MT1 Gene Research Articles

Pineal melatonin deprivation alters the mRNA expression of melatonin and steroidogenic-related receptor genes in rat oviduct and uterus during the estrus stage of estrous cycle

The effects of pinealectomy and melatonin replacement therapy on the mRNA expression of melatonin (Mt1, Mt2 and Rora), steroidogenic (Era - Estrogen a, Erb - Estrogen b, Ar - Androgen, Pgr - Progesterone, Lhr - LH and Oxr - Oxytocin receptors) -related receptors and melatonin-synthetic enzyme genes (Tph1, Asmt and Aanat) in oviduct and uterus of Wistar rats were studied. Tissues were collected from sham-pinealectomized (Control), pinealectomized (Pinx) and Pinx plus melatonin- supplemented (Pinx-Mel) females during the estrus stage of both the light (ZT6) and dark (ZT18) phases of the light-dark cycle. In oviduct, Pinx altered the mRNA expression of Mt1, Mt2, Rora, Era, Pgr, Oxr, Asmt and Aanat genes. In uterus, Pinx altered the mRNA expression of Mt1, Mt2, Rora, Lhr, Pgr, Oxr and Tph1 genes. Melatonin treatment partially or completely restored the Mt2, Rora, Era, Oxr, Asmt and Aanat mRNA expressions in oviduct and the Mt1, Mt2 and Rora mRNA expressions in uterus. These alterations varied according to the phase of light-dark cycle. The results suggest that pineal melatonin regulates the daily mRNA expression of melatonin and steroidogenic-related receptors in the rat oviduct and uterus during the estrus stage of estrous cycle. Melatonin also regulates the daily mRNA expression of Asmt and Aanat in oviduct and Tph1 in uterus.

Activation of melatonin receptor 1 by CRISPR-Cas9 activator ameliorates cognitive deficits in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of neurotoxic beta-amyloid (Aβ) in the brain. Melatonin receptors have been reported to associate with aging and AD, and their expression decreased with the progression of AD. As an alternative to AD treatment, overexpression of melatonin receptors may lead to melatonin-like effects to treat alleviate the symptoms of AD. Here, we successfully activated the type 1 melatonin receptor (Mt1) in vivo brain using a Cas9 activator as a novel AD therapeutic strategy. The Cas9 activator efficiently activated the endogenous Mt1 gene in the brain. Activation of Mt1 via Cas9 activators modulated anti-amyloidogenic and anti-inflammatory roles in 5xFAD AD mice brain. Moreover, activation of Mt1 with the CRISPR/Cas9 activator improved cognitive deficits in an AD model. These results demonstrated the therapeutic potential of melatonin receptor activation via CRISPR/Cas9 activator for AD.

Dietary licorice enhances in vivo cadmium detoxification and modulates gut microbial metabolism in mice.

Mass cadmium (Cd) poisoning is a serious health problem in many parts of the world. We propose that dietary intervention can be a practical solution to this problem. This study aimed to identify effective dietary products from traditional Chinese herbs that can detoxify Cd. Five candidate herbal foods with detoxifying potential were selected and subjected to mouse toxicological tests. The chemical composition and dose-response effects of licorice on mouse hepatocytes were determined. Licorice was selected for further tests to examine its effects on growth, tissue Cd accumulation, and gut and liver fitness of mice. The expression of hepatic metallothionein (Mt) genes was quantified in vitro in hepatocytes and in vivo in liver tissues of mice. The results showed that licorice dietary intervention was effective in reducing blood Cd by >50% within 1 month. Cd was also substantially reduced in the heart and lung tissues, but increased 2.1-fold in the liver. The liver of Cd poisoned mice improved with licorice intervention. Licorice treatment significantly induced Cd accumulation and expression of the Mt1 gene in hepatic cells both in vitro and in vivo. Licorice intake substantially altered gut microbial structure and enriched Parabacteroides distasonis. Omics results showed that licorice improved gut metabolism, particularly the metabolic pathways for glycyrrhizin, bile acids, and amino acids. Dietary licorice effectively reduced mouse blood Cd and had a profound impact on liver and gut fitness. We conclude that herbal licorice can be used as a dietary intervention for mass Cd poisoning.

Prolonged Administration of Melatonin Ameliorates Liver Phenotypes in Cholestatic Murine Model.

