Thallium(I) induces a prolonged inhibition of (6-4)photoproduct binding and UV damage excision repair activities in zebrafish (Danio rerio) embryos via protein inactivation.

Cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4 PP) are two major types of UV-induced DNA lesion and 6-4 PP is more mutagenic than CPD. Activated by lesion detection, nucleotide excision repair (NER) eliminates CPDs and 6-4 PPs. Thallium (Tl) is a toxic metal existing primarily as Tl+ in the aquatic environment. Ingestion of Tl+-contaminated foods and water is a major route of human poisoning. As Tl+ may inhibit enzyme activities via binding to sulfhydryl groups, this study explored if Tl+ could intensify UV mutagenicity by inactivating NER-linked damage recognition factors using zebrafish (Danio rerio) embryo as a model system. Incubation of Tl+ (as thallium nitrate) at 0.1-0.4 µg/mL with zebrafish extracts for 20 min caused a concentration-dependent inhibition of 6-4 PP binding activities as shown by a photolesion-specific band shift assay, while CPD binding activities were insensitive to Tl+. The ability of Tl+ to suppress 6-4 PP detection was stronger than that of Hg2+. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to Tl+ at 0.4-1 µg/mL for 9 or 71 h also specifically inhibited 6-4 PP detection, indicating that Tl+ induced a prolonged inhibition of 6-4 PP sensing ability primarily via its direct interaction with damage recognition molecules. Tl+-mediated inhibition of 6-4 PP binding in embryos at distinct stages resulted in a suppression of NER capacity monitored by a transcription-based DNA repair assay. Our results revealed the potential of Tl+ to enhance UV mutagenicity by disturbing the removal of 6-4 PP through repressing the lesion detection step of NER.

Toxic effects of thallium (Tl+) on prokaryotic alga Microcystis aeruginosa: Short and long-term influences by potassium and humic acid.

Thallium (Tl) is a priority pollutant regulated by the US EPA. It is also a critical element commonly used in high technology industries; with an increasing demand for semiconductors nowadays, wastewater discharges from manufacturing plants or metal mining activities may result in elevated levels of thallium in receiving water harming aquatic organisms. Regarding the impact of thallium on freshwater algae, little attention has been paid to prokaryotic physiology through various exposure periods. In this bench-scale study, prokaryotic alga Microcystis aeruginosa PCC 7806 was cultured in modified BG11 medium and exposed to Tl+ (TlNO3) ranging from 250 to 1250 µg/L for 4 and 14 days. Throughout the experiment using flow cytometry assays, algal population, cell membrane integrity, oxidation stress level, and chlorophyll fluorescence were exacerbated following the exposure to 750 µg Tl/L (approximately 4-day effective concentration of Tl+ for reducing 50% of algal population). Potassium and humic acid (HA) (1-5 mg/L) were added to study their influences on the thallium toxicity. With the additions of potassium, thallium toxicities to algal population and physiology were not significantly changed within 4 days, while they were alleviated within 14 days. With the addition of HA at 1 mg/L, cell membrane integrity was significantly attenuated within 4 days; ameliorating effects on algal population and oxidative stress were not observed until day 14. Thallium toxicities on oxidative stress level and photosynthesis activity were exacerbated in the presence of HA at 3-5 mg/L. The study provides useful information for further studies on the mode of toxic action of Tl+ in prokaryotic algae; it also demonstrates the necessity of considering short and long-term exposure durations while incorporating water chemistry into assessment of thallium toxicity to algae.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

Thallium-mediated NO signaling induced lipid accumulation in microalgae and its role in heavy metal bioremediation.

