U-iIOs exhibit intestinal barriers indicated by blocking FITC-dextran permeation and uptaking of the specific substrate rhodamine 123. Our study provides a novel platform for patient-specific intestinal organoid generation, which may lead to precision treatment of intestinal diseases and facilitate drug discovery.

Integrated assessment of trace elements in a marine ranching area based on multi-species and multi-level biomarkers: a case study in China’s national-level marine ranching demonstration area

The goal of this study was to evaluate the trace element pollution in a marine ranching area in China based on molecular responses (expression of metallothionein and antioxidant enzyme genes), and biochemical biomarkers (metallothionein content, antioxidant enzyme activities, and malonaldehyde level) in four indicator species. We collected samples of two fish (Lateolabrax maculatus and Sebastes schlegelii), one crustacean (Charybdis japonica), and one gastropod (Rapana venosa) from the western Furong Island marine ranching area and from an adjacent area in March 2022 and measured the trace element content in these indicator species as well as in the seawater and sediment. We found that the bioaccumulation characteristics of trace elements and the response patterns of biomarkers were species specific. Moreover, not every biomarker was significantly correlated with environmental trace element content. We then established two biomarker combinations indicative of trace element pollution in seawater and sediment, respectively, based on the correlation between biomarkers and trace element contents. The selected biomarkers were integrated using integrated biomarker response version 2 (IBRv2). IBRv2 values in the studied marine ranching area were lower than those in the adjacent area. Additionally, these values were consistent with the bioaccumulation of trace elements in the indicator species, the integrated trace element pollution index for seawater, and the potential risk index for sediment. These results show that this multi-biomarker and multi-species IBRv2 approach provided a comprehensive diagnosis of trace element pollution in the marine ranching area. Therefore, its application may be beneficial for marine environmental monitoring and management in view of the ecotoxicological impact of pollutants on organisms.

Leaching and cytotoxicity of bismuth oxide in ProRoot MTA - A laboratory investigation.

The present study examined the leaching and cytotoxicity of bismuth from ProRoot MTA and aimed to identify whether bismuth leaching was affected by the cement base and the immersion regime used. The leaching profile of bismuth was examined from ProRoot MTA and compared with hydroxyapatite containing 20% bismuth oxide as well as hydroxyapatite and tricalcium silicate to investigate whether bismuth release changed depending on the cement base. Bismuth leaching was determined after 30 and 180 days of ageing immersed in Dulbecco's modified Eagle's medium (DMEM) using mass spectroscopy (ICP-MS). The media were either unchanged or regularly replenished. The pH, surface microstructure and phase changes of aged materials were assessed. Wistar rat femoral bone marrow stromal cells (BMSCs) and cutaneous fibroblasts were isolated, cultured and seeded for cell counting (trypan blue live/dead) after exposure to non-aged, 30- and 180-days-aged samples in regularly replenished DMEM. Aged DMEM in contact with materials was also used to culture BMSCs to investigate the effect of material leachates on the cells. Gene expression analysis was also carried out after direct exposure of cells to non-aged materials. Differences between groups were statistically tested at a significance level of 5%. All materials exhibited alterations after immersion in DMEM and this increased with longer exposure times. The bismuth leached from ProRoot MTA as detected by ICP-MS. Aged ProRoot MTA samples exhibited a black discolouration and surface calcium carbonate deposition. ProRoot MTA influenced cell counts after direct exposure and its 180-days leachates reduced BMSC viability. After direct BMSC contact with non-aged ProRoot MTA an upregulation of metallothionein (MT1 and MT2A) expression and down-regulation of collagen-1a (Col-1a) and bone sialoprotein (BSP) expression was identified. Bismuth leaching was observed throughout 180-days observation period from all materials containing bismuth oxide. This negatively influenced cell viability and gene expression associated with bismuth exposure. This is the first study to report that metallothionein gene expression was influenced by exposure to ProRoot MTA.