Background & AimsPrimary sclerosing cholangitis (PSC) is characterized by biliary senescence and hepatic fibrosis. Melatonin exerts its effects by interacting with Melatonin receptor 1 and 2 (MT1/MT2) melatonin receptors. Short-term (1 wk) melatonin treatment reduces a ductular reaction and liver fibrosis in bile duct–ligated rats by down-regulation of MT1 and clock genes, and in multidrug resistance gene 2 knockout (Mdr2-/-) mice by decreased miR200b-dependent angiogenesis. We aimed to evaluate the long-term effects of melatonin on liver phenotype that may be mediated by changes in MT1/clock genes/miR200b/maspin/glutathione-S transferase (GST) signaling.MethodsMale wild-type and Mdr2-/- mice had access to drinking water with/without melatonin for 3 months. Liver damage, biliary proliferation/senescence, liver fibrosis, peribiliary inflammation, and angiogenesis were measured by staining in liver sections, and by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay in liver samples. We confirmed a link between MT1/clock genes/miR200b/maspin/GST/angiogenesis signaling by Ingenuity Pathway Analysis software and measured liver phenotypes and the aforementioned signaling pathway in liver samples from the mouse groups, healthy controls, and PSC patients and immortalized human PSC cholangiocytes.ResultsChronic administration of melatonin to Mdr2-/- mice ameliorates liver phenotypes, which were associated with decreased MT1 and clock gene expression.ConclusionsMelatonin improves liver histology and restores the circadian rhythm by interaction with MT1 through decreased angiogenesis and increased maspin/GST activity.

Metabolomic profiling of plasma from middle-aged and advanced-age male mice reveals the metabolic abnormalities of carnitine biosynthesis in metallothionein gene knockout mice.

Metallothionein (MT) is a family of low molecular weight, cysteine-rich proteins that regulate zinc homeostasis and have potential protective effects against oxidative stress and toxic metals. MT1 and MT2 gene knockout (MTKO) mice show shorter lifespans than wild-type (WT) mice. In this study, we aimed to investigate how MT gene deficiency accelerates aging. We performed comparative metabolomic analyses of plasma between MTKO and WT male mice at middle age (50-week-old) and advanced age (100-week-old) using liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). The concentration of N6,N6,N6-trimethyl-L-lysine (TML), which is a metabolic intermediate in carnitine biosynthesis, was consistently higher in the plasma of MTKO mice compared to that of WT mice at middle and advanced age. Quantitative reverse transcription PCR (RT-PCR) analysis revealed remarkably lower mRNA levels of Tmlhe, which encodes TML dioxygenase, in the liver and kidney of male MTKO mice compared to that of WT mice. L-carnitine is essential for β-oxidation of long-chain fatty acids in mitochondria, the activity of which is closely related to aging. Our results suggest that reduced carnitine biosynthesis capacity in MTKO mice compared to WT mice led to metabolic disorders of fatty acids in mitochondria in MTKO mice, which may have caused shortened lifespans.

Analysis of MT1 and ZIP1 gene expression in the liver of rats with chronic poisoning with cadmium chloride

Introduction. Cadmium is a toxic heavy metal with devastating effects on most organ systems. After absorption, cadmium is transported throughout the body, primarily by binding to proteins by metallothioneins. It is believed that the mechanisms of cadmium-induced transformation arise due to the disruption of zinc-dependent cellular processes. This part is due to the structural and physical similarities between zinc and cadmium. More than half of the incoming cadmium is deposited in the liver and kidneys. The rest part is distributed throughout other organs and their systems. Materials and methods. In total, 40 white outbred rats of both sexes weighing 170-230 g were used in the experiment; they were formed into four experimental groups of 10 animals each, depending on the dose of the injected toxicant. Liver tissue samples were used as research materials, in the homogenate of which the quantitative content of Cd and Zn was determined, as well as the mRNA level of the MT1 and ZIP1 genes. Results. It was found that the most pronounced activity of the MT1 gene in liver tissues was achieved when animals were administered cadmium chloride at a dose of 0.1 mg/kg (2.69 ± 0.37; p = 0.017), while the multiplicity of expression of the ZIP1 gene showed the maximum value of the level of transcripts with the minimum dose of toxin (2.70 ± 0.37; p = 0.007). It was also revealed that the highest concentration of zinc in the liver tissue was observed with the introduction of cadmium chloride at a dose of 0.1 mg/kg (33.84 ± 0.53; p <0.001), and the concentration of cadmium increased along with an increase in the dose of the toxicant (0, 0049 ± 0.0003; 0.0203 ± 0.0024; 0.664 ± 0.007; 0.76 ± 0.0089). Conclusion. Thus, a comprehensive study of the expression of genes for metallothioneins and zinc transporters can be used as a biomarker of poisoning with cadmium and its compounds.