Thallium (Tl+) is a trace metal with extreme toxicity and is highly soluble in water, posing a great risk to ecological and human safety. This work aimed to investigate the role played by Tl+ in regulating lipid accumulation in microalgae and the removal efficiency of Tl+. The effect of Tl+ on the cell growth, lipid production and Tl+ removal efficiency of Parachlorella kessleri R-3 was studied. Low concentrations of Tl+ had no significant effect on the biomass of microalgae. When the Tl+ concentration exceeded 5 µg L-1, the biomass of microalgae showed significant decrease. The highest lipid content of 63.65% and lipid productivity of 334.55 mg L-1 d-1 were obtained in microalgae treated with 10 and 5 µg L-1 Tl+, respectively. Microalgae can efficiently remove Tl+ and the Tl+ removal efficiency can reach 100% at Tl+ concentrations of 0-25 µg L-1. The maximum nitric oxide (NO) level of 470.48 fluorescence intensity (1 × 106 cells)-1 and glutathione (GSH) content of 343.51 nmol g-1 (fresh alga) were obtained under 5 µg L-1 Tl+ stress conditions. Furthermore, the exogenous donor sodium nitroprusside (SNP) supplemented with NO was induced in microalgae to obtain a high lipid content (59.99%), lipid productivity (397.99 mg L-1 d-1) and GSH content (430.22 nmol g-1 (fresh alga)). The corresponding analysis results indicated that NO could participate in the signal transduction pathway through modulation of reactive oxygen species (ROS) signaling to activate the antioxidant system by increasing the GSH content to eliminate oxidative damage induced by Tl+ stress. In addition, NO regulation of ROS signaling may enhance transcription factors associated with lipid synthesis, which stimulates the expression of genes related to lipid synthesis, leading to increased lipid biosynthesis in microalgae. Moreover, it was found that the change in Tl+ had little effect on the fatty acid components and biodiesel properties. This study showed that Tl+ stress can promote lipid accumulation in microalgae for biodiesel production and simultaneously effectively remove Tl+, which provided evidence that NO was involved in signal transduction and antioxidant defense, and improved the understanding of the interrelation between NO and ROS to regulate lipid accumulation in microalgae.

In vitro and in vivo removal efficacy of insoluble Prussian blue in combination with calcium polystyrene sulfonate for thallium.

The similarities between thallium and potassium have suggested the use of calcium polystyrene sulfonate (CPS), an oral ion exchange resin, as a potential agent against thallium intoxication. Therefore, the study was an attempt to evaluate the efficacy of CPS and Prussian blue when given alone or in combination against thallium toxicity. The effect on binding capacity was investigated in terms of contact time, amount of CPS, influence of pH, simulated physiological solutions and interference of potassium ions. Also, rats were given single dose of thallium chloride (20 mg kg-1) and the treatment with PB and CPS was given for 28 days as CPS 30 g kg-1, orally, twice a day, PB 3 g kg-1, orally, twice a day and their combination. The effect of antidotal treatment was evaluated by calculating the thallium levels in various organs, blood, urine and feces. The results of the in vitro study indicated exceedingly quick binding in the combination of CPS and PB as compared to PB alone. Also, it was found that the binding capacity at pH 2.0 was considerably increased for PB with CPS (184.656 mg g-1) as compared to PB (37.771 mg g-1). Furthermore, statistically significant results were obtained in the in vivo study as after 7th day, thallium levels in blood of rats treated with combination were reduced by 64% as compared to control group and 52% as compared to alone PB treated group. Also, Tl retention in liver, kidney, stomach, colon and small intestine of combination treated rats was significantly reduced to 46%, 28%, 41%, 32% and 33% respectively, as compared to alone PB treated group. These findings demonstrate this as a good antidotal option against thallium intoxication.

Thallium exposure interfered with heart development in embryonic zebrafish (Danio rerio): From phenotype to genotype.

