Metallothionein Induction Research Articles

Metallothionein ameliorates airway epithelial apoptosis upon particulate matter exposure: role of oxidative stress and ion homeostasis

PurposeTo investigate the mechanism underlying particulate matter (PM) exposure-induced oxidative stress and potential rescue strategies against pulmonary damage in this context.MethodsA combination of omics technology and bioinformatic analysis were used to uncover mechanisms underlying cellular responses to PM exposure in human bronchial epithelia (HBE) cells and imply the potential rescue.ResultsOur results implicated that oxidative stress, metal ion homeostasis, and apoptosis were the major cellular responses to PM exposure in HBE cells. PM exposure disrupted oxidative phosphorylation (OXPHOS)-related gene expressions in HBE cells. Rescuing the expression of these genes with supplemental coenzyme Q10 (Co Q10) inhibited reactive oxygen species (ROS) generation; however, it only partially protected HBEs against PM exposure-induced apoptosis. Further, metallothionein (MT)-encoding genes associated with metal ion homeostasis were significantly induced in HBE cells, which was transcriptionally regulated by specificity protein 1 (SP1). SP1 knock-down (KD) aggravated PM-induced apoptosis in HBE cells, suggesting it plays a role in MT induction. Subsequent studies corroborated the protective role of MT by showing that exogenous MT supplement demonstrated effective protection against PM-induced oxidative stress and apoptosis in HBE cells. Importantly, exogenous MT supplement was shown to reduce ROS generation and apoptosis in airway epithelia in both HBE cells and a PM-inhaled murine model.ConclusionThis study demonstrates that the impact of MT on airway epithelia by suppressing oxidative stress and maintaining metal ion homeostasis is beneficial in attenuating damage to pulmonary cells undergoing PM exposure.

Evaluating combined effects of chronic, low-dose exposures of cadmium (CLEC) and hyperglycemia on insulin signaling dysfunction in a hepatocellular model

The pathophysiological effects of chronic heavy metal exposures on human health remains uncertain. In this study, we developed a novel chronic, low-dose exposure of Cadmium (CLEC) model using the hepatocellular cell lines, HepG2 and HUH7. We modulated cell culture conditions to mimic human normoglycemic (5.6 mM) and hyperglycemic (15 mM) states with concomitant cadmium (Cd) exposures for 24 weeks. CLEC cells undergo non-trivial alterations in glucose signaling and metabolic characteristics within our model. We observe elevated baseline reactive oxygen species (ROS) production and decreased 2-NBDG uptake indicative of glucose metabolic dysfunction. Additionally, induction of metallothionein (MT) expression, increased activation of Akt signaling (via phosphorylation) and reduced IRS-2 protein expression are observed in CLEC cells. Cell line specific changes are observed with HepG2 showing a much higher MT gene induction compared to HUH7 cell line which impacts glucose metabolic dysfunction. Hyperglycemic culture conditions (representing type II diabetes) significantly modulate CLEC effects on cells. In conclusion, pathophysiologically relevant models of chronic heavy metal exposures are urgently needed to gain an in-depth, mechanistic understanding of the long-term impacts of toxic metals (e.g., Cd) on human metabolic health.

Abstract We038: Metal-organic frameworks as next-generation cardiac therapeutics

Background: Zinc (Zn) is an essential trace element for the normal functioning of the heart. Long-term dietary supplementation of Zn has been shown to improve cardiac function and prevent myocardial damage caused by ischemia-reperfusion injury. However, the major limitation in delivering Zn to the cardiac tissue is the off-targeted absorption and the duration of treatment to achieve a therapeutic effect. Hypothesis: Cardiac-targeted delivery of trace elements like Zn may attenuate myocardial damage caused by ischemia-reperfusion injury, improve cardiac function, and exert a cardioprotective effect. Methods: Zn-based metal-organic frameworks (ZIF-NPs) were fabricated and tested on Microvascular Endothelial Cells (MVECs), and human-induced pluripotent derived cardiomyocytes (hiCMs) for cytoprotective effect against hypoxic stress using Live/dead assay and multi-electrode array (MEA) analysis respectively. The in vivo efficacy of ZIF-NPs was tested in the rodent myocardial infarction (MI) model and cardiac function was assessed via M-mode echocardiography. Results: The fabricated ZIF-NPs were biocompatible and showed significant intracellular uptake causing the induction of cytoprotective metallothioneins. ZIF-NP treatment showed enhanced cell survival in serum-starved MVECs and exhibited pro-angiogenic effects in vitro. Similarly, hiCMs treated with ZIF-NPs displayed enhanced endurance and electrophysiological function, when subjected to hypoxic stress and protected the cells against ROS-induced DNA damage. Intramyocardial delivery of ZIF-NPs in the cardiac tissue improved cardiac function and attenuated fibrosis post-MI. Conclusions: ZIF-NPs exerted a pro-survival, cytoprotective response in both in vitro and in vivo conditions. Our study highlights the significance of cardiac targeted Zn delivery by ZIF-NPs for cardioprotection as next-generation cardiac therapeutics.