Efficiency of Escherichia coli Jm109 and genetical engineering strains (E. coli MT2, E. coli MT3) in cadmium removal from aqueous solutions

Metallothioneins (MTs) are proteins with high metal binding affinity that exist in many organisms. Recently, this feature of MTs has been used in many areas, including environmental studies. In this study, the human MT2, and MT3 genes were cloned into Escherichia coli Jm109 to remove cadmium (Cd) from aqueous solutions. Minimum inhibition concentrations (MIC) of Cd for E. coli Jm109, E. coli MT2, and E. coli MT3 were determined as 154, 212, and 250 mg L −1 , respectively. 0.5, 5, and 25 mg L −1 Cd were applied to the strains. The maximum Cd removal was at the concentration of 0.5 mg L −1 . The optimal conditions for temperature, pH, and contact time were 37 °C, 7, and 24 h, respectively. E. coli MT3 was the most effective strain in Cd bioremoval. Scanning electron microscope (SEM) images showed that the morphological structures of the Cd treated cells were disrupted. The Energy dispersive spectroscopy (EDS) peaks of the Cd treated cells clearly showed Cd absorption into the cells. Scanning transmission electron microscope (STEM) images showed that there were clusters reflecting the accumulation of Cd in the MT3 cells. The appearance of attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) spectrum peaks of hydroxyl, carboxyl, amine, and sulfoxide groups in MT3 cells exposed to Cd confirmed that these groups bind Cd. As a result, it can be said that the MT3 gene can be used as an effective tool in Cd removal from waters. • The Cd removal capacities of genetically modified Escherichia coli were high. • E. coli MT3 was more effective in Cd removal than E. coli Jm109 and E. coli MT2. • SEM and STEM images showed that E. coli MT3 absorbs more Cd than other strains. • ATR-FTIR spectrum peaks confirmed that MT3 cells bind Cd. • MT3 can be used as an effective tool in Cd removal from water.

Coadministration of Melatonin and Insulin Improves Diabetes-Induced Impairment of Rat Kidney Function

Introduction: The present study was designed to evaluate the therapeutic efficacy of melatonin and insulin coadministration in diabetes-induced renal injury in rats. Research Design and Methods: Diabetes was achieved by giving streptozotocin (15 mg/kg) for 6 consecutive days. The diabetic condition was confirmed by assessing the blood glucose level; animals having blood glucose levels above 250 mg were considered as diabetic. Following the confirmation, animals were randomly divided into different experimental groups, viz group I served as the control (CON), group II diabetic (D), group III D+melatonin (MEL), group IV D+insulin (INS), group V D+MEL+INS, group VI D+glibenclamide (GB), group VII CON+MEL, group VIII CON+INS, and group IX CON+GB. Following the completion of the experimental period, animals were sacrificed, blood was collected via a retro-orbital puncture, and kidneys were harvested. Diabetic rats exhibited a significant increment in blood glucose and biochemical indexes of renal injury (tubular disruption, swollen glomeruli with loss of glomerular spaces, and distortion of the endothelial lining) including augmented levels of serum creatinine, urea, uric acid, Na+, and K+, and inhibition/suppression of the activity of glutathione (GSH) peroxidase, GSH reductase, glucose-6-phosphate dehydrogenase, and GSH-S-transferase in the renal cortex. Results: By examining thiobarbiturate reactive substances, reduced GSH, superoxide dismutase activity, and catalase activity in the renal cortex of control and diabetic rats, it was documented that treatment with melatonin or insulin alone or in combination showed a significant ad integrum recovery of GSH-dependent antioxidative enzymatic activities. Melatonin and insulin coadministration caused greater reductions in circulating tumor necrosis factor-α, tumor growth factor-β1, interleukin (IL)-1β, and IL-6 levels in diabetic rats, whereas IL-10 levels increased, as compared to each treatment alone. Diabetic rats showed a significant increase in the expression of both MT1 and MT2 melatonin receptor genes. Melatonin or insulin treatment alone or in combination resulted in significant restoration of the relative expression of both melatonin receptors in the renal cortex. Conclusion: The coadministration of exogenous melatonin and insulin abolished many of the deleterious effects of type 1 diabetes on rat renal function.

