Upregulation Of Metallothionein Research Articles

Macrophage metallothioneins participate in the antileishmanial activity of antimonials.

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing.

Placental BCRP transporter reduces cadmium accumulation and toxicity in immortalized human trophoblasts

Cadmium (Cd) is a ubiquitous environmental metal detectable in most pregnant women. Animal and human studies demonstrate that in utero exposure to Cd reduces birth weight and impairs perinatal growth due to placental toxicity. BCRP is a prominent transporter that can efflux xenobiotics from the placenta. This study sought to investigate Cd transport and toxicity in cultured human BeWo trophoblasts with reduced expression and function of the placental barrier transporter BCRP. Knockdown (KD) of BCRP protein expression and function in BeWo trophoblasts increased the intracellular accumulation of Cd by 100% following treatment with 1 μM CdCl2. No change in the expression of Cd uptake transporters was observed between control and BCRP-KD cells. Reduced BCRP expression impaired viability of BeWo cells exposed to CdCl2 for 48 hr (BCRP-KD IC50: 11 μM, control cells IC50: 18 μM). Moreover, BCRP-KD cells were more sensitive to CdCl2-induced cytotoxicity compared to control BeWo cells. CdCl2 treatment strongly induced the expression of the metal-binding protein metallothionein (MT) in both control and BCRP-KD cells, with significantly greater MT upregulation in Cd-treated BCRP-KD cells. These data suggest that the BCRP transporter reduces Cd accumulation in syncytiotrophoblasts, which may be one mechanism to reduce subsequent toxicity to the placenta and developing fetus.

Dihydroartemisinin-induced ferroptosis in acute myeloid leukemia: links to iron metabolism and metallothionein

Artemisinin is an anti-malarial drug that has shown anticancer properties. Recently, ferroptosis was reported to be induced by dihydroartemisinin (DHA) and linked to iron increase. In the current study, we determined the effect of DHA in leukemic cell lines on ferroptosis induction and iron metabolism and the cytoprotective effect triggered in leukemic cells. We found that treatment of DHA induces early ferroptosis by promoting ferritinophagy and subsequent iron increase. Furthermore, our study demonstrated that DHA activated zinc metabolism signaling, especially the upregulation of metallothionein (MT). Supportingly, we showed that inhibition MT2A and MT1M isoforms enhanced DHA-induced ferroptosis. Finally, we demonstrated that DHA-induced ferroptosis alters glutathione pool, which is highly dependent on MTs-driven antioxidant response. Taken together, our study indicated that DHA activates ferritinophagy and subsequent ferroptosis in AML and that MTs are involved in glutathione regenerating and antioxidant response.

Upregulation of Metallothionein 1 G (MT1G) Negatively Regulates Ferroptosis in Clear Cell Renal Cell Carcinoma by Reducing Glutathione Consumption.

Ferroptosis is characterized by lipid peroxidation and iron accumulation, closely associated with clear cell renal cell carcinoma (ccRCC). It is of great significance for prognostic prediction and treatment of ccRCC to find biomarkers related to ferroptosis. We conducted several bioinformatic analyses using the transcriptome data and clinical information derived from online databases. Firstly, we identified the differentially expressed target genes in ccRCC. Then, t test and COX analysis were used to determine whether it was an independent prognostic factor combined with clinical information. String and gene set enrichment analysis (GSEA) were used to predict its function. Finally, we used ccRCC cells: 769-P and KAKI-1 in vitro to verify the regulation of target genes on cell proliferation apoptosis, iron metabolism, and GSH metabolism, which were used to judge the effect of target genes on ferroptosis. The study showed that MT1G is downregulated in ccRCC tissues compared with normal renal tissues. However, the ccRCC patients with higher expression relatively had higher malignancy and advanced stages. MT1G is an independent adverse factor for the prognosis of ccRCC. The protein interaction network analysis and GSEA showed that MT1G was closely related to GSH metabolism-related proteins (GSR) and lipid oxidation-related proteins (PLA2G2A). Samples with high expression of MT1G were enriched in “glutathione metabolism,” “oxidative phosphorylation,” and “proteasome,” whose function was involved in GSH metabolism and lipid peroxidation. The term associated with the occurrence and development of tumors included “P53 signaling pathway.” Furthermore, in vitro experiments showed that MT1G partially blocked ferroptosis induced by erastin and sorafenib-induced ccRCC cell lines (769-P and CAKI-1). The mechanism may be that MT1G affects ferroptosis by regulating GSH consumption in ccRCC cells. MT1G may be a negative regulator of ferroptosis in ccRCC cells and a biomarker of poor prognosis.