Retracted: Investigation on Immune-Related Protein (Heat Shock Proteins and Metallothionein) Gene Expression Changes and Liver Histopathology in Cadmium-Stressed Fish.

[This retracts the article DOI: 10.1155/2022/2075791.].

ScRNA-seq analysis discovered suppression of immunomodulatory dependent inflammatory response in PMBCs exposed to silver nanoparticles

The assessment of AgNPs toxicity in vitro and in vivo models are frequently conflicting and inaccurate. Nevertheless, single cell immunological responses in a heterogenous environment have received little attention. Therefore, in this study, we have performed in-depth analysis which clearly revealed cellular-metal ion association as well as specific immunological response. Our study didn’t show significant population differences in PMBC between control and AgNPs group implying no toxicological response. To confirm it further, deep profiling identified differences in subsets and differentially expressed genes (DEGs) of monocytes, B cells and T cells. Notably, monocyte subsets showed significant upregulation of metallothionein (MT) gene expression such as MT1G, MT1X, MT1E, MT1A, and MT1F. On the other hand, downregulation of pro-inflammatory genes such as IL1β and CCL3 in both CD16 + and CD16- monocyte subsets were observed. This result indicated that AgNPs association with monocyte subsets de-promoted inflammatory responsive genes suggesting no significant toxicity observed in AgNPs treated group. Other cell types such as B cells and T cells also showed negligible differences in their subsets suggesting no toxicity response. Further, AgNPs treated group showed upregulation of cell proliferation, ribosomal synthesis, downregulation of cytokine release, and T cell differentiation inhibition. Overall, our results conclude that treatment of AgNPs to PMBC cells didn’t display immunological related cytotoxicity response and thus motivate researchers to use them actively for biomedical applications.

Novel biomarkers and interferon signature in secondary progressive multiple sclerosis

Multiple sclerosis (MS) exhibits poor immune regulation and subnormal interferon (IFN-β) signaling. Secondary Progressive MS displays waning exacerbations, relentless neurodegeneration, and diminished benefit of therapy. We find dysregulated serum protein balance (Th1/Th2) and excessive gene expression in Relapsing-Remitting MS vs. healthy controls (8700 differentially-expressed genes, DEG) and intermediate levels in SPMS (3900 DEG). Olfactory receptor genes (chemosensing), and WNT/ß-catenin (anti-inflammatory, repair) and metallothionein (anti-oxidant) gene pathways, have less expression in SPMS than RRMS. IFN-β treatment decreased pro-inflammatory and increased metallothionein gene expression in SPMS. These gene expression biomarkers suggest new targets for immune regulation and brain repair in this neurodegenerative disease.

Effect of salinity on the toxicokinetics, oxidative stress, and metallothionein gene expression in Meretrix meretrix exposed to cadmium

To understand the effect of salinity on the toxicokinetics, oxidative stress, and detoxification of cadmium-exposed Meretrix meretrix, M. meretrix were acclimatized to different salinities (8, 14, 20, 26, and 32 ppt) for 14 d, exposed to 10 μg/L Cd for 7 d, followed by a 28-day depuration period. The internal Cd concentration was determined, and the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST)), and the malondialdehyde (MDA) content were measured. The mRNA expression levels of antioxidant enzyme (Cu/Zn SOD, CAT) and detoxification-related genes metallothionein (MT) were analyzed. The mean concentrations of Cd in M. meretrix tissues were in the order gill > digestive gland > mantle > axe foot. The Cd uptake rate in the four tissues decreased with increasing salinity (range: 14–26 ppt). The Cd elimination half-lives were the highest at 8 ppt and 14 ppt salinity. Cadmium activated the four oxidative stress-related related enzymes in the gills. At the end of accumulation period, Cd exposure at 20 ppt salinity significantly increased the expression of Cu/Zn SOD. CAT expression was significantly inhibited at 20 ppt salinity, but was induced at 32 ppt. MT mRNA expression was only induced under Cd at 20 ppt salinity. At the end of depuration period, Cu/Zn SOD expression was inhibited at salinities of 8, 14, and 26 ppt. The results indicated that SOD, CAT, GST, MDA, Cu/Zn SOD, CAT, and MT were sensitive to cadmium in a water environment, and can be used as indicators of marine heavy metal pollution.