The effects of metallothionein in paraquat-induced Parkinson disease model of zebrafish

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease caused by selective degeneration of dopaminergic neurons in the substantia nigra. Metallothionein has been shown to act as a neuroprotectant in various brain injury. Thus, this study aims to identify the effects of full-length human metallothionein 2 peptide (hMT2) in paraquat-induced brain injury in the zebrafish. Methodology A total of 80 adult zebrafish were divided into 4 groups namely control, paraquat-treated, pre-hMT2-treated, and post-hMT2-treated groups. Fish were treated with paraquat intraperitoneally every 3 days for 15 days. hMT2 were injected intracranially on day 0 (pre-treated group) and day 16 (post-treated group). Fish were sacrificed on day 22 and the brains were collected for qPCR, ELISA and immunohistochemistry analysis. Results qPCR analysis showed that paraquat treatment down-regulated the expression of genes related to dopamine activity and biosynthesis (dat and th1) and neuroprotective agent (bdnf). Paraquat treatment also up-regulated the expression of the mt2, smtb and proinflammatory genes (il-1α, il-1β, tnf-α and cox-2). hMT2 treatment was able to reverse the effects of paraquat. Lipid peroxidation decreased in the paraquat and pre-hMT2-treated groups. However, lipid peroxidation increased in the post-hMT2-treated group. Paraquat treatment also led to a reduction of dopaminergic neurons while their numbers showed an increase following hMT2 treatment. Conclusion Paraquat has been identified as one of the pesticides that can cause the death of dopaminergic neurons and affect dopamine biosynthesis. Treatment with exogenous hMT2 could reverse the effects of paraquat in the zebrafish brain.

Evaluation of the hair cell regeneration and claudin b and phoenix gene expression during exposure to low concentrations of cadmium and zinc in early developing zebrafish larvae

Zebrafish possess hair cells on the body surface similar to that of mammals inner hear, in particular in the neuromasts, and due to its ability in regenerating damaged hair cells, is regularly used as a powerful animal model to study in vivo cytotoxicity.Among the factors leading to hair cell disruption, metal ions are of particular concern since they are important environmental pollutants. To date, several studies on zebrafish hair cell regeneration after metal exposure exist, while no data on regeneration during continuous metal exposure are available. In the present study, neuromast hair cell disruption and regeneration were assessed in zebrafish larvae for the first time during zinc (Zn) and cadmium (Cd) continuous exposure and a visual and molecular approach was adopted.Fluorescent vital dye DASPEI was used to assess hair cell regeneration and the gene expression of claudin b (cldnb) and phoenix (pho), was analyzed. Metallotionein-2 (mt2) gene expression was used as standard molecular marker of metal toxicity and confirmed the higher toxicity of Cd compared to Zn. In addition, Cd caused a delay in hair cell regeneration compared to Zn. Molecular analysis showed cldnb gene expression increased in relation to the metal concentrations used, confirming the involvement of this gene in hair cell regeneration. On the contrary, a dramatic decrease of pho gene expression was observed in Cd exposed groups, suggesting a negative impact of Cd on pho expression, thus negatively interfering with hair cell regeneration in zebrafish larvae exposed to this metal.

Ethyl Acetate Fraction of Amomum villosum var. xanthioides Attenuates Hepatic Endoplasmic Reticulum Stress-Induced Non-Alcoholic Steatohepatitis via Improvement of Antioxidant Capacities.

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), affects 25% of the global population. Despite the prevalence of NAFLD worldwide, effective therapeutics are currently lacking. Amomum villosum var. xanthioides (Wall. ex Baker) T.L.Wu & S.J.Chen (AX) is a medicinal herb traditionally used for treating digestive tract disorders in countries across Asia. We aimed to examine the pharmacological effects of the ethyl acetate fraction of AX (AXEF) against tunicamycin (TM)-induced ER stress in a NASH mouse model using C57/BL6J male mice. Following TM injections (2 mg/kg), the mice were orally administrated AXEF (12.5, 25, or 50 mg/kg), silymarin (50 mg/kg), or distilled water daily for 5 days, and the outcomes for fatty liver, inflammation, and oxidative stress were measured in serum or liver tissue levels. AXEF drastically attenuated hepatic ER stress-induced NASH as indicated by decreases in lipid droplet accumulations, serum liver enzymes, hepatic inflammations, and cell death signals in the hepatic tissue and/or serum levels. Interestingly, AXEF showed potent antioxidant effects by quenching reactive oxidative stress and its final product lipid peroxide in the hepatic tissue, specifically an increase in metallothionein (MT). To confirm the underlying actions of AXEF, we observed that AXEF increases MT1 gene promoter activities in the physiological levels. Collectively, AXEF showed antioxidant properties on TM-induced ER stress in a NASH mice model through the improvement of MTs.