High Temperature-Induced Oxidative Stress Affects Systemic Zinc Homeostasis in Broilers by Regulating Zinc Transporters and Metallothionein in the Liver and Jejunum.

To investigate the change in zinc homeostasis of broilers under heat stress, 512 broiler chickens were raised to the age of 28 days. The broilers were then assigned to heat stress and normal temperature (36.0°C vs. 26.0°C) groups for 7 days. The results showed that oxidative stress induced by high temperature had a negative effect on the growth performance of broilers. Heat stress altered zinc homeostasis and led to a redistribution of zinc in broilers, which was reflected in increased zinc concentrations in the jejunum, liver, and tibia. Upregulation of the expression of the zinc exporter ZnT1 and importers ZIP8 and ZIP14 in the jejunum indicated that more zinc was absorbed and transported from the jejunum into the blood, while the liver increased its capacity to hold zinc through upregulation of metallothionein (MT) expression, which was achieved by reducing ZnT1 expression and upregulating the expression of the importer ZIP3. The pathway was mediated by zinc transporters, but the capacity of MT to chelate and release zinc ions also played a crucial role. The mechanism of alterations in zinc homeostasis under heat stress was revealed by the changes in zinc transporters and MT levels in the intestine and liver. Heat stress also altered cecal microbial diversity and reduced the relative abundances of Bilophila and Dialister. In conclusion, broilers altered systemic zinc homeostasis through the regulation of zinc transporters and MT in the liver and jejunum to resist oxidative stress induced by high temperature.