Blockage of metallothionein synthesis via adrenaline β receptor activation invalidates dehydroeffusol-mediated prevention of amyloid β1-42 toxicity

Dehydroeffusol, a major phenanthrene in Juncus effusus, protects neurodegeneration induced by intracellular Zn2+ ferried by extracellular amyloid β1-42 (Aβ1-42). Here we focused on adrenaline β receptor activation and the induction of metallothioneins (MTs), intracellular Zn2+-binding proteins to test the protective mechanism of dehydroeffusol. Isoproterenol, an agonist of adrenergic β receptors elevated the level of MTs in the dentate granule cell layer 1 day after intracerebroventricular (ICV) injection. When Aβ1-42 was injected 1 day after isoproterenol injection, pre-injection of isoproterenol protected Aβ1-42 toxicity via reducing the increase in intracellular Zn2+ after ICV injection of Aβ1-42. On the basis of the effect of increased MTs by isoproterenol, dehydroeffusol (15 mg/kg body weight) was orally administered to mice once a day for 2 days. On day later, dehydroeffusol elevated the level of MTs and prevented Aβ1-42 toxicity via reducing Aβ1-42-mediated increase in intracellular Zn2+. In contrast, propranolol, an antagonist of adrenergic β receptors reduced the level of MTs increased by dehydroeffusol, resulting in invalidating the preventive effect of dehydroeffusol on Aβ1-42 toxicity. The present study indicates that blockage of MT synthesis via adrenaline β receptor activation invalidates dehydroeffusol-mediated prevention of Aβ1-42 toxicity. It is likely that MT synthesis via adrenaline β receptor activation is beneficial to neuroprotection and that oral intake of dehydroeffusol preventively serves against the Aβ1-42 toxicity.

Protection of cultured vascular endothelial cells against cadmium cytotoxicity by simultaneous treatment or pretreatment with manganese.

Cadmium is a heavy metal that pollutes the environment and foods and is a risk factor for vascular disorders. We have previously demonstrated that pretreatment of vascular endothelial cells with zinc and copper protects the cells against cadmium cytotoxicity. In contrast, cadmium cytotoxicity was potentiated in cells following exposure to lead, thereby indicating that in vascular endothelial cells, cadmium cytotoxicity can be differentially modified by the co-occurrence of other heavy metals. In this study, we revealed that simultaneous treatment or pretreatment with manganese protects vascular endothelial cells against cadmium cytotoxicity. Intracellular accumulation of cadmium was observed to be reduced by simultaneous treatment with manganese, although not by pretreatment. The mRNA expression of metal transporters that regulate the uptake of both cadmium and manganese (ZIP8, ZIP14, and DMT1) remained unaffected by either simultaneous treatment or pretreatment with manganese, and simultaneous treatment with manganese suppressed the cadmium-induced expression of metallothionein but pretreatment with manganese did not exhibit such suppressive effect. Thus, the protection of vascular endothelial cells against cadmium cytotoxicity conferred by simultaneous treatment with manganese is assumed to be partially attributed to a reduction in the intracellular accumulation of cadmium, whereas the effects of pretreatment with manganese are independent of both the reduced intracellular accumulation of cadmium and the induction of metallothionein. These observations accordingly indicate that the protective effects of manganese are mediated via alternative (as yet unidentified) mechanisms.