Importance of Genetic Polymorphisms in MT1 and MT2 Genes in Metals Homeostasis and Their Relationship with the Risk of Acute Pancreatitis Occurrence in Smokers-Preliminary Findings.

This study was aimed at evaluating the changes in metallothionein (MT) concentration in the blood of patients with acute pancreatitis (AP) and healthy subjects, taking into account the extracellular (plasma) and intracellular (erythrocyte lysate) compartments. The impact of single-nucleotide polymorphisms (SNPs) in the MT1A (rs11640851), MT1B (rs964372) and MT2A (rs10636) genes on MT concentration and their association with the concentration of metals (Cu, Zn, Cd) and ceruloplasmin as Cu-related proteins were analyzed. The concentration of a high-sensitivity C-reactive protein (hs-CRP) and IL-6 as markers of inflammation, and malonyldialdehyde (MDA), superoxide dismutase (SODs) activity and the value of total antioxidant capacity (TAC) as parameters describing the pro/antioxidative balance were also assessed. In the AP patient groups, an increased MT concentration in erythrocyte lysate compared to healthy subjects was shown, especially in individuals with the GG genotype for rs964372 in the MT1B gene. A Zn concentration was especially decreased in the blood of smoking AP patients with the AA genotype for SNP rs11640851 in the MT1A gene and the GC genotype for SNP rs10636 in MT2A, compared to non-smokers with AP, which was accompanied by an increase in the value of the Cu/Zn ratio. The exposure to tobacco smoke xenobiotics increased the risk of AP occurrence in subjects with the CC genotype for SNP rs11640851 in the MT1A gene by more than fourfold. The investigated polymorphisms, rs11640851 in the MT1A gene, rs964372 in the MT1B gene and rs10636 in the MT2A gene, seem to be an important factor in maintaining homeostasis in an organism under oxidative stress conditions.

Metallic Orthodontic Materials Induce Gene Expression and Protein Synthesis of Metallothioneins.

Background: Due to the long-term contact with metallic elements of orthodontic appliances, the potential influence of released metal ions on living organisms and the type of induced changes was investigated. Materials and Methods: Twenty-four young domestic pigs classified in two groups (experimental and control) were chosen as the object of this study. In the experimental group of animals, two metal plates consisting of orthodontic bands representing the mass of orthodontic appliance were mounted on the internal side of the cheek for six months. The liver, lung, and brain samples were taken post mortem from animals of both groups. The gene expression of two isoforms of metallothionein (MT-1 and MT-2) were investigated using the qPCR technique. Protein expression was confirmed by the Western blot and ELISA techniques. Results: The differences in metallothionein concentrations were observed in the lung and brain in the group of experimental animals, but not in the liver. The expression of MT-1 and MT-2 genes in the experimental vs. control group (respectively) was as follows: lung MT-1 1.04 vs. 1.11, MT-2 0.96 vs. 1.05, liver MT-1 0.89 vs. 0.91 vs. 1.12, MT-2 0.91 vs. 1.05, brain MT-1 1.24 vs. 1.20, and MT-2 0.955 vs. 0.945. These results were confirmed by gene activity, which was tested by qPCR. This increased the activity of metallothionein genes in the lungs and brain as a consequence of the release of metal ions into these tissues. The possible effects of detected change in metallothionein-2 gene expression could be the alteration of physiological functions of lung tissue. Conclusions: The effect of long-term exposure to metal orthodontic appliances on metallothioneins gene expression, as well as the induction of protein synthesis was proved.