The Paradoxical Efficacy of KDM6 Inhibition in Germinal Centre B-Cell Lymphomas

IntroductionGerminal centre (GC) lymphomas Follicular Lymphoma (FL) and Diffuse Large B Cell Lymphoma (DLBCL) are strikingly dependent on mutations affecting chromatin regulators. The majority of FL tumours harbour multiple mutated chromatin-regulatory genes, including loss-of-function mutations in KMT2D (80%), CREBBP (60%) and gain-of-function mutations in EZH2 (25%), whilst these are also frequently mutated within the GC B-cell (GCB) subtype of DLBCL. The FDA approval of EZH2-inhibitor Tazemetostat for relapsed/refractory FL exemplifies the potential and the need to broaden our search for novel avenues targeting epigenetic reprogramming in GC lymphomas. Hence, in this study, we sought to identify novel epigenetic vulnerabilities and biological interactions in GC B cell lymphomas.Methods and Results:25 compounds targeting 18 histone binding/modifying enzymes were tested across 12 GCB-DLBCL cell lines harbouring differing degrees of epigenetic dysregulation. The two most potent cytotoxic compounds (GSK-J4 and KDOBA67) both target the H3K27me3 demethylase, KDM6. The KDM6 family consists of KDM6A (UTX) and KDM6B (JMJD3), both of which demethylate H3K27me3/H3K27me2 to lower methylation states, and KDM6C (UTY) which has negligible enzymatic activity. Indeed, KDM6 inhibition (KDM6i) with GSK-J4 increased levels of the inhibitory chromatin mark H3K27me3 and simultaneously reduced H3K27me1/me2, while we observed no global changes in H3K4me3.The induction of apoptosis in the cell lines following either inhibition of the ‘eraser’ KDM6 (leading to increased H3K27me3) or the opposing ‘writer’ EZH2 (lowering H3K27me3) appears paradoxical at first glance, given the EZH2 gain-of-function mutations seen in FL/DLBCL and known efficacy of EZH2 inhibitors. However, RNA-sequencing analysis of GSK-J4-treated cells revealed no significant overlap with published EZH2 mutation or EZH2 inhibition signatures from GC lymphoma models, implying that KDM6i-regulated H3K27me3 peaks do not overlay gene promoters targeted by EZH2 in the GC reaction. The most significant gene regulation by KDM6i was a significant up-regulation of 6 members of the metallothionein (MT) family within 4-hours of treatment, which was sustained for 72-hours (MT1E, MT1F, MT1G, MT1H, MT1X and MT2A at 4 hours log2FoldChange 5.1-12.1, average p adj 3.4E-06). These metal-binding proteins cluster on chromosome 16 and their up-regulation correlated with up-regulation of the zinc transporter SLC30A1, suggesting a potential biological relationship between KDM6 enzymes and zinc homeostasis, with both isoforms containing a zinc-binding domain. Targeted validation across 6 DLBCL cell lines also showed MT up-regulation correlated with sensitivity to GSK-J4, whilst siRNA-mediated knockdown of KDM6A or KDM6B individually recapitulated the up-regulation of MT genes, albeit to a lower extent. ChIP-qPCR profiling at the MT loci for H3K27me3 and H3K4me3 revealed no significant changes at 4 or 24 hours; considering that expression of MT genes increase within 60 minutes of drug exposure, suggesting these changes may be induced by an indirect mechanism of KDM6A and/or KDM6B activity. Using ENCODE data of 4 cell lines included in our series (DOHH-2, OCI-LY-1, SU-DHL-6, KARPAS-422), we observe a link between GSK-J4 sensitivity, up-regulation of MT genes and significantly lower basal enrichment for H3K27ac at the MT loci. This distinction may reflect altered 3D chromatin conformation that render only sensitive cell lines permissive to regulation by KDM6i, with H3K27ac having recently been shown to modulate enhancer positioning and interactions in DLBCL cell lines.Conclusion:Screening epigenetic-targeting compounds in GC lymphomas has identified KDM6 as a particularly promising candidate target. Overall, we describe up-regulation of MT genes as one potential mechanism of action, which correlates with response in DLBCL cell lines and suggests that KDM6 members may regulate H3K27me3 in a manner different from EZH2. In our current model we propose, that in GC-lymphomas KDM6A may have tumour suppressive roles whilst KDM6B is required for cell survival. Given KDM6A is frequently lost in cancer and Kabuki syndrome, a congenital developmental disorder caused by germline mutations in KMT2D or KDM6A, our results highlight the importance of establishing what roles the KDM6 family play in lymphoma pathogenesis. DisclosuresFitzgibbon: Epizyme: Research Funding.

Arsenic (III) induces oxidative stress and inflammation in the gills of common carp, which is ameliorated by zinc (II)

Arsenic (As) is widely present in the environment in form of arsenite (AsIII) and arsenate (AsV). Oxidative stress and inflammation are believed to be the dominant mechanisms of AsIII toxicity in vivo and in vitro. The aim of this study was to investigate whether zinc (Zn2+) alleviates exogenous gill toxicity in carp induced by AsIII and to gain insight into the underlying mechanisms. Exposure of carp to 2.83 mg As2O3/L for 30 days reduced superoxide dismutase activity by 4.0%, catalase by 41.0% and glutathione by 19.8%, while the concentration of malondialdehyde was increased by 16.4% compared to the control group, indicating oxidative stress. After the exposure of carp to AsIII the expression of inflammatory markers, such as interleukin-6, interleukin-8, tumor necrosis factor α and inducible nitric oxide synthase in gill tissue were significantly increased. In addition, the phosphorylation of nuclear factor kappa-B (NF-κB) was increased by 225%. 1 mg ZnCl2/L can relieve the toxicity of AsIII based on histopathology, antioxidase activity, qRT-PCR and western results. Zn2+ attenuated AsIII-induced gill toxicity that suppressed intracellular oxidative stress and NF-κB pathway by an upregulation of metallothionein. Therefore, the toxic effect of AsIII on the gill cells of carp was reduced. This study provides a theoretical basis for exploring the alleviation of the toxic effects of metalloids on organisms by heavy metals and the biological assessment of the effects.

CT23 knockdown attenuating malignant behaviors of hepatocellular carcinoma cell is associated with upregulation of metallothionein 1.

The cancer-testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progressof hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, theRNA interfering (RNAi) method was used to silence CT23 expression in BEL-7404 cells. Microarray analysis was performed on mRNAextracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two-fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformaticanalysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attainedthe maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non-neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL-7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight intosearching for a novel therapeutic target for HCC.