The induction of metallothionein by sulforaphane reduces iron toxicity via Nrf2

The induction of metallothionein by sulforaphane reduces iron toxicity via Nrf2

Arsenic induces metabolome remodeling in mature human adipocytes

Human lifetime exposure to arsenic through drinking water, food supply or industrial pollution leads to its accumulation in many organs such as liver, kidneys, lungs or pancreas but also adipose tissue. Recently, population-based studies revealed the association between arsenic exposure and the development of metabolic diseases such as obesity and type 2 diabetes. To shed light on the molecular bases of such association, we determined the concentration that inhibited 17% of cell viability and investigated the effects of arsenic acute exposure on adipose-derived human mesenchymal stem cells differentiated in vitro into mature adipocytes and treated with sodium arsenite (NaAsO2, 10 nM to 10 µM). Untargeted metabolomics and gene expression analyses revealed a strong dose-dependent inhibition of lipogenesis and lipolysis induction, reducing the cellular ability to store lipids. These dysregulations were emphasized by the inhibition of the cellular response to insulin, as shown by the perturbation of several genes and metabolites involved in the mentioned biological pathways. Our study highlighted the activation of an adaptive oxidative stress response with the strong induction of metallothioneins and increased glutathione levels in response to arsenic accumulation that could exacerbate the decreased insulin sensitivity of the adipocytes. Arsenic exposure strongly affected the expression of arsenic transporters, responsible for arsenic influx and efflux, and induced a pro-inflammatory state in adipocytes by enhancing the expression of the inflammatory interleukin 6 (IL6). Collectively, our data showed that an acute exposure to low levels of arsenic concentrations alters key adipocyte functions, highlighting its contribution to the development of insulin resistance and the pathogenesis of metabolic disorders.

Sex-specific differences in metallothionein induction by cadmium in different organs of Donax trunculus Linnaeus, 1758

ABSTRACT To investigate the impact of heavy metals on the physiology of aquatic organisms, previous studies have focused on selected biomarker measurements in samples. However, most studies do not take into account toxicity effects on both males and females. The present paper aims to examine the effect of sex on the detoxification response using metallothioneins (MTs), a biomarker of metallic contamination. MTs were measured in different organs (mantle, digestive gland and gonad) of both males and females of Donax trunculus L. (Mollusca, Bivalvia) exposed to cadmium (Cd). The samples were collected at El Battah beach, far from any source of pollution, and reared in the laboratory for four days. Cd was added to the rearing water at two previously determined sublethal concentrations (LC10 and LC25). Measurements of MTs were made at (0, 48 and 96 h). Cd exposure caused a significant induction (P < 0.05) of MTs in all studied organs in both sexes with a marked effect in females. The digestive gland had the highest MT levels, followed by the gonad and then mantle. Female and male individuals of this species differ in their susceptibility to Cd. This study supports the importance of sex and tissue in ecotoxicological studies.

Diabetic cardiomyopathy – Zinc preventive and therapeutic potentials by its anti-oxidative stress and sensitizing insulin signaling pathways

Oxidative stress and insulin resistance are two key mechanisms for the development of diabetic cardiomyopathy (DCM, cardiac remodeling and dysfunction). In this review, we discussed how zinc and metallothionein (MT) protect the heart from type 1 or type 2 diabetes (T1D or T2D) through its anti-oxidative function and insulin-mediated PI3K/Akt signaling activation. Both T1D and T2D-induced DCM, shown by cardiac structural remodeling and dysfunction, in wild-type mice, but not in cardiomyocyte-specific overexpressing MT mice. In contrast, mice with global MT gene deletion were more susceptible to the development of DCM. When we used zinc to treat mice with either T1D or T2D, cardiac remodeling and dysfunction were significantly prevented along with increased cardiac MT expression. To support the role of zinc homeostasis in insulin signaling pathways, treatment of diabetic mice with zinc showed the preservation of phosphorylation levels of insulin-mediated glucose metabolism-related Akt2 and GSK-3β and even rescued cardiac pathogenesis induced by global deletion of Akt2 gene in a MT-dependent manner. These results suggest the protection by zinc from DCM is through both the induction of MT and sensitization of insulin signaling. Combined our own and other works, this review comprehensively summarized the roles of zinc homeostasis in the development and progression of DCM and its therapeutic implications. At the end, we provided pre-clinical and clinical evidence for the preventive and therapeutic potential of zinc supplementation through its anti-oxidative stress and sensitizing insulin signaling actions. Understanding the intricate connections between zinc and DCM provides insights for the future interventional approaches.

Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Evidences of metabolic alterations and cellular damage in different tissues of scallops Aequipecten tehuelchus exposed to cadmium

Scallops Aequipecten tehuelchus (Patagonia, Argentina) were exposed to 0, 2, 5 and 12 μg Cd/L for 7 and 14 days, causing in digestive gland a significant production of reactive oxygen and nitrogen species (RONS), induction of catalase (CAT) and glutathione S-transferase (GST) activities and metallothioneins (MT) synthesis. In gills, there was inhibition of GST and induction of CAT, MT and α-tocopherol (α-Toc). In muscle, a significant increment of MT was also registered and inhibition of CAT. Lipid peroxidation, measured as TBARS, was not promoted in any tissue. More significant effects were observed in digestive gland than in gills and muscle, evidencing the critical role of digestive gland in Cd accumulation and metabolisation. This research would evidence dose-dependent effects of Cd on MT, GST, CAT and α-Toc in the three organs assayed, as well as a time-dependent effect of Cd on the response of CAT, GST and TBARS in digestive gland.

Cadmium effects on the freshwater teleost Prochilodus lineatus: Accumulation and biochemical, genotoxic, and behavioural biomarkers

In order to evaluate the effects of Cd, juveniles of the Neotropical fish Prochilodus lineatus were exposed to 1 and 10 μg L-1 Cd, for 24 and 96 h. Fish exposed to Cd showed metal accumulation in the gills, kidney, and liver, an increase in DNA damage in erythrocytes, and an increase in lipid peroxidation (LPO) in the kidney. Cd exposure also caused a reduction in catalase activity, metallothionein induction, and LPO in the liver. Cd stimulated the swimming activity of exposed fish, resulting in longer swimming times and distances travelled, especially for the shortest exposure time. Changes in acetylcholinesterase activity (AChE) in the muscle and brain are probably related to these behavioural responses. These results show that Cd affects the functioning of several organs in P. lineatus, which is indicated by the genotoxic damage and changes in the AChE and swimming pattern of the exposed fish.

Role of the luminal composition on intestinal metal toxicity, bioavailability and bioreactivity: An in vitro approach based on the cell line RTgutGC

The bioavailability of metal complexes is poorly understood. To evaluate bioavailability and toxicity of neutral and charged complexes as well as free metal ions, Visual Minteq, a chemical equilibrium model, was used to design media containing different metal species. Two non-essential (silver and cadmium) and two essential (copper and zinc) metals were selected. The rainbow trout (Oncorhynchus mykiss) gut cell line (RTgutGC) was used to investigate bioavailability, bioreactivity and toxicity of the different metal species. Toxicity was measured using a multiple endpoint cytotoxicity assay, bioavailability by measuring intracellular metal concentration, and bioreactivity by quantification of mRNA level of the metal responsive genes, metallothionein (MT), glutathione reductase (GR) and zinc transporter 1 (ZnT1). Speciation calculations showed that silver and cadmium preferentially bind chloride, copper phosphate and bicarbonate, and zinc remained primarily as a free ion. Cysteine avidly complexed with all metals reducing toxicity, bioavailability and bioreactivity. Silver and copper toxicity was not affected by inorganic metal speciation, whereas cadmium and zinc toxicity was decreased by chloride complexation. Moreover, reduction of calcium concentration in the medium increased toxicity and bioavailability of cadmium and zinc. Bioavailability of silver and zinc was reduced by low chloride while cadmium bioavailability was increased by low chloride and in presence of bicarbonate. Copper bioavailability was not affected by the medium composition. Cadmium and silver were more bioreactive, independently from the medium composition, in comparison to copper and zinc (i.e., higher induction of MT and GR). Cadmium was the only metal able to induce MT in presence of cysteine. ZnT1 was induced by cadmium in low-chloride, by zinc in low-chloride low-calcium and by cadmium and copper in the bicarbonate media. Overall, this study demonstrates that metal complexation alone is not sufficient to explain metal toxicity, and that anion exchange mechanisms play a role in metal uptake and bioreactivity.

Development of an in Vitro Digestion System to Study the Bioavailability and Bioreactivity of Zinc Sulfate and Zn-Bioplex in Fish Using the RTgutGC Cell Line

Here, we report the development of a novel in vitro digestion system that mimics the digestive process in fish and uses the RTgutGC cell line, a model of the rainbow trout (Oncorhynchus mykiss) intestine, to study the bioavailability, bioreactivity, and toxicity of Zn added as an inorganic (ZnSO4) and organic zinc compound (Zn-Bioplex). Our results showed that, before digestion, Zn-Bioplex was more bioavailable than ZnSO4. However, after in vitro digestion, ZnSO4 and Zn-Bioplex accumulated and were transported equally across the RTgutGC cell epithelium. However, Zn-Bioplex was shown to be less bioreactive than ZnSO4, suggesting that peptide complexation might reduce the intracellular release of zinc as shown by a reduced induction of metallothionein and ZnT1 mRNA levels. This novel method shows promise as a standardized in vitro fish digestion model to study the interactions occurring at the lumen of the intestine and the bioavailability and toxicity of digested compounds in the fish gut.