ANALYSIS OF MT2A AND MT3 GENE EXPRESSION IN RAT'S LIVER AND KIDNEY IN RESPONSE TO CADMIUM CHLORIDE POISONING

The aim of this study was to investigate the expression of metallothionein genes in the liver and kidneys of rats with acute cadmium poisoning.Simulation of poisoning with cadmium chloride was carried out on white outbred female rats, divided into 4 groups depending on the dose of the injected toxicant. RNA samples isolated from rat liver and kidneys were used as research materials.The multiplicity of expression of the MT3 gene in the kidneys increased at the lowest dose of CdCl2 , which was used in this experiment (0.029 mg / kg); with increasing dosage, the expression level decreased, but not lower than the control values. Analysis of the expression of the same gene in the liver showed a tendency towards a decrease in the content of transcripts with increasing dose. The frequency of expression of the MT2A gene at higher doses of CdCl2 increased both in the liver and in the kidneys.In the present work, statistically significant dose-dependent changes in the expression multiplicity of metallothionein genes were detected 24 hours after CdCl2 administration. The revealed differences in the level of transcriptional activity of metallothionein genes require further investigation, since there are probably differences in the level of gene expression at earlier and later periods of toxicant action.

Screening Dietary Herbs for Health Intervention on Mass Cadmium Poisoning

Objective: Mass cadmium (Cd) poisoning is a serious health problem in many parts of the world. We propose that dietary intervention can be a practical solution to this problem. This study aimed to identify effective dietary products from traditional Chinese herbs that can detoxify Cd. Design: Five candidate herbal foods with detoxifying potential were selected and subjected to mouse toxicological tests. The chemical composition and dose-response effects of licorice on mouse hepatocytes were determined. Licorice was selected for further tests to examine its effects on growth, tissue Cd accumulation, and gut and liver fitness of mice. The expression of hepatic metallothionein (Mt) genes was quantified in vitro in hepatocytes and in vivo in liver tissues of mice. Results: The results showed that licorice dietary intervention was effective in reducing blood Cd by > 50% within 1 month. Cd was also substantially reduced in the heart and lung tissues, but increased 2.1-fold in the liver. The liver of Cd poisoned mice improved with licorice intervention. Licorice treatment significantly induced Cd accumulation and expression of the Mt1 gene in hepatic cells both in vitro and in vivo. Licorice intake substantially altered gut microbial structure and enriched Parabacteroides distasonis. Omics results showed that licorice improved gut metabolism, particularly the metabolic pathways for glycyrrhizin, bile acids, and amino acids. Conclusion: Dietary licorice effectively reduced mouse blood Cd and had a profound impact on liver and gut fitness. We conclude that herbal licorice can be used as a dietary intervention for mass Cd poisoning. Funding: This work was supported by the Hebei Provincial Science Fund or Distinguished Young Scholars (No. D2018503005), the National Key Research and Development Program of China (No.2018YFD0800306), and the National Natural Science Foundation of China (Nos. 41877414 and 31700228). Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Animal Care and Use Committee of the Hebei Science and Technical Bureau, China

Cadmium distribution and metallothionein expression in rat organs following acute intoxication

Introduction. This article presents the results of experimental simulation of the acute toxic effect of cadmium on the rat organism, its distribution in the liver, and kidneys. Activation of the protective mechanism against toxic metal through the metallothionein protein has to reduce the bioavailability of free cadmium. Material and methods. The study was conducted on rats weighing 140-190 g, which was once intragastrically injected with cadmium chloride in an amount of 1/20 LD50. We studied the time intervals: before intoxication, 1, 2, 4, 6, 24, 48, and 96 hours after inoculation. The accumulation of cadmium in the liver and kidneys was measured by atomic absorption spectrometry. The expression of the metallothionein gene (МТ1, МТ2А, МТ3) was determined using RT-PCR on RNA isolated from the same organs. Results. Quantitative differences in the metal content in the liver and kidneys are observed 1 hour after intoxication, with a cadmium content of 250 and 125 times higher than in the control groups, respectively. There is an accumulation of cadmium in the liver with a maximum after 6 hours, and then its redistribution to the kidneys. The pronounced expression of metallothionein with a single acute exposure to cadmium is tissue-specific, so the expression of the MT1 and MT2A genes was greatest in the liver and the MT3 gene in the kidneys. Discussion. After administration cadmium is mainly localized in hepatocytes and its concentration may exceed the ability of metallothionein to bind cadmium ions, which leads to histopathological changes in the liver. In response to the intake of metal in the cell, the expression pattern of many genes, including those associated with the activation of protective reactions, changes. Conclusion. Our data show a single exposure to cadmium to lead to an increase in the content of MT transcript in the liver and kidneys, simultaneously with the accumulation of metal in them. The nature of this accumulation depends on the organ, on the time of exposure, and gene expression also on the form of MT.