Thallium (Tl) is a rare trace metal element but increasingly detected in wastewater produced by coal-burning, smelting, and more recently, high-tech manufacturing industries. However, the adverse effects of Tl, especially cardiotoxicity, on aquatic biota remain unclear. In this study, zebrafish model was used to elucidate the effects and mechanisms of Tl(I) cardiotoxicity in developing embryos. Exposure of embryonic zebrafish to low-dose Tl(I) (25-100 µg/L) decreased heart rate and blood flow activity, and subsequently impaired swim bladder inflation and locomotive behavior of larvae. Following high-level Tl(I) administration (200-800 µg/L), embryonic zebrafish exhibited pericardial edema, incorrect heart looping, and thinner myocardial layer. Based on RNA-sequencing, Tl(I) altered pathways responsible for protein folding and transmembrane transport, as well as negative regulation of heart rate and cardiac jelly development. The gene expression of nppa, nppb, ucp1, and ucp3, biomarkers of cardiac damage, were significantly upregulated by Tl(I). Our findings demonstrate that Tl(I) at environmentally relevant concentrations interfered with cardiac development with respect to anatomy, function, and transcriptomic alterations. The cardiotoxic mechanisms of Tl(I) provide valuable information in the assessment of Tl-related ecological risk in freshwater environment.

Integrated physiological, biochemical, and transcriptomic analysis of thallium toxicity in zebrafish (Danio rerio) larvae.

Although several studies have evaluated the effects of Thallium (Tl) in adult species of fish, the developmental toxicity of Tl has not been previously explored. In this study, zebrafish embryos (<4 h post fertilization (hpf)) were exposed to Tl at concentrations from 0.8 to 400 µg L-1 for 7 d. The results showed that the decreased hatching rate and increased malformation rate were observed in the larvae. The swimming velocity of larvae from 200 and 400 µg L-1 treatments was respectively reduced by ~26 % and 15 %. Histopathological analysis of liver indicated the number of cells of karyolysis (143 % and 202 %) and pyknosis (170 % and 131 %) were respectively increased in 200 and 400 µg L-1 Tl treatments. Meanwhile, the Tl body burden and metallothionein (MT) levels in the larvae were increased with elevated Tl concentrations. The level of malondialdehyde (MDA) was increased by ~20 to 51 % in all Tl treatments and total antioxidant capacity (TAC) was decreased by ~12 % at 200 µg L-1. The activities of Na+/K+-ATPase and protease were inhibited in 200 and 400 µg L-1 Tl treatments. Moreover, the transcripts of genes (Nrf2, HO-1, TNF-α, IL-1ß, IL-8, IL-10, TGF) were significantly altered. In addition, a total of 930 differentially expressed genes (DEGs) and 1549 DEGs were found in the 200 and 400 µg L-1 treatments with 458 overlapped DEGs by transcriptomic analysis. The protein digestion and absorption, ECM-receptor interaction, and complement and coagulation cascades pathways were shown to be the most significantly enriched pathways. This study helps better understand the molecular mechanisms of Tl toxicity in fish.

Aluminum, Arsenic, Beryllium, Cadmium, Chromium, Cobalt, Copper, Iron, Lead, Mercury, Molybdenum, Nickel, Platinum, Thallium, Titanium, Vanadium, and Zinc: Molecular Aspects in Experimental Liver Injury.