Understanding the molecular mechanism of arsenic and ammonia toxicity and high-temperature stress in Pangasianodon hypophthalmus.

The current investigation explores the mechanisms of ammonia and arsenic toxicity, along with high-temperature stress, which other researchers rarely addressed. Pangasianodon hypophthalmus was exposed to low doses of ammonia and arsenic (1/10th of LC50, 2.0 and 2.68mg L-1, respectively) and high temperature (34°C) for 105days. The following treatments were applied: control (unexposed), arsenic (As), ammonia (NH3), ammonia + arsenic (NH3 + As), ammonia + temperature (NH3 + T), and NH3 + As + T. Cortisol levels significantly increased with exposure to ammonia (NH3), arsenic (As), and high temperature (34°C) compared to the unexposed group. Heat shock protein (HSP 70), inducible nitric oxide synthase (iNOS), and metallothionein (MT) gene expressions were notably upregulated by 122-210%, 98-122%, and 64-238%, respectively, compared to the control. Neurotransmitter enzymes (acetylcholine esterase, AChE) were significantly inhibited by NH3 + As + T, followed by other stressor groups. The apoptotic (caspase, Cas 3a and 3b) and detoxifying (cytochrome P450, CYP P450) pathways were substantially affected by the NH3 + As + T group. Immune (total immunoglobulin, Ig; tumor necrosis factor TNFα; and interleukin IL) and growth-related genes (growth hormone, GH; growth hormone regulator, GHR1 and GHRβ; myostatin, MYST and somatostatin, SMT) were noticeably upregulated by NH3 + As + T, followed by other stress groups, compared to the control group. Weight gain %, protein efficiency ratio, feed efficiency ratio, specific growth rate, and other growth attributes were significantly affected by low doses of ammonia, arsenic, and high-temperature stress. Albumin, total protein, globulin, A:G ratio, and myeloperoxidase (MPO) were highly affected by the As + NH3 + T group. Blood profiling, including red blood cells (RBC), white blood count (WBC), and hemoglobin (Hb), were also impacted by stressor groups compared to the control group. Genotoxicity, as DNA damage, was significantly higher in groups exposed to NH3 + As + T (89%), NH3 + T (78%), NH3 (73), NH3 + As (71), and As (68%). The bioaccumulation of arsenic was substantially higher in liver and kidney tissues. The present study contributes to understanding the toxicity mechanisms of ammonia and arsenic, as well as high-temperature stress, through different gene expressions, biochemical attributes, genotoxicity, immunological status, and growth performance of P. hypophthalmus.

Prognostic and Immunological Role of Cuproptosis-Related Gene MTF1 in Pan-Cancer.

Metal regulatory transcription factor 1 (MTF1) has been reported to induce the expression of metallothionein and other genes involved in metal homeostasis. However, the role of MTF1 in pan-cancer and tumor immunity remains unclear. In this study, we conducted a series of bioinformatics analyses to investigate the clinical significance and potential functions of MTF1 across various types of cancer. By employing bioinformatics algorithms and immunofluorescence assays, we analyzed the associations between MTF1 and immune infiltration in the tumor microenvironment as well as the expression levels of immune-related molecules. Our findings revealed dysregulation of MTF1 in pan-cancer along with its correlation with certain clinicopathological features, suggesting its diagnostic and prognostic value for multiple cancer types. Furthermore, our immune-associated analyses and assays demonstrated strong correlations between MTF1 expression and plasmacytoid dendritic cells (pDC), central memory T cells (Tcm), as well as several immune biomarkers. Subsequent in vitro assays indicated that MTF1 reduced the sensitivity of cancer cells to cuproptosis. Overall, our study highlights that MTF1 may serve as a promising biomarker for prognosis assessment and a potential therapeutic target for more effective treatment strategies against various cancers.

