Toxic Effects of Two Redox States of Thallium on Immortalised Hypothalamic GT1-7 Neuronal Cells.

Thallium (Tl), is a highly toxic heavy metal that exists in monovalent (Tl(I)) and trivalent (Tl(III)) ionic states. This study aimed to compare the toxicities of Tl(I) and Tl(III) in a mouse hypothalamic GT1-7 neuronal cell line. Decreased viability and increased cytotoxicity were observed in the GT1-7 cells 16 h after Tl(I) or Tl(III) treatment. Tl(III) was more cytotoxic, than Tl(I), as indicated by extracellular lactate dehydrogenase levels. Both treatments induced caspase 3 activity, DNA fragmentation, malondialdehyde (MDA) production, and superoxide dismutase activity in the cells. MDA production was higher after Tl(III) than after Tl(I) treatment. Moreover, co-treatment with antioxidants, such as mannitol, ascorbic acid, or tocopherol, significantly attenuated the Tl-induced decrease in GT1-7 cell numbers. Therefore, both treatments induced oxidative stress-related apoptosis. Furthermore, Tl(III) reduced the cell viability more subtly than Tl(I) after 1 and 3 h of treatment. This effect was enhanced by co-treatment with maltol or citric acid, which promoted the influx of metallic elements into the cells. Thus, Tl(III) entered GT1-7 cells later than Tl(I) and had a delayed onset of toxicity. However, Tl(III) likely produces more extracellular lipid peroxides, which may explain its stronger cytotoxicity.

Tl(I) and Tl(III) induce reticulum stress in MDCK cells.

The effects of the exposure of proliferating MDCK cells to thallium [Tl(I) or Tl(III)] on cell viability and proliferation were investigated. Although Tl stopped cell proliferation, the viability was > 95%. After 3 h, two autophagy markers (SQSTM-1 expression and LC3ß localization) were altered, and at 48 h increased expression of SQSTM-1 (60%) and beclin-1 (50-100%) were found. At 24 h, the expression of endoplasmic reticulum (ER) stress markers ATF-6 and IRE-1 were increased in 100% and 150%, respectively, accompanied by XBP-1 splicing and nuclear translocation. At 48 h, major ultrastructure abnormalities were found, including ER enlargement and cytoplasmic vacuolation which was not prevented by protein synthesis inhibition. Increased PHB (85% and 40% for Tl(I) and Tl(III), respectively) and decreased ß-tubulin (45%) expression were found which may be related to the promotion of paraptosis. In summary, Tl(I) and Tl(III) promoted ER stress and probably paraptosis in MDCK cells, impairing their proliferation.

A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl+-Induced Mitochondrial Permeability Transition Pore.

Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.

Maternal exposure to metal mixtures during early pregnancy and fetal growth in the Jiangsu Birth Cohort, China.

Previous epidemiological studies have reported that prenatal exposure to metals might have influence on fetal growth. Most studies assessed the effect of individual metals, while the investigation on the relationship between multiple metal exposure and fetal growth is sparse. The objective of the present study is to assess the joint impact of metal mixtures on fetal growth during pregnancy. A total of 1275 maternal-infant pairs from the Jiangsu Birth Cohort (JBC) Study were included to investigate the effect of maternal metal exposure on fetal biometry measures at 22-24, 30-32, and 34-36 weeks of gestation. Lead (Pb), arsenic (As), cadmium (Cd), mercury (Hg), chromium (Cr), vanadium(V), thallium (Tl) and barium (Ba) were measured by inductively coupled plasma mass spectrometry (ICP-MS) in maternal urine samples collected in the first trimester. We used general linear models and restricted cubic splines to test dose-response relationships between single metals and fetal growth. The weighted quantile sum (WQS) models were then applied to evaluate the overall effect of all these metals. We observed inverse associations of exposure to Pb, V and Cr with estimated fetal weight (EFW) at 34-36 weeks of gestation. Notably, maternal exposure to metal mixtures was significantly associated with reduced EFW at 34-36 weeks of gestation after adjusting for some covariates and confounders (aß -0.05 [95% CI: 0.09, -0.01], P = 0.023), and this association was mainly driven by Cr (30.41%), Pb (23.92%), and Tl (15.60%). These findings indicated that prenatal exposure to metal mixtures might impose adverse effects on fetal growth.