D609 Protects Corneal Epithelial from Oxidative Stress-mediated Injury via Metallothionein Upregulation in Dry Eye

D609 Protects Corneal Epithelial from Oxidative Stress-mediated Injury via Metallothionein Upregulation in Dry Eye

Ex-situ induction of Metallothionein gene in Lybiodrilus violaceous post cadmium and zinc exposure

BackgroundEarthworms are good indicators used in evaluating the health status of the terrestrial ecosystem and metallothionein (Mt) is a biomarker monitored during environmental studies. There is however a dearth of information on metallothioneins in African earthworms. Libyodrilus violaceous is a West African earthworm well investigated and reported including its tolerance to metal pollution but the presence of Mt gene in L. violaceous is not established. MethodsThe presence of Mt. in L. violaceous was determined to validate its potential role in metal tolerance. L. violaceous sampled from Ije-Ododo soil were exposed to varying concentrations of Cadmium (Cd) and Zinc (Zn) in a potted experimental set-up and the metallothionein (MT) expressions were determined. The presence of Mt gene and metallothionein expression were determined using whole transcriptome sequencing and real-time polymerase chain reaction (PCR), respectively after extracting Ribonucleic acid (RNA) from samples on the 1 st, 3rd and 7th days. The genetic relationship of the detected MT protein sequence was compared to existing MT protein sequences and a phylogenetic tree was constructed. ResultsWe established the presence of the MT gene and the phylogenetic tree showed that it had a relationship with other earthworms' MTs. Up-regulation of Mt. expression was observed on the 7th day in L.violaceous exposed to concentrations of Cd and Zn. ConclusionOne of the modes of survival of L. violaceous to metal pollution is by the up-regulation of metallothioneins. This earthworm metallothionein protein sequence is the first report of species in the Eudriloides family and the first report in African earthworms deposited in the GenBank repository for reference. The use of L.violaceous for Cd and Zn remediation in soils could be exploited.

Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Effects of Enteric Environmental Modification by Coffee Components on Neurodegeneration in Rotenone-Treated Mice.

Epidemiological studies have shown that coffee consumption decreases the risk of Parkinson’s disease (PD). Caffeic acid (CA) and chlorogenic acid (CGA) are coffee components that have antioxidative properties. Rotenone, a mitochondrial complex I inhibitor, has been used to develop parkinsonian models, because the toxin induces PD-like pathology. Here, we examined the neuroprotective effects of CA and CGA against the rotenone-induced degeneration of central dopaminergic and peripheral enteric neurons. Male C57BL/6J mice were chronically administered rotenone (2.5 mg/kg/day), subcutaneously for four weeks. The animals were orally administered CA or CGA daily for 1 week before rotenone exposure and during the four weeks of rotenone treatment. Administrations of CA or CGA prevented rotenone-induced neurodegeneration of both nigral dopaminergic and intestinal enteric neurons. CA and CGA upregulated the antioxidative molecules, metallothionein (MT)-1,2, in striatal astrocytes of rotenone-injected mice. Primary cultured mesencephalic or enteric cells were pretreated with CA or CGA for 24 h, and then further co-treated with a low dose of rotenone (1–5 nM) for 48 h. The neuroprotective effects and MT upregulation induced by CA and CGA in vivo were reproduced in cultured cells. Our data indicated that intake of coffee components, CA and CGA, enhanced the antioxidative properties of glial cells and prevents rotenone-induced neurodegeneration in both the brain and myenteric plexus.

Integrative Analysis of Global Gene Expression Identifies Opposite Patterns of Reactive Astrogliosis in Aged Human Prefrontal Cortex.

