Modeling principles of protective thyroid blocking.

PURPOSE: In the case of a nuclear incident, the release of radioiodine must be expected. Radioiodine accumulates in the thyroid and by irradiation enhances the risk of cancer. Large doses of stable (non-radioactive) iodine may inhibit radioiodine accumulation and protect the thyroid ('thyroid blocking'). Protection is based on a competition at the active carrier site in the cellular membrane and an additional temporary inhibition of the organification of iodide (Wolff-Chaikoff effect). Alternatively, other agents like e.g. perchlorate that compete with iodide for the uptake into the thyrocytes may also confer thyroidal protection against radioiodine exposure.Biokinetic models for radioiodine mostly describe exchanges between compartments by first order kinetics. This leads to correct predictions only for low (radio)iodide concentrations. These models are not suited to describe the kinetics of iodine if administered at the dosages recommended for thyroid blocking and moreover does not permit to simulate either the protective competition mechanism at the membrane or the Wolff-Chaikoff effect. Models adapted for this purpose must be used. Such models may use a mathematical relation between the serum iodide concentration and a relative uptake suppression or a dependent rate constant determining total thyroidal radioiodine accumulation. Alternatively, the thyroidal uptake rate constant may be modeled as a function of the total iodine content of the gland relative to a saturation amount. Newer models integrate a carrier-mechanism described by Michalis-Menten kinetics in the membrane and in analogy to enzyme kinetics apply the rate law for monomolecular irreversible enzyme reactions with competing substrates to model the competition mechanism. An additional total iodide uptake block, independent on competition but limited in time, is used to simulate the Wolff-Chaikoff effect. CONCLUSION: The selection of the best model depends on the issue to be studied. Most models cannot quantify the relative contributions of the competition mechanism at the membrane and the Wolff-Chaikoff effect. This makes it impossible or exceedingly difficult to simulate prolonged radioiodine exposure and the effect of repetitive administrations of stable iodine. The newer thyroid blocking models with a separate modeling of competition and Wolff-Chaikoff effect allow better quantitative mechanistic insights and offer the possibility to simulate complex radioiodine exposure scenarios and various protective dosage schemes of stable iodine relatively easily. Moreover, they permit to study the protective effects of other competitors at the membrane carrier site, like e.g. perchlorate, and to draw conclusions on their protective efficacy in comparison to stable iodine.

Ions in the Deep Subsurface of Earth, Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA-Ligand Binding.

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl2, MgSO4, and Mg(ClO4)2) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.

In vitro effects of bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 on the physiology and interaction process of Leishmania amazonensis.

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC50 of 3.91 µM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.

Medical treatment of thyrotoxicosis.

Medical treatment is the primary therapeutic option for thyrotoxicosis/hyperthyroidism. Two groups of causes of thyrotoxicosis (i.e. thyrotoxicosis with hyperthyroidism and thyrotoxicosis without hyperthyroidism) need to be considered for therapeutic reasons. Herein we provide an updated review on the role of conventional medical therapies (i.e. ß-blockers, antithyroid drugs [ATDs], corticosteroids, inorganic iodide, perchlorate, cholecystographic agents, lithium, cholestyramine) in the main causes of thyrotoxicosis, starting from the rationale subtending their clinical application.

A comparison of thyroidal protection by stable iodine or perchlorate in the case of acute or prolonged radioiodine exposure.

In the case of a nuclear power plant accident, repetitive/prolonged radioiodine release may occur. Radioiodine accumulates in the thyroid and by irradiation enhances the risk of cancer. Large doses of non-radioactive iodine may protect the thyroid by inhibiting radioiodine uptake into the gland (iodine blockade). Protection is based on a competition at the active carrier site in the cellular membrane and the Wolff-Chaikoff effect, the latter being, however, only transient (24-48 h). Perchlorate may alternatively provide protection by a carrier competition mechanism only. Perchlorate has, however, a stronger affinity to the carrier than iodide. Based on an established biokinetic-dosimetric model developed to study iodine blockade, and after its extension to describe perchlorate pharmacokinetics and the inhibition of iodine transport through the carrier, we computed the protective efficacies that can be achieved by stable iodine or perchlorate in the case of an acute or prolonged radioiodine exposure. In the case of acute radioiodine exposure, perchlorate is less potent than stable iodine considering its ED50. A dose of 100 mg stable iodine has roughly the same protective efficacy as 1000 mg perchlorate. For prolonged exposures, single doses of protective agents, whether stable iodine or perchlorate, offer substantially lower protection than after acute radioiodine exposure, and thus repetitive administrations seem necessary. In case of prolonged exposure, the higher affinity of perchlorate for the carrier in combination with the fading Wolff-Chaikoff effect of iodine confers perchlorate a higher protective efficacy compared to stable iodine. Taking into account the frequency and seriousness of adverse effects, iodine and perchlorate at equieffective dosages seem to be alternatives in case of short-term acute radioiodine exposure, whereas preference should be given to perchlorate in view of its higher protective efficacy in the case of longer lasting radioiodine exposures.