The Joint Influence of Tl+ and Thiol-Modifying Agents on Rat Liver Mitochondrial Parameters In Vitro.

Recent data have shown that the mitochondrial permeability transition pore (MPTP) is the complex of the Ca2+-modified adenine nucleotide translocase (ANT) and the Ca2+-modified ATP synthase. We found in a previous study that ANT conformational changes may be involved in Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria. In this study, the effects of thiol-modifying agents (eosin-5-maleimide (EMA), fluorescein isothiocyanate (FITC), Cu(o-phenanthroline)2 (Cu(OP)2), and embelin (Emb)), and MPTP inhibitors (ADP, cyclosporine A (CsA), n-ethylmaleimide (NEM), and trifluoperazine (TFP)) on MPTP opening were tested simultaneously with increases in swelling, membrane potential (ΔΨmito) decline, decreases in state 3, 4, and 3UDNP (2,4-dinitrophenol-uncoupled) respiration, and changes in the inner membrane free thiol group content. The effects of these thiol-modifying agents on the studied mitochondrial characteristics were multidirectional and showed a clear dependence on their concentration. This research suggests that Tl+-induced MPTP opening in the inner membrane of calcium-loaded mitochondria may be caused by the interaction of used reagents (EMA, FITC, Emb, Cu(OP)2) with active groups of ANT, the mitochondrial phosphate carrier (PiC) and the mitochondrial respiratory chain complexes. This study provides further insight into the causes of thallium toxicity and may be useful in the development of new treatments for thallium poisoning.

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.

Rhenium and yttrium ions as antimicrobial agents against multidrug resistant Klebsiella pneumoniae and Acinetobacter baumannii biofilms.

Antimicrobial resistance presents major global concerns to patient health. In this study, metal ions of molybdenum, rhenium, yttrium and thallium were tested against bacteria in planktonic and biofilm form using one strain of Klebsiella pneumoniae and Acinetobacter baumannii. The antimicrobial efficacy of the metal ions was evaluated against the planktonic bacterial strains using minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations, whilst the efficacy of the metal ions against biofilms was tested using a crystal violet biofilm assay. Live Dead staining was used to visualize the antimicrobial activity elicited by the metal ions on the bacterial cell. The results showed that higher concentrations of the metals were required to inhibit the growth of biofilms (72·9 mg l-1 to 416·7 mg l-1 ), in comparison to their planktonic counterparts. MICs of the metal ions (<46·9 mg l-1 ) (planktonic cells) did not affect biofilm formation. Overall, rhenium and yttrium were effective antimicrobial agents. Molybdenum demonstrated the greatest level of biotoxicity. When taking into account these results and the known toxicity of thallium, it is possible that rhenium or yttrium ions could be developed as effective biocidal formulations in order to prevent transmission in healthcare environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The metal ions, molybdenum, rhenium, thallium and yttrium were tested against both Klebsiella pneumoniae and Acinetobacter baumannii in planktonic and biofilm forms. This research demonstrated that all the metal ions may be effective antimicrobial agents. However, molybdenum induced high levels of cytotoxicity, whilst, there was no significant difference in the toxicity of the other metal ions tested. When considering the results for the antimicrobial efficacy and biotoxicity of the metal ions, in conjunction with the known toxicity of thallium in certain chemical compositions, it was concluded that overall rhenium or yttrium ions may be effective antimicrobial agents, one potential application may be utilizing these metal ions in hospital surface cleaning formulations.

Spectrophotometric investigation of Glutathione modulation by Thallium chloride in aqueous medium.

Thallium has been shown to significantly influence various tissues of living organisms; Exposure to Thallium can disturb mitochondrial function, degenerate neurons, and interfere with the function of critical metabolic enzymes and co-enzymes. Glutathione (GSH) an essential biomarker is considered a key factor in harnessing the thallium toxicity. In the present study the interaction of Thallium (Thallium Chloride) and glutathione was investigated spectro-photo-metrically in aqueous media. The renowned Elman's experimental protocol was followed at a wavelength of 412nm for Glutathione quantification in each sample. The pH of each sample was maintained at 7.6 using Phosphate buffer during the entire course of the experiment. A concentration as well as time dependent depletion of glutathione after exposure to various concentration of Thallium metal was observed, revealing chemical interaction between the metal and glutathione. The exact mechanism of interaction of Thallium and glutathione is still to be investigated. However, this piece of research suggests that a decrease in the concentration of Glutathione may be due to Thallium-GSH abduct or oxidize glutathione (GSSG) formation. This study was performed in-vitro as a model of in vivo.