The prefrontal cortex (PFC) is one of the brain regions with more prominent changes in human aging. The molecular processes related to the cognitive decline and mood changes during aging are not completely understood. To improve our knowledge, we integrated transcriptomic data of four studies of human PFC from elderly people (58–80 years old) compared with younger people (20–40 years old) using a meta-analytic approximation combined with molecular signature analysis. We identified 1817 differentially expressed genes, 561 up-regulated and 1256 down-regulated. Pathway analysis revealed down-regulation of synaptic genes with conservation of gene expression of other neuronal regions. Additionally, we identified up-regulation of markers of astrogliosis with transcriptomic signature compatible with A1 neurotoxic astrocytes and A2 neuroprotective astrocytes. Response to interferon is related to A1 astrocytes and the A2 phenotype is mediated in aging by activation of sonic hedgehog (SHH) pathway and up-regulation of metallothioneins I and genes of the family ERM (ezrin, radixin, and moesin). The main conclusions of our study are the confirmation of a global dysfunction of the synapses in the aged PFC and the evidence of opposite phenotypes of astrogliosis in the aging brain, which we report for the first time in the present article.

A calcium channel blocker nifedipine distorts the effects of nano-zinc oxide on metal metabolism in the marsh frog Pelophylax ridibundus.

Global decline of amphibian populations causes particular concern about their vulnerability to novel environmental pollutants, including engineering nanomaterials and pharmaceutical products. We evaluated the bioavailability of nanoform of zinc oxide (n-ZnO) in frog Pelophylax ridibundus and determined whether co-exposure to a common pharmaceutical, a calcium-channel blocker nifedipine (Nfd) can affect this bioavailability. Male frogs were exposed for 14 days to the tap water (Control) and n-ZnO (3.1 μM), Zn2+ (3.1 μM, as a positive control for n-ZnO exposures), Nfd (10 μM), and combination of n-ZnO and Nfd (n-ZnO + Nfd) in environmentally-relevant concentration. Exposure to Zn2+ or n-ZnO led to up-regulation of metal-binding proteins, metallothioneins (MTs) in the liver and Zn-carrying vitellogenin-like proteins in the blood plasma. Notably, upregulation of MTs by Zn2+ or n-ZnO exposures combined with increased binding of Zn and Cu to MTs. This was associated with the more reducing conditions in the liver tissue indicated by elevated lactate to pyruvate ratio. Nfd suppressed the binding of Zn and Cu to MTs and led to a decrease in Lactate/Pyruvate ratio and elevated protein carbonylation indicating pro-oxidant conditions. Redox status parameters were not directly related to DNA fragmentation, nuclear abnormalities or suppression of cholinesterase activity indicating that factors other than oxidative stress are involved in cytotoxicity of different pollutants and their combinations. Furthermore, activity of Phase I biotransformation enzyme (CYP450 oxidase measured as EROD) was elevated in Nfd-containing exposures and in Zn2+ exposed frogs. Tyrosinase-like activity in the frog liver was strongly stimulated by Zn2+ but suppressed by n-ZnO, Nfd and n-ZnO + Nfd. These findings show that Nfd modulates homeostasis of essential metals in amphibians and emphasize that physiological consequences of combined n-ZnO and Nfd exposures are difficult to predict based on the mechanisms of single stressors.

Zinc gluconate toxicity in wild-type vs. MT1/2-deficient mice

Previous studies have suggested that oral zinc supplementation can help reduce the duration of the common cold; however, the use of intranasal (IN) zinc is strongly associated with anosmia, or the loss of the sense of smell, in humans. Prior studies from this lab showed that upregulation of metallothioneins (MT) is a rapid and robust response to zinc gluconate (ZG). Therefore, we assessed the role of MT in the recovery of nasal epithelial damage resulting from IN zinc administration. The main studies in this investigation used a high dose of ZG (170mM) to ensure ablation of the olfactory mucosa, so that the progression of histological and functional recovery could be assessed. In vivo studies using wild-type, MT1/2 knockout mice (MT KO), and heterozygotes administered ZG by IN instillation showed profound loss of the olfactory mucosa in the nasal cavity. Recovery was monitored, and a lower percentage of the MT KO mice were able to smell 28 d after treatment; however, no significant difference was observed in the rate of cell proliferation in the basal layer of the olfactory epithelium between MT KO and wild-type mice. A lower concentration of ZG (33mM), equivalent to that found in homeopathic IN ZG preparations, also caused olfactory epithelial toxicity in mice. These studies suggest that the use of zinc in drug formulations intended for IN administration in humans must be carefully evaluated for their potential to cause olfactory functional deficits.

Toxicant mixtures in sediment alter gene expression in the cysteine metabolism of Chironomus tepperi.