A comparison of thyroid blockade strategies in paediatric 123I-meta-iodobenzylguanidine scanning: a dual centre study.

OBJECTIVE: The aim of this study was to evaluate three thyroid blockade regimes to determine which protocol provides the optimal level of thyroidal protection for paediatric 123-I-meta-iodobenzylguanidine (mIBG) imaging and estimate the relative radiation dose inferred from unbound radioiodine. METHODS: A total of 231 patients were retrospectively evaluated for thyroid uptake and categorised into five subgroups depending upon the protocol of thyroid blockade received. Efficacy of thyroid blockade was established by visual scoring and image quantitation with comparison against a control group. RESULTS: Visual Likert scale responses were subjected to the Mann-Whitney U and Kruskal-Wallis tests, respectively. Statistical significance was reached for observed thyroid uptake in potassium perchlorate recipients (U = 1107, P = 0.001). No statistically significant difference was observed in thyroid uptake for iohexol blockade (U = 176, P = 0.71) or potassium iodate blockade despite variations in iodate dosage and duration (χ(2) = 0.203, P = 0.93). The analyses were repeated for the image quantitation data. A statistically significantly higher absorbed thyroid dose was observed using potassium perchlorate blockade compared with the control group (U = 719, P = 0.001). The Mann-Whitney U did not reach statistical significance in absorbed thyroid dose for iohexol blockade (U = 126, P = 0.209, r = -0.13). The Kruskal-Wallis test, conducted across the potassium iodate groups, did not reach statistical significance (χ(2) = 0.513, P = 0.774). The median absorbed thyroid dose across the iodate groups ranged from 3.58 to 3.91 mGy indicating comparable blockade effectiveness for single-dose potassium iodate. CONCLUSION: Potassium iodate blockade is more efficacious compared with potassium perchlorate within the cohort observed. Both visual and quantitative data indicate that potassium iodate given at 30-60 min before I-mIBG injection provides comparable blockade effectiveness to lengthier administrations, suggesting that a single dose is well tolerated and practical.

NMR assignments of human linker histone H1x N-terminal domain and globular domain in the presence and absence of perchlorate.

Human linker histone H1 plays a seminal role in eukaryotic DNA packaging. H1 has a tripartite structure consisting of a central, conserved globular domain, which adopts a winged-helix fold, flanked by two variable N- and C-terminal domains. Here we present the backbone resonance assignments of the N-terminal domain and globular domain of human linker histone H1x in the presence and absence of the secondary structure stabilizer sodium perchlorate. Analysis of chemical shift changes between the two conditions is consistent with induction of transient secondary structural elements in the N-terminal domain of H1x in high ionic strength, which suggests that the N-terminal domain adopts significant alpha-helical conformations in the presence of DNA.

Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction.

Hydrogen sulfide produced by sulfate-reducing microorganisms (SRM) poses significant health and economic risks, particularly during oil recovery. Previous studies identified perchlorate as a specific inhibitor of SRM. However, constant inhibitor addition to natural systems results in new selective pressures. Consequently, we investigated the ability of Desulfovibrio alaskensis G20 to evolve perchlorate resistance. Serial transfers in increasing concentrations of perchlorate led to robust growth in the presence of 100 mM inhibitor. Isolated adapted strains demonstrated a threefold increase in perchlorate resistance compared to the wild-type ancestor. Whole genome sequencing revealed a single base substitution in Dde_2265, the sulfate adenylyltransferase (sat). We purified and biochemically characterized the Sat from both wild-type and adapted strains, and showed that the adapted Sat was approximately threefold more resistant to perchlorate inhibition, mirroring whole cell results. The ability of this mutation to confer resistance across other inhibitors of sulfidogenesis was also assayed. The generalizability of this mutation was confirmed in multiple evolving G20 cultures and in another SRM, D. vulgaris Hildenborough. This work demonstrates that a single nucleotide polymorphism in Sat can have a significant impact on developing perchlorate resistance and emphasizes the value of adaptive laboratory evolution for understanding microbial responses to environmental perturbations.

Liensinine perchlorate inhibits colorectal cancer tumorigenesis by inducing mitochondrial dysfunction and apoptosis.