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 resulted in the Tl+-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

Thallium stimulates ethanol production in immortalized hippocampal neurons.

Lactate and ethanol (EtOH) were determined in cell culture medium (CCM) of immortalized hippocampal neurons (HN9.10e cell line) before and after incubation with Thallium (Tl). This cell line is a reliable, in vitro model of one of the most vulnerable regions of central nervous system. Cells were incubated for 48 h with three different single Tl doses: 1, 10, 100 µg/L (corresponding to 4.9, 49 and 490 nM, respectively). After 48 h, neurons were "reperfused" with fresh CCM every 24/48 h until 7 days after the treatment and the removed CCM was collected and analysed. Confocal microscopy was employed to observe morphological changes. EtOH was determined by head space-solid phase microextraction -gas chromatography -mass spectrometry (HS-SPME-GCMS), lactate by RP-HPLC with UV detection. Tl exposure had significant effects on neuronal growth rate and morphology. The damage degree was dose-dependent. In not exposed cells, EtOH concentration was 0.18 ± 0.013 mM, which represents about 5% of lactate concentration (3.4 ± 0.10 mM). After Tl exposure lactate and EtOH increased. In CCM of 100 and 10 µg/L Tl-treated cells, lactate increased 24 h after reperfusion up to 2 and 3.3 times the control value, respectively. In CCM of 10 and 100 µg/L Tl-treated cells 24 h after reperfusion, EtOH increased up to 0.3 and 0.58 mmol/L. respectively. These results are consistent with significant alterations in energy metabolism, despite the low doses of Tl employed and the relatively short incubation time.

Y3+, La3+, and some bivalent metals inhibited the opening of the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria.

We showed earlier that diminution of 2,4-dinitrophenol (DNP)-stimulated respiration and increase of both mitochondrial swelling and electrochemical potential (ΔΨmito) dissipation in medium containing TlNO3 and KNO3 were caused by opening of Tl(+)-induced mitochondrial permeability transition pore (MPTP) in the inner membrane of Ca(2+)-loaded rat liver mitochondria. The MPTP opening was studied in the presence of bivalent metal ions (Sr(2+), Ba(2+), Mn(2+), Co(2+) and Ni(2+)), trivalent metal ions (Y(3+) and La(3+)), and ruthenium red. We found that these metal ions (except Ba(2+) and Co(2+)) as well as ruthenium red inhibited to the MPTP opening that manifested in preventing both diminution of the DNP-stimulated respiration and increase of the swelling and of the ΔΨmito dissipation in medium containing TlNO3, KNO3, and Ca(2+). Inhibition of the MPTP opening by Sr(2+) and Mn(2+) is suggested because of their interaction with high affinity Ca(2+) sites, facing the matrix side and participating in the MPTP opening. The inhibitory effects of metal ions (Y(3+), La(3+), and Ni(2+)), and ruthenium red are accordingly discussed in regard to competitive and noncompetitive inhibition of the mitochondrial Ca(2+)-uniporter. High concentrations (50µM) of Y(3+) and La(3+) favored of MPTP opening in the inner membrane of rat liver mitochondria in Ca(2+) free medium containing TlNO3. The latter MPTP opening was markedly eliminated by MPTP inhibitors (cyclosporine A and ADP).

Tl(+) induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria.

Effects of Tl(+) were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO3 and a nitrate salt (KNO3 or NH4NO3) or TlNO3 and sucrose. Tl(+) increased permeability of the inner membrane of the RHM to K(+) and H(+). This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K(+) < NH4 (+), respectively. After succinate administration, the swollen RHM contracted. The Tl(+)-induced opening of the mitochondrial permeability pore (MPTP) in Ca(2+)-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl(+) were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoKATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoKATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl(+) effects on muscle cell mitochondria.

Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells.