RETRACTED ARTICLE: Metallothionein ameliorates airway epithelial apoptosis upon particulate matter exposure: role of oxidative stress and ion homeostasis

PurposeTo investigate the mechanism underlying particulate matter (PM) exposure-induced oxidative stress and potential rescue strategies against pulmonary damage in this context.MethodsA combination of omics technology and bioinformatic analysis were used to uncover mechanisms underlying cellular responses to PM exposure in human bronchial epithelia (HBE) cells and imply the potential rescue.ResultsOur results implicated that oxidative stress, metal ion homeostasis, and apoptosis were the major cellular responses to PM exposure in HBE cells. PM exposure disrupted oxidative phosphorylation (OXPHOS)-related gene expressions in HBE cells. Rescuing the expression of these genes with supplemental coenzyme Q10 (Co Q10) inhibited reactive oxygen species (ROS) generation; however, it only partially protected HBEs against PM exposure-induced apoptosis. Further, metallothionein (MT)-encoding genes associated with metal ion homeostasis were significantly induced in HBE cells, which was transcriptionally regulated by specificity protein 1 (SP1). SP1 knock-down (KD) aggravated PM-induced apoptosis in HBE cells, suggesting it plays a role in MT induction. Subsequent studies corroborated the protective role of MT by showing that exogenous MTs supplement demonstrated effective protection against PM-induced oxidative stress and apoptosis in HBE cells. Importantly, exogenous MTs supplement was shown to reduce ROS generation and apoptosis in airway epithelia in both HBE cells and a PM-inhaled murine model.ConclusionThis study demonstrates that the impact of MTs on airway epithelia by suppressing oxidative stress and maintaining metal ion homeostasis is beneficial in attenuating damage to pulmonary cells undergoing PM exposure.

Metals and Metallothionein Expression in Relation to Progression of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka.

Chronic kidney disease of unknown etiology was investigated for metal relations in an endemic area by a cross-sectional study with CKD stages G1, G2, G3a, G3b, G4, G5 (ESRD), and endemic and nonendemic controls (EC and NEC) as groups. Subjects with the medical diagnosis were classified into groups by eGFR (SCr, CKD-EPI) and UACR of the study. It determined 24 metals/metalloids in plasma (ICPMS) and metallothionein (MT) mRNA in blood (RT-PCR). MT1A at G3b and MT2A throughout G2–G5 showed increased transcription compared to NEC (ANOVA, p < 0.01). Both MT1A and MT2A remained metal-responsive as associations emerged between MT2A and human MT inducer Cr (in EC: r = 0.54, p < 0.05, n = 14), and between MT1A and MT2A (in EC pooled with G1–G5: r = 0.58, p < 0.001, n = 110). Human MT (hMT)-inducers, namely Zn, Cu, As, Pb, and Ni; Σ hMT-inducers; 14 more non-inducer metals; and Σ MT-binding metals remained higher (p < 0.05) in EC as compared to NEC. Declining eGFR or CKD progression increased the burden of Be, Mg, Al, V, Co, Ni, Rb, Cs, Ba, Mn, Zn, Sr, Σ hMT-inducers, and Σ MT-binding metals in plasma, suggesting an MT role in the disease. MT1A/2A mRNA followed UACR (PCA, Dendrogram: similarity, 57.7%). The study provides evidence that proteinuric chronic renal failure may increase plasma metal levels where blood MT2A could be a marker.

Impacts of tailings of Fundão dam (Brazil) rupture on marine fish: Metals bioaccumulation and physiological responses

This study evaluated the impacts of the mining tailings after the rupture of the Fundão dam on fish communities on the Atlantic Ocean southeast coast. Four sample collections were carried out over two years (2018–2020), in seasonal periods. Omnivorous/herbivorous and carnivorous fish were collected for analysis of metal bioaccumulation, multibiomarkers of environmental contamination and histopathology. Metal bioaccumulation was stronger correlated in carnivorous fish in the dry-2018 collection, besides higher activity of antioxidant enzymes, energy metabolism and higher morphological damage; however, there was less oxidative damage and less metallothioneins induction, and these variables were strongly associated with the wet-2020 collection. In a temporal view, it was possible to observe a reduction in metal levels in fish, except in the mouth of the Doce River. These events can be explained by seasonal natural events, which tend the resuspension and boost metal levels, mainly in the mouth region during the rainy season.