Transcriptional activity of metallothionein genes in acute poisoning caused by cadmium chloride

Introduction. Cadmium is one of the most toxic heavy metals widely distributed in the environment. It has a long half-life, leading to its accumulation in various organs, which in turn is the cause of many adverse effects on human health. Material and methods. Acute toxic poisoning with cadmium chloride was simulated on white outbred male rats, divided into groups depending on the exposure time. Samples of liver and kidney tissues were used as study materials, in the homogenate of which the level of mRNA of metallothionein genes was determined. Results. It was found that the maximum value of the multiplicity of MT1 gene expression in the liver was reached after 6 hours (16.36 ± 0.77; p <0.001), and in the kidneys one day after intoxication with cadmium chloride (6.12 ± 0.43; p <0.001). The activity of the MT2 gene in the liver was most pronounced in the range of 2-4 hours (14.35 ± 1.73; 14.78 ± 1.44; p <0.001), while in the renal tissues an increase in the amount of mRNA was recorded after 24 hours (7.32 ± 0.63; p <0.001). The level of MT3 gene transcripts in liver tissues was decreased throughout the experiment, however, the opposite picture was observed in the kidney tissue, where the maximum was reached one day after the administration of the toxicant (6.14 ± 0.31; p <0.001). Conclusion. Thus, an increase in metallothionein expression in response to the presence of heavy metal ions in the body can be used as a genetic marker in case of poisoning with various compounds.

CpG Site-Specific Regulation of Metallothionein-1 Gene Expression.

Metal-binding inducible proteins called metallothioneins (MTs) protect cells from heavy-metal toxicity. Their transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF1), which is strongly recruited to MREs in the MT promoters, in response to Zn and Cd. Mouse Mt1 gene promoter contains 5 MREs (a–e), and MTF1 has the highest affinity to MREd. Epigenetic changes like DNA methylation might affect transcription and, therefore, the cytoprotective function of MT genes. To reveal the CpG site(s) critical for Mt1 transcription, we analyzed the methylation status of CpG dinucleotides in the Mt1 gene promoter through bisulfite sequencing in P1798 mouse lymphosarcoma cells, with high or low MT expression. We found demethylated CpG sites near MREd and MREe, in cells with high expression. Next, we compared Mt1 gene-promoter-driven Lucia luciferase gene expression in unmethylated and methylated reporter vectors. To clarify the effect of complete and partial CpG methylation, we used M.SssI (CG→5mCG) and HhaI (GCGC→G5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited Mt1 gene expression. Our results suggest that the methylation status of this site is important for the regulation of Mt1 gene expression.

Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms.

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA-enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.

Transcriptome alterations and genotoxic influences in zebrafish larvae after exposure to dissolved aluminum and aluminum oxide nanoparticles

Manufactured nanoparticles (NPs) can potentially cause negative effects on molecular (proteins and nucleic acids), subcellular, cellular, tissue, organ, and organism due to their unusual physicochemical characteristics. Ionizable NPs in water (e.g., Al2O3-NPs) may create toxic effects on aquatic animals. The present research determined the influences of Al2O3-NPs and appropriate concentrations of ionizing Al(III) using water-soluble AlCl3 in zebrafish larvae (72 h post-fertilization, Danio rerio) by analyzing transcriptional alterations of stress-associated genes (rad51, p53, mt2) with quantitative real-time PCR (qRT-PCR). In addition, genotoxic effects of Al(III) and Al2O3-NPs were evaluated. The lethal concentrations that cause death of 50% (LC50) of zebrafish larvae when exposed to 0–50 mg/l Al(III) and 0-500 mg/l Al2O3-NPs were 3.26 ± 0.38 and 130.19 ± 5.59 mg/l, respectively, for 96 h. A concentration-dependent increase was observed in the genotoxicity in cells of larvae exposed to Al(III) and Al2O3-NPs for 96 h. DNA damage was more severe in larvae exposed to Al(III) (41.0% tail) than that of Al2O3-NPs (21.8% tail). A complex induction of stress-associated genes was observed in fish and this induction was not directly related to the concentrations of Al(III) and Al2O3-NPs, although a significant induction was detected in mt2 gene of larvae exposed to Al(III) and Al2O3-NPs relative to control group. The induction levels of mt2 were 4.13 ± 0.1 and 2.13 ± 0.1-fold change (mean ± S.E.M.) in larvae at 15 mg/l of Al(III) and 100 mg/l of Al2O3-NP concentrations, respectively.