The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 µM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

Influence of Tl(+) on mitochondrial permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

The Tl(+)-induced opening of the MPTP in Ca(2+)-loaded rat liver mitochondria energized by respiration on the substrates succinate or glutamate plus malate was recorded as increased swelling and dissipation of mitochondrial membrane potential as well as decreased state 4, or state 3, or 2,4-dinitrophenol-stimulated respiration. These effects of Tl(+) increased in nitrate media containing monovalent cations in the order of Li(+) < NH (4) (+) ≤ Na(+) < K(+). They were potentiated by inorganic phosphate and diminished by the MPTP inhibitors (ADP, CsA, Mg(2+), Li(+), rotenone, EGTA, and ruthenium red) both individually and more potently in their combinations. Maximal swelling of both non-energized and energized Ca(2+)-loaded mitochondria in rotenone-free media is an indication of Ca(2+) uptake driven by respiration on mitochondrial endogenous substrates. It is suggested that Tl(+) (distinct from Cd(2+), Hg(2+), and other heavy metals and regardless of the used respiratory substrates) can stimulate opening of the MPTP only in the presence of Ca(2+). We discuss the possible participation of Ca(2+)-binding sites, located near the respiratory complex I and the adenine nucleotide translocase, in inducing opening of the MPTP.

Value of stress myocardial perfusion scanning in diagnosis of severe coronary artery disease in liver transplantation candidates.

BACKGROUND: The significant potential for perioperative and late cardiovascular complications makes careful preoperative cardiac risk assessment imperative in liver transplantation candidates. OBJECTIVE: To determine the sensitivity and specificity of myocardial perfusion scanning for detection of coronary artery disease (CAD) in liver transplantation candidates. PATIENTS AND METHODS: We prospectively evaluated 93 liver transplantation candidates. Patients with known CAD were excluded. All patients, regardless of symptoms and risk factors, underwent myocardial perfusion scanning and coronary angiography. RESULTS: Results of myocardial perfusion scanning were abnormal in 64 patients (68.8%) and normal in 29 patients (31.2%). Of patients with abnormal scans, only 6 (9.4%) had severe CAD at coronary angiography. None of the 29 patients with normal perfusion scans and the 24 patients with fixed defects had severe CAD; however, 6 of 40 patients (15.0%) with reversible perfusion defects had severe CAD at coronary angiography (P = .005). Alcoholic liver disease, reversible perfusion defects at myocardial perfusion scanning, left ventricular systolic dysfunction, and higher low-density lipoprotein (LDL) cholesterol and triglyceride levels were significantly associated with CAD. Defining reversible perfusion defects as a sign of ischemia, and fixed defects and normal perfusion as nonischemic, myocardial perfusion scanning had 100% sensitivity but 61% specificity for severe CAD. The test's accuracy was low (38%). CONCLUSIONS: The results of reversible perfusion defects on myocardial perfusion scanning were sensitive but not specific for CAD in liver transplantation candidates. The high number of false-positive results decreased the test's accuracy.

Effects of Tl(+) on ion permeability, membrane potential and respiration of isolated rat liver mitochondria.

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K(+), Na(+), H(+)) but high to Tl(+). Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (DeltaPsi(mito)) of rat liver mitochondria were studied in media containing 0-75 mM TlNO(3) either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO(3), or NaNO(3), or NH(4)NO(3)). Tl(+) increased permeability of the inner mitochondrial membrane to K(+), Na(+), and H(+), that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of DeltaPsi(mito). These effects of Tl(+) increased in the order of sucrose

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1.

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.

Fluorescence-based Tl(+)-influx assays as a novel approach for characterization of small-conductance Ca(2+)-activated K (+) channel modulators.

Small-conductance Ca(2+)-activated potassium (SK) channels constitute a family of ion channels that are regulated by the cytosolic Ca(2+) concentration. Increases in the intracellular Ca(2+) concentration ([Ca(2+)](i)) result in opening of the channels, which in turn will lead to changes in the membrane potential. As the name implies, the channels are of small conductance, but even so, they are known to play a crucial role in several physiological processes, such as modulation of neurotransmitter and hormone secretion, as well as memory and learning (e.g.,see Curr Med Chem 14:1437-1457, 2007). Owing to the central role of SK channels, they have attracted much attention as potential drug targets, both with respect to identification of activators and blockers of SK channel activity for indications such as, e.g., epilepsy, pain, and urinary incontinence (see Curr Med Chem 14:1437-1457, 2007; Curr Pharm Des 12:397-406, 2006). Thus, great efforts have been put into the development of robust high-throughput assays for detection and characterization of modulators of SK channel activity. In the present chapter, we describe two fluorescence-based Tl(+)influx assays for detection of positive and negative SK channel modulators.