Experimental liver injury with hepatocelluar necrosis and abnormal liver tests is caused by exposure to heavy metals (HMs) like aluminum, arsenic, beryllium, cadmium, chromium, cobalt, copper, iron, lead, mercury, molybdenum, nickel, platinum, thallium, titanium, vanadium, and zinc. As pollutants, HMs disturb the ecosystem, and as these substances are toxic, they may affect the health of humans and animals. HMs are not biodegradable and may be deposited preferentially in the liver. The use of animal models can help identify molecular and mechanistic steps leading to the injury. HMs commonly initiate hepatocellular overproduction of ROS (reactive oxygen species) due to oxidative stress, resulting in covalent binding of radicals to macromolecular proteins or lipids existing in membranes of subcellular organelles. Liver injury is facilitated by iron via the Fenton reaction, providing ROS, and is triggered if protective antioxidant systems are exhausted. Ferroptosis syn pyroptosis was recently introduced as mechanistic concept in explanations of nickel (Ni) liver injury. NiCl2 causes increased iron deposition in the liver, upregulation of cyclooxygenase 2 (COX-2) protein and mRNA expression levels, downregulation of glutathione eroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), nuclear receptor coactivator 4 (NCOA4) protein, and mRNA expression levels. Nickel may cause hepatic injury through mitochondrial damage and ferroptosis, defined as mechanism of iron-dependent cell death, similar to glutamate-induced excitotoxicity but likely distinct from apoptosis, necrosis, and autophagy. Under discussion were additional mechanistic concepts of hepatocellular uptake and biliary excretion of mercury in exposed animals. For instance, the organic anion transporter 3 (Oat3) and the multidrug resistance-associated protein 2 (Mrp2) were involved in the hepatic handling of mercury. Mercury treatment modified the expression of Mrp2 and Oat3 as assessed by immunoblotting, partially explaining its impaired biliary excretion. Concomitantly, a decrease in Oat3 abundance in the hepatocyte plasma membranes was observed that limits the hepatic uptake of mercury ions. Most importantly and shown for the first time in liver injury caused by HMs, titanium changed the diversity of gut microbiota and modified their metabolic functions, leading to increased generation of lipopolysaccharides (LPS). As endotoxins, LPS may trigger and perpetuate the liver injury at the level of gut-liver. In sum, mechanistic and molecular steps of experimental liver injury due to HM administration are complex, with ROS as the key promotional compound. However, additional concepts such as iron used in the Fenton reaction, ferroptosis, modification of transporter systems, and endotoxins derived from diversity of intestinal bacteria at the gut-liver level merit further consideration.

Thallium(I and III) exposure leads to liver damage and disorders of fatty acid metabolism in mice.

Thallium (Tl), a highly toxic and priority pollutant heavy metal, exposure can damage mitochondria and disrupt their function. The liver is the central organ that controls lipid homeostasis and contains a large number of mitochondria. So far, there is no study investigating the effects of Tl exposure on hepatic fatty acid metabolism. Here, we showed that 10 ppm of Tl(I) and Tl(III) exposures for two weeks did not significantly affect the body weight and water/food intake in mice. However, it decreased the ratio of liver/weight and induced hepatic sinus congestion and hepatocyte necrosis. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed Tl accumulation in the liver. Gas chromatography-mass spectrometry (GC-MS) results showed that Tl(I) exposure significantly increased hepatic C18:0 concentration, while significantly decreased the concentrations of C16:1n-7, C20:1n-9, C18:3n-6, and C20:2n-9. Tl(III) exposure significantly reduced hepatic concentrations of C20:0, C22:0, C20:1n-9, C18:3n-6, and C20:3n-6. In addition, Tl(I) exposure upregulated the genes related to antioxidation (HO-1, GPX1, and GPX4), fatty acid synthesis (FADS2 and Elovl2), and fatty acid oxidation pathway (PPARα, ACADM, ACADVL, ACAA2, and CPT1A) in the liver. Tl(III) exposure did not significantly affect the transcript levels of liver antioxidative/metabolic enzymes and fatty acid synthesis-related genes, but upregulated fatty acid oxidation pathway-related genes (CYP4A10 and CPT1A). These results suggest that Tl(I) and Tl(III) exposures can cause liver damage and disrupt hepatic fatty acid metabolism, which provide new insights into Tl exposure-induced energy depletion from the perspective of fatty acid metabolism.

A High-Throughput Screening Assay to Identify Drugs that Can Treat Long QT Syndrome Caused by Trafficking-Deficient KV11.1 (hERG) Variants.

Loss-of-function (LOF) variants in the KV11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl+) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') scores comparing vehicle to E-4031, a drug that increases KV11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs+) to the Tl+ flux assay buffer resulted in an outstanding Z' of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase KV11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS KV11.1 variants. We report two new ways to increase target-specific activity in trafficking assays-genetic modification and channel activation-that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic KV11.1 variants. SIGNIFICANCE STATEMENT: This manuscript reports the development of a high-throughput assay (thallium flux) to identify drugs that can increase function in KV11.1 variants that are trafficking-deficient. Two key aspects that improved the resolving power of the assay and could be transferable to other ion channel trafficking-related assays include genetic modification and channel activation.