Evaluation of copper-tolerant fungi isolated from Sarcheshmeh copper mine of Iran.

Mycoremediation, a subset of bioremediation, is considered an advanced method to eliminate environmental contaminations. To identify tolerant fungi to copper contamination and study the related gene expression, sampling was carried out from the soil of "Sarcheshmeh Copper Mine," which is one of the biggest open-cast copper mines in the world. A total of 71 fungal isolates were obtained and purified. Afterward, the inhibitory effect of different concentrations (1000, 1500, 3500, 4000, and 5500 ppm) of copper sulfate on mycelial growth was evaluated. Results indicated that only 5500 ppm of copper sulfate inhibited fungal growth compared to the control. Based on the bioassay experiments, three isolates including S3-1, S3-21, and S1-7, which were able to grow on solid and broth medium containing 5500 ppm of copper sulfate at different pH conditions, were selected and identified using molecular approaches. Also, laccase and metallothionein gene expression has been assessed in these isolates. According to the molecular identification using ITS1-5.8S- ITS2 region, isolates S3-1 and S1-7 were identified as Pleurotus eryngii, and isolate S3-21 belonged to the genus Sarocladium. In addition, P. eryngii showed laccase gene expression reduction after 8 days of exposure to copper sulfate. While in the genus Sarocladium, it increased (almost 2 times) from 6 to 8 days. Besides, metallothionein gene expression has increased from 6 to 8 days of copper sulfate treatment compared to the control which reveals its role in copper tolerance of all studied isolates. In this study, Pleurotus eryngii and Sarocladium sp. are introduced as heavy metal tolerant fungi and the related gene expression to copper tolerance was studied for the first time in Iran.

Genetic evidence behind the Cd resistance of wild Metaphire californica: The global RNA regulation rather than specific mutation of well-known gene

Heavy metal contamination presents a profound threat to terrestrial biodiversity, yet the genetic adaptation and evolution of field organisms under persistent stress are poorly understood. In this study, the Cd-resistant earthworms Metaphire californica collected from the control (Meihua, MHC) and elevated-pollution (Lupu, LPC) pairwise sites were used to elucidate the underlying genetic mechanism. A 48-h acute test showed that LPC worms exhibited 2.34 times higher LC50 (50% lethal concentration values) compared to MHC ones. The Cd bioaccumulation, metallothionein (MT) protein contents, and MT gene expression of LPC M.californica were all significantly higher than those of MHC worms. The well-known MT gene of M.californica was successfully cloned and identified, however, the encoding nucleotide and amino acids displayed non-observable mutations and the phylogenetic tree also revealed that different populations clustered together. Additionally, the results of transcriptomics sequencing demonstrated 173 differentially expressed genes between LPC and MHC worms, primarily involved in stress-response and detoxification pathways, including signal transduction, material metabolism, and protein exports. The above results confirmed that the crucial MT gene did not undergo genetic mutations but rather exhibited global mRNA regulation responsible for the Cd resistance of M.californica. The current study partially disclosed the stress adaptation and evolution of organisms under long-term in situ contamination, which provides insights into maintaining biodiversity under adverse environment.

Single and joint toxic effects of waterborne exposure to copper and cadmium on Coregonus ussuriensis Berg.