Azepine-Indole Alkaloids From Psychotria nemorosa Modulate 5-HT2A Receptors and Prevent in vivo Protein Toxicity in Transgenic Caenorhabditis elegans.

Nemorosine A (1) and fargesine (2), the main azepine-indole alkaloids of Psychotria nemorosa, were explored for their pharmacological profile on neurodegenerative disorders (NDs) applying a combined in silico–in vitro–in vivo approach. By using 1 and 2 as queries for similarity-based searches of the ChEMBL database, structurally related compounds were identified to modulate the 5-HT2A receptor; in vitro experiments confirmed an agonistic effect for 1 and 2 (24 and 36% at 10 μM, respectively), which might be linked to cognition-enhancing properties. This and the previously reported target profile of 1 and 2, which also includes BuChE and MAO-A inhibition, prompted the evaluation of these compounds in several Caenorhabditis elegans models linked to 5-HT modulation and proteotoxicity. On C. elegans transgenic strain CL4659, which expresses amyloid beta (Aβ) in muscle cells leading to a phenotypic paralysis, 1 and 2 reduced Aβ proteotoxicity by reducing the percentage of paralyzed worms to 51%. Treatment of the NL5901 strain, in which α-synuclein is yellow fluorescent protein (YFP)-tagged, with 1 and 2 (10 μM) significantly reduced the α-synuclein expression. Both alkaloids were further able to significantly extend the time of metallothionein induction, which is associated with reduced neurodegeneration of aged brain tissue. These results add to the multitarget profiles of 1 and 2 and corroborate their potential in the treatment of NDs.

Differential Gene Expression Profiles between N-Terminal Domain and Ligand-Binding Domain Inhibitors of Androgen Receptor Reveal Ralaniten Induction of Metallothionein by a Mechanism Dependent on MTF1.

Simple SummaryInhibition of the androgen receptor (AR) remains the mainstay treatment for prostate cancer. All current therapies involving AR inhibition either directly or indirectly target its ligand-binding domain (LBD). We have developed the first novel compounds which target the N-terminal domain, (NTD) a region which is essential for AR transcriptional activity. First-generation ralaniten (NCT02606123), and second-generation EPI-7386 (NCT04421222) remain the only AR-NTD inhibitors to progress to clinical trials. Here we aim to characterize differences between different classes of AR antagonists targeting the AR-LBD and the AR-NTD, as well as next generation AR-NTD inhibitors. An incidental finding was that ralaniten was uniquely associated with increased metallothionein expression which was independent of AR activity. Instead, expression of metallothionein genes was driven by MTF-1 indicating a potential off-target effect. Neither AR-LBD inhibitor enzalutamide nor second-generation AR-NTD inhibitor EPI-7170 had this effect. This work has important implications for the development of novel AR-NTD inhibitors.Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein–protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.

Effect of temperature on the toxicokinetics and gene expression of the pacific cupped oyster Crassostrea gigas exposed to cadmium

In this study, we investigated the influence of temperature on the bioaccumulation and depuration of Crassostrea gigas exposed to Cd associated with its molecular responses. Oysters were acclimatized to different temperatures (10 °C, 15 °C, 20 °C, 25 °C, and 30 °C) for 14 d and then exposed to 10 μg/L Cd for 28 d, followed by a depuration period of 35 d. Oysters were sampled for chemical analysis by inductively coupled plasma mass spectrometry (ICP-MS) and for mRNA quantification by qPCR. In the digestive gland, gill, and mantle, the cadmium concentration at 10 °C was significantly lower than that at 25 °C and 30 °C in both the whole experiments. The use of a two-compartment model showed that the uptake rate k1 in the above three tissues increased with increasing temperatures ranging from 15 to 25 °C. The fastest elimination rates and shortest half-lives were observed at 15–25 °C. The induction of metallothionein (MT) only occurred in the digestive gland at 15 °C and 20 °C at the end of the accumulation phase. In the mantle and gills, the expression of P-glycoprotein (P-gp) was significantly induced at the end of the accumulation phase and significantly inhibited at the end of the depuration phase. In the digestive gland, the expression of P-gp was induced at the end of both the accumulation and depuration phases. Heat shock protein (hsp70) expression exhibited an overall increasing trend throughout the experiment.