Urinary metals and maternal circulating extracellular vesicle microRNA in the MADRES pregnancy cohort.

Exposure to metals increases risk for pregnancy complications. Extracellular vesicle (EV) miRNA contribute to maternal-foetal communication and are dysregulated in pregnancy complications. However, metal impacts on maternal circulating EV miRNA during pregnancy are unknown. Our objective was to investigate the impact of multiple metal exposures on EV miRNA in maternal circulation during pregnancy in the MADRES Study. Associations between urinary concentrations of nine metals and 106 EV miRNA in maternal plasma during pregnancy were investigated using robust linear regression (N = 231). Primary analyses focused on metal-miRNA associations in early pregnancy (median: 12.3 weeks gestation). In secondary analyses, we investigated associations with late pregnancy miRNA counts (median: 31.8 weeks gestation) in a subset of participants (N = 184) with paired measures. MiRNA associated with three or more metals (PFDR<0.05) were further investigated using Bayesian Kernel Machine Regression (BKMR), an environmental mixture method. Thirty-five miRNA were associated (PFDR<0.05) with at least one metal in early pregnancy. One association (an inverse association between cobalt and miR-150-5p) remained statistically significant when evaluating late pregnancy miRNA counts. Eight miRNA (miR-302b-3p, miR-199a-5p, miR-188-5p, miR-138-5p, miR-212-3p, miR-608, miR-1272, miR-19b-3p) were associated with three metals (barium, mercury, and thallium) in early pregnancy, and their predicted target genes were enriched in pathways important for placental development. Results were consistent when using BKMR. Early pregnancy exposure to barium, mercury, and thallium may have short-term impacts on a common set of EV miRNA which target pathways important for placental development.

Aprepitant is a novel, selective activator of the K2P channel TRAAK.

TRAAK (KCNK4, K2P4.1) is a mechanosensitive two-pore domain potassium (K2P) channel. Due to its expression within sensory neurons and genetic link to neuropathic pain it represents a promising potential target for novel analgesics. In common with many other channels in the wider K2P sub-family, there remains a paucity of small molecule pharmacological tools. Specifically, there is a lack of molecules selective for TRAAK over the other members of the TREK subfamily of K2P channels. We developed a thallium flux assay to allow high throughput screening of compounds and facilitate the identification of novel TRAAK activators. Using a library of ∼1200 drug like molecules we identified Aprepitant as a small molecule activator of TRAAK. Aprepitant is an NK-1 antagonist used to treat nausea and vomiting. Close structural analogues of Aprepitant and a range of NK-1 antagonists were also selected or designed for purchase or brief chemical synthesis and screened for their ability to activate TRAAK. Electrophysiology experiments confirmed that Aprepitant activates both the 'long' and 'short' transcript variants of TRAAK. We also demonstrated that Aprepitant is selective and does not activate other members of the K2P superfamily. This work describes the development of a high throughput assay to identify potential TRAAK activators and subsequent identification and confirmation of the novel TRAAK activator Aprepitant. This discovery identifies a useful tool compound which can be used to further probe the function of TRAAK K2P channels.

The acute systemic toxicity of thallium in rats produces oxidative stress: attenuation by metallothionein and Prussian blue.

Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental contamination by Tl has been reported in several countries, urging the need for studies to determine the impact of endogenous and exogenous mechanisms preventing thallium toxicity. The cytoprotective effect of metallothionein (MT), a protein with high capacity to chelate metals, at two doses (100 and 600 µg/rat), was tested. Prussian blue (PB) (50 mg/kg) was administered alone or in combination with MT. A dose of Tl (16mg/kg) was injected i.p. to Wistar rats. Antidotes were administered twice daily, starting 24h after Tl injection, for 4 days. Tl concentrations diminished in most organs (p < 0.05) by effect of PB, alone or in combination with MT, whereas MT alone decreased Tl concentrations in testis, spleen, lung and liver. Likewise, brain thallium also diminished (p < 0.05) by effect of PB and MT alone or in combination in most of the regions analyzed (p < 0.05). The greatest diminution of Tl was achieved when the antidotes were combined. Plasma markers of renal damage increased after Tl administration, while PB and MT, either alone or in combination, prevented the raise of those markers. Only MT increased the levels of reduced glutathione (GSH) in the kidney. Finally, increased Nrf2 was observed in liver and kidney, after treatment with MT alone or in combination with PB. Results showed that MT alone or in combination with PB is cytoprotective after thallium exposure.