Heavy metal contamination severely affects the aquatic environment and organisms. Copper (Cu) and cadmium (Cd) are two of the most common heavy metal contaminants that impair the survival, development, and reproduction of aquatic organisms. With the growth of agriculture and industry, there is a possibility of heavy metal pollution in Coregonus ussuriensis Berg's water source. However, there are no published studies on the toxicity to C. ussuriensis. Acute toxicity experiments in C. ussuriensis revealed the 96-h median lethal concentrations of copper and cadmium to be 0.492 mg·L-1 (95% confidence interval: 0.452-0.529) and 1.548 mg·L-1 (95% confidence interval: 1.434-1.657), respectively, and safe concentrations of 4.92 µg·L-1 and 15.48 µg·L-1, respectively. C. ussuriensis was then treated for 96 h with Cu (20% of 96 h LC50), Cd (20% of 96 h LC50), and a combination of Cu and Cd (20% of Cu 96 h LC50 + 20% of Cd 96 h LC50). The histological damage caused by the three different exposure modes to the liver and gills of C. ussuriensis was verified using hematoxylin and eosin staining. All three exposure modes caused different degrees of vacuolization, nuclear consolidation, and necrosis in the liver tissue of C. ussuriensis and edema, hyperplasia, laminar fusion, and epithelial elevation in the gill tissue compared with the reference group. The severity of the damage increased with increasing exposure time. Anti-oxidant activity in the gill and liver tissues were measured using enzyme activity assay kits to reflect oxidative stress induced by copper and cadmium exposure alone and in combination. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were substantially higher than those in the reference groups. However, the activities of the enzymes decreased with increasing exposure time. Malondialdehyde (MDA) activity significantly increased during exposure in relation to that in the reference group. Analysis of immune gene expression in C. ussuriensis gill and liver tissues was executed using real-time inverse transcript polymerase chain response (RT-PCR). The expression levels of the pro-inflammatory cytokines interleukin one beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were positively correlated with exposure time and were significantly upregulated with increasing exposure time. Metallothionein (MT) gene expression levels were significantly upregulated in the short term after exposure compared to the reference group but decreased with increasing exposure time. Our results indicate that exposure to aqueous copper and cadmium solutions, either alone or in combination, causes histopathological damage, oxidative stress, and immunotoxicity in C. ussuriensis gill and liver tissue. This study investigated the toxic effects of copper and cadmium on C. ussuriensis to facilitate the monitoring of heavy metals in water sources for healthy aquaculture.

Induction of metallothionein expression by supplementation of zinc induces resistance against platinum-based treatment in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Currently, multimodality treatment including chemotherapy with cisplatin or carboplatin in combination with pemetrexed offers the best options. Detoxification of heavy metals in the cell by metallothioneins (MT) is associated with early failure to platin-based chemotherapy. The induction of MTs gene expression or its enzyme results in saturation by exposure to metal ions such as zinc or cadmium. Its therapeutically effect is still not analyzed in depth. In our study, we investigated three MPM cell lines and one fibroblast cell line in the course of cisplatin treatment and supplementation of zinc. Cell state analyses via an enzyme-activity based assay were performed. With this, we were able to analyze apoptosis, necrosis and viability of cells. Additionally, we tested treated cells for changes in metallothionein IIA (MT2A) expression by using quantitative realtime polymerase chain reaction. Zinc supplementation induces gene expression of MT2A. Overall, a zinc dose-dependent induction of apoptosis under platin-based treatment could be observed. This effect could be verified in all analyzed cell lines in varying intensity. MT expression is induced by zinc in a dose-dependent manner and inhibits a successful cisplatin therapy. Therefore, heavy metal exposure during cisplatin therapy, e.g., via cigarette smoke, might be an important factor. This should be considered in further therapeutic approaches.