Sediment contamination can pose risks to the environment, and sediment toxicity tests have been developed to isolate the impact of sediment from other factors. Mixtures of contaminants often occur in sediments, and traditional endpoints used in toxicity testing, such as growth, reproduction, and survival, cannot discern the cause of toxicity from chemical mixtures because of complex interactions. In urban waterways, the synthetic pyrethroid bifenthrin and the metal copper are commonly found in mixtures, so the present study was designed to investigate how these contaminants cause toxicity in mixtures. To investigate this, Chironomus tepperi was exposed to environmentally relevant concentrations of copper and bifenthrin-spiked sediments in a 2-way factorial mixture for 5 d. Growth and expression profiles of cysteine metabolism genes were measured after exposure. Growth increased at low copper concentrations, decreased at high copper concentrations, and was unaffected by bifenthrin exposures. Copper exposures induced possible cellular repair by upregulating S-adenosylmethionine synthetase expression and downregulating expression of S-adenosylhomocysteine hydrolase and cystathionine-β-synthase. Metallothionein upregulation was also observed. Bifenthrin exposure altered cysteine metabolism to a lesser extent, downregulating cystathionine-β-synthase and γ-glutamylcysteine synthase. Synergistic, antagonistic, and dose-dependent interactions were observed, and there was evidence of conflicting modes of action and limited substrate production. These findings demonstrate how contextual gene expression changes can be sensitive and specific identifiers of toxicant exposure in mixtures. Environ Toxicol Chem 2017;36:691-698. © 2016 SETAC.

Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.

Zinc‐induced upregulation of metallothionein (MT)‐2A is predicted by gene expression of zinc transporters in healthy adults

Marginal zinc deficiency has been suggested to influence multiple physiological systems due to the extensive involvement of zinc in numerous structural and biochemical functions of the body. Recent advancements in molecular techniques have enabled the use of novel approaches in the study of zinc metabolism and homeostasis, especially within the cell. The usefulness of zinc transporter and metallothionein (MT) gene expressions to detect changes in zinc intake remains unclear. This study aims to determine the effects of zinc supplementation on zinc transporters and metallothioneins (MT) and their relationships with physical activity level. Healthy adults (n = 39) were randomised to a zinc group (20 mg Zn/day; n = 19) or no treatment (n = 20). Blood samples were collected on Days 0, 2, 7, 14 and 21. The IPAQ‐short form was used to assess physical activity level at baseline. RNA was extracted from peripheral blood mononuclear cells and gene expression of zinc transporters and MT were analysed using real‐time PCR. Using repeated measures ANOVA, MT‐2A gene expression was higher in the zinc group (P < 0.05) with a significant increase at Day 2 (40 ± 18%, mean ± SE, P < 0.05). In a multiple regression model exploring the changes in all gene expressions between Days 0 and 21, the change in MT‐2A gene expression was correlated with changes in zinc transporter expressions (r2 = 0.54, P = 0.029) with ZIP1 expression emerging as a univariate predictor (P = 0.003). Total physical activity level was positively correlated with ZIP7 gene expression at baseline (r = 0.36, P = 0.029). The influence of physical activity and zinc supplementation on the expression of ZIP7 and MT‐2A respectively demonstrates the effects of external stimuli and mediators of cellular zinc homeostasis, with potential implications for signalling pathways and related cellular functions.Support or Funding InformationR&D Human Nutrition Grant, Meat and Livestock Australia

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

Reduced zinc cytotoxicity following differentiation of neural stem/progenitor cells into neurons and glial cells is associated with upregulation of metallothioneins

We investigated zinc cytotoxicity in mouse neural stem/progenitor cells (NSPCs) and their differentiated progeny (neuronal/glial cells) in correlation with expression of metallothionein (MT) gene. Differentiated cells were less sensitive than NSPCs to ZnCl2 (IC50: 128μM vs. 76μM). Differentiation of immature NSPCs to the differentiated cells led to an increase in expression of MT family genes (Mt1, Mt2, Mt3, and Mt4). Zinc exposure induced a dose-dependent increase in expression level of Mt1 and that of Mt2 in both NSPCs and the differentiated cells. Our results showed that the reduced cytotoxicity of zinc associated with differentiation from NSPCs into their progeny was related to the upregulation of MTs.