Effects of Tl+ on the inner membrane thiol groups, respiration, and swelling in succinate-energized rat liver mitochondria were modified by thiol reagents.

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.

Thallium-sensitive fluorescent assay reveals loperamide as a new inhibitor of the potassium channel Kv10.1.

BACKGROUND: Ion channels have been proposed as therapeutic targets for different types of malignancies. One of the most studied ion channels in cancer is the voltage-gated potassium channel ether-à-go-go 1 or Kv10.1. Various studies have shown that Kv10.1 expression induces the proliferation of several cancer cell lines and in vivo tumor models, while blocking or silencing inhibits proliferation. Kv10.1 is a promising target for drug discovery modulators that could be used in cancer treatment. This work aimed to screen for new Kv10.1 channel modulators using a thallium influx-based assay. METHODS: Pharmacological effects of small molecules on Kv10.1 channel activity were studied using a thallium-based fluorescent assay and patch-clamp electrophysiological recordings, both performed in HEK293 stably expressing the human Kv10.1 potassium channel. RESULTS: In thallium-sensitive fluorescent assays, we found that the small molecules loperamide and amitriptyline exert a potent inhibition on the activity of the oncogenic potassium channel Kv10.1. These results were confirmed by electrophysiological recordings, which showed that loperamide and amitriptyline decreased the amplitude of Kv10.1 currents in a dose-dependent manner. Both drugs could be promising tools for further studies. CONCLUSIONS: Thallium-sensitive fluorescent assay represents a reliable methodological tool for the primary screening of different molecules with potential activity on Kv10.1 channels or other K+ channels.

Development of a Cell-Based Assay for Identifying KCa3.1 Inhibitors Using Intestinal Epithelial Cell Lines.

Inhibition of the KCa3.1 potassium channel has therapeutic potential in a variety of human diseases, including inflammation-associated disorders and cancers. However, KCa3.1 inhibitors with high therapeutic promise are currently not available. This study aimed to establish a screening assay for identifying inhibitors of KCa3.1 in native cells and from library compounds derived from natural products in Thailand. The screening platform was successfully developed based on a thallium flux assay in intestinal epithelial (T84) cells with a Z' factor of 0.52. The screening of 1352 compounds and functional validation using electrophysiological analyses identified 8 compounds as novel KCa3.1 inhibitors with IC50 values ranging from 0.14 to 6.57 µM. These results indicate that the assay developed is of excellent quality for high-throughput screening and capable of identifying KCa3.1 inhibitors. This assay may be useful in identifying novel KCa3.1 inhibitors that may have therapeutic potential for inflammation-associated disorders and cancers.

A "Target Class" Screen to Identify Activators of Two-Pore Domain Potassium (K2P) Channels.

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a "target class" approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration-response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology.

Repeated measures of prenatal thallium exposure and placental inflammatory cytokine mRNA expression: The Ma'anshan birth cohort (MABC) study.