Bioactive copper(II) agents and their potential involvement in the treatment of copper deficiency-related orphan diseases

The deregulation of copper homoeostasis can promote various diseases such as Menkes disease or hypertrophic cardioencephalomyopathy. We have recently synthesized solid copper(II) complexes ([Cu(His)2Cl2] and [Cu(Ser)2]), stable in physiological media and with potential as therapeutic agents. This report describes: i) the biocompatibility of these complexes at concentrations up to 100 μM using a differentiated Caco-2 cells model; ii) their transport across the intestinal epithelium using a transepithelial resistance assay and monitoring the amount of copper complexes at the apical and basolateral sides of the cells. The results suggest that the flow occurs through paracellular routes. The intracellular copper retention was <2.7% with no significant differences in intracellular copper content between 6 h and 48 h, suggesting an early copper retention process. Furthermore, this is the first evidence that demonstrates [Cu(His)2Cl2] and [Cu(Ser)2] induce transcriptional downregulation of the four major copper transporters (CTR1, DMT1, ATP7A, ATP7B), and the upregulation of the metallothionein gene expression. A remarkable finding was the increase in cytochrome c oxidase activity observed after the treatment of differentiated Caco-2 cells with copper(II) complexes at concentrations of 50–100 μM. The understanding of the transport mechanisms of these copper(II) complexes across the intestinal epithelium and of their subsequent biological activities could contribute to the development of optimal pharmaceutical formulations for the therapy of copper deficiency-related diseases.

Zinc alleviates cadmium-induced reproductive toxicity via regulating ion homeostasis, metallothionein expression, and inhibiting mitochondria-mediated apoptosis in the freshwater crab Sinopotamon henanense

Cadmium (Cd) is a carcinogenic environmental pollutant that harms male reproductive systems by lowering sperm quality, impairing spermatogenesis, and causing apoptosis. Although zinc (Zn) has been reported to alleviate Cd toxicity, the underlying mechanisms have not been fully elucidated. The aim of this work was to investigate the mitigating effects of Zn on Cd-induced male reproductive toxicity in the freshwater crab Sinopotamon henanense. Cd exposure not only resulted in its accumulation but also in Zn deficiency, decreased sperm survival rate, poor sperm quality, altered ultrastructure, and increased apoptosis in the testis of the crabs. Morever, Cd exposure increased the expression and distribution of metallothionein (MT) in the testis. However, Zn supplementation effectively mitigated the aforementioned effects of Cd, as demonstrated by preventing Cd accumulation, increasing Zn bioavailability, alleviating apoptosis, increasing mitochondrial membrane potential, decreasing reactive oxygen species (ROS) levels, and restoring MT distribution. Moreover, Zn also significantly reduced the expression of apoptosis-related (p53, Bax, CytC, Apaf-1, Caspase-9, Caspase-3), metal transporter-related ZnT1, metal-responsive transcription factor 1 (MTF1), and the gene and protein expression of MT, while increasing the expression of ZIP1 and Bcl-2 in the testis of Cd-treated crabs. In conclusion, Zn alleviates Cd-induced reproductive toxicity via regulating ion homeostasis, MT expression, and inhibiting mitochondria-mediated apoptosis in the testis of S. henanense. The information obtained in this study may serve as the foundation for further investigation into the development of mitigation strategies for adverse ecological and human health outcomes associated with Cd contamination or poisoning.

Design of Specific Primer for Methallothionein Gene of Tor Fish (Tor tambra)

The use of heavy metal biomarkers is needed to detect and integrate heavy metals at the molecular level. Metallothionein gene is a gene that is expressed in fish organs that have been heavily polluted. The aim of the study was to find specific primers for methallothionein of tor fish. Detection of the metallothionein gene expression in PCR instruments requires a primer in the form of a short chain DNA sequence as a specific target DNA identifier. The primer design was performed in silico using the NCBI site and multiple-aligned using Geneious Prime bioinformatic software. Primers were designed according to the conserved region of these genes. The primers specificity was checked using Primer BLAST tools in NCBI. The results showed that the forward primer 1 (5'- GAT TGC GCC AAG ACT GGA ACT –3') and reverse primer 1 (5' – ATC ACG TTG ACC TCC TCA CTG -3') qualified as good primers with an amplicon size 186 bp.