Thallium (Tl), a ubiquitous environmental toxicant, can cross the placental barrier during pregnancy. However, the effects of prenatal Tl exposure on placental function are currently unclear. Based on the Ma'anshan Birth Cohort study, we examined whether long-term prenatal Tl exposure was associated with placental inflammation. Tl concentrations were quantified in serum samples (n = 7050) from 2515 pregnancy during each trimester, placental inflammatory cytokine mRNA expression was assessed in 2519 placenta tissues. Geometric mean values of serum Tl concentrations were 63.57, 63.63 and 48.71 ng/L for the first, second and third trimesters, respectively. After adjustment for potential confounders, serum Tl concentration was positively associated with CD68 (ß: 0.30; 95% CI: 0.05, 0.56) in the first trimester and TNF-α (ß: 0.12; 95% CI: 0.01, 0.23), IL-6 (ß: 0.15; 95% CI: 0.05, 0.25) and CD68 (ß: 0.25; 95% CI: 0.10, 0.39) in the third trimester, however was negatively associated with IL-4 (ß: -0.21; 95% CI: -0.41, -0.01) and CD206 (ß: -0.23; 95% CI: -0.45, -0.02) in the first trimester. Repeated measures analysis showed that TNF-α, IL-6 and CD68 increased by 0.11 (95% CI: 0.01, 0.21), 0.12 (0.15, 95% CI: 0.05, 0.25), 0.22 (95% CI: 0.10, 0.39), respectively, with each 1ln-transformed Tl increase in total samples. Gender-specific analyses revealed that these associations were largely driven by male offspring. In addition, immunohistochemistry revealed that nuclear NF-κB p65 expression increased in placenta tissue. The results of this prospective cohort study provide longitudinal evidence that prenatal Tl exposure induces a placental inflammatory response in the Chinese population.

Hyperaccumulation and transport mechanism of thallium and arsenic in brake ferns (Pteris vittata L.): A case study from mining area.

Both thallium (Tl) and arsenic (As) bear severe toxicity. Brake fern (Pteris vittata L.) is well-known for its hyperaccumulation capacity of As, yet its role on Tl accumulation remains unknown. Herein, brake ferns growing near an As tailing site in Yunnan, Southwestern China are for the first time discovered as a co-hyperaccumulator of both Tl and As. The results showed that the brake ferns extracted both As and Tl efficiently from the soils into the fronds. The studied ferns growing on Tl and As co-polluted soils were found to accumulate extremely high levels of both As (7215-11110 mg/kg) and Tl (6.47-111 mg/kg). Conspicuously high bio-accumulation factor (BCF) was observed for As (7.8) and even higher for Tl (28.4) among these co-hyperaccumulators, wherein the contents of As and Tl in contaminated soils were 1240 ± 12 and 3.91 ± 0.01 mg/kg, respectively. The applied advanced characterization techniques (e.g. transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS)) indicated a preferential uptake of Tl(I) while simultaneous accumulation of As (III) and As(V) from the Tl(I)/Tl(III)-As (III)/As(V) co-existent rhizospheric soils. The findings benefit the phytoremediation practice and pose implications for managing and restoring Tl-As co-contaminated soils in other countries.

Thallium stable isotope fractionation in white mustard: Implications for metal transfers and incorporation in plants.

We studied thallium (Tl) isotope fractionation in white mustard grown hydroponically at different Tl doses. Thallium isotope signatures in plants indicated preferential incorporation of the light 203Tl isotope during Tl uptake from the nutrient solution. Negative isotope fractionation was even more pronounced in dependence on how much the available Tl pool decreased. This finding corresponds to the concept of isotope overprinting related to a high contamination level in the growing media (solution or soil). Regarding Tl translocation in plants, we observed a large Tl isotope shift with an enrichment in the heavy 205Tl isotope in the shoots relative to the roots in treatments with low/moderate solution Tl concentrations (0.01/0.05 mg Tl/L), with the corresponding α205/203Tl fractionation factors of ˜1.007 and 1.003, respectively. This finding is probably a consequence of specific (plant) reactions of Tl replacing K in its cycle. The formation of the S-coordinated Tl(I) complexes, potentially affecting both Tl accumulation and Tl isotope fractionation in plants, however, was not proven in our plants, since we did not have indication for that on the basis of X-ray absorption spectroscopy, suggesting that Tl was mainly present as free/hydrated Tl+ ion or chemically bound to O-containing functional groups.