SCOPE: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. METHODS AND RESULTS: Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. CONCLUSION: This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.

Developmental expression profiles and thyroidal regulation of cytokines during metamorphosis in the amphibian Xenopus laevis.

Early life-stages of amphibians rely on the innate immune system for defense against pathogens. While thyroid hormones (TH) are critical for metamorphosis and later development of the adaptive immune system, the role of TH in innate immune system development is less clear. An integral part of the innate immune response are pro-inflammatory cytokines - effector molecules that allow communication between components of the immune system. The objective of this study was to characterize the expression of key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), throughout amphibian development and determine the impacts of thyroidal modulation on their expression. Xenopus laevis were sampled at various stages of development encompassing early embryogenesis to late prometamorphosis and cytokine expression was measured by real-time PCR. Expression of TNFα and IL-1ß were transient over development, increasing with developmental stage, while IFN-γ remained relatively stable. Functionally athyroid, premetamorphic tadpoles were exposed to thyroxine (0.5 and 2 µg/L) or sodium perchlorate (125 and 500 µg/L) for seven days. Tadpoles demonstrated characteristic responses of advanced development with thyroxine exposure and delayed development (although to a lesser extent) and increased thyroid gland area and follicular cell height with sodium perchlorate exposure. Exposure to thyroxine for two days resulted in decreased expression of IL-1ß in tadpole trunks. Sodium perchlorate had negligible effects on cytokine expression. Overall, these results demonstrate that cytokine transcript levels vary with stage of tadpole development but that their ontogenic regulation is not likely exclusively influenced by thyroid status. Understanding the direct and indirect effects of altered hormone status may provide insight into potential mechanisms of altered immune function during amphibian development.

Determination of Thresholds of Radioactive Iodine Uptake Response With Clinical Exposure to Perchlorate: A Pooled Analysis.

OBJECTIVE: To conduct a more robust examination of perchlorate exposure on iodide uptake inhibition (IUI) using pooled data from four clinical studies of perchlorate exposure. METHODS: To establish a response threshold for IUI, data were analyzed using segmented linear regression and benchmark dose (BMD) analysis. RESULTS: Segmented linear regression applied to data for 69 subjects representing nine doses identified a breakpoint corresponding to a change in the slope of the dose-response relationship of 3.0 mg/d perchlorate. The estimated BMD for a 20% decrease in iodine uptake was 2.3 mg/d, with a lower 95% confidence interval limit of 1.6 mg/d. CONCLUSIONS: A threshold dose for IUI from perchlorate exposure of 1.6 to 3.0 mg/d (0.021 to 0.038 mg/kg d) was estimated using two modeling approaches. These estimates are slightly higher than the lowest observed effect level of 0.02 mg/kg d from the Greer Study.

An Antarctic Extreme Halophile and Its Polyextremophilic Enzyme: Effects of Perchlorate Salts.

Effects of perchlorate salts prevalent on the surface of Mars are of significant interest to astrobiology from the perspective of potential life on the Red Planet. Halorubrum lacusprofundi, a cold-adapted halophilic Antarctic archaeon, was able to grow anaerobically on 0.04 M concentration of perchlorate. With increasing concentrations of perchlorate, growth was inhibited, with half-maximal growth rate in ca. 0.3 M NaClO4 and 0.1 M Mg(ClO4)2 under aerobic conditions. Magnesium ions were also inhibitory for growth, but at considerably higher concentrations, with half-maximal growth rate above 1 M. For a purified halophilic ß-galactosidase enzyme of H. lacusprofundi expressed in Halobacterium sp. NRC-1, 50% inhibition of catalytic activity was observed at 0.88 M NaClO4 and 0.13 M Mg(ClO4)2. Magnesium ions were a more potent inhibitor of the enzyme than of cell growth. Steady-state kinetic analysis showed that Mg(ClO4)2 acts as a mixed inhibitor (KI = 0.04 M), with magnesium alone being a competitive inhibitor (KI = 0.3 M) and perchlorate alone acting as a very weak noncompetitive inhibitor (KI = 2 M). Based on the estimated concentrations of perchlorate salts on the surface of Mars, our results show that neither sodium nor magnesium perchlorates would significantly inhibit growth and enzyme activity of halophiles. This is the first study of perchlorate effects on a purified enzyme. Key Words: Halophilic archaea-Perchlorate-Enzyme inhibition-Magnesium. Astrobiology 18, 412-418.

A mitochondrial protein increases glycolytic flux.

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.

Improving enzymatic hydrolysis of corn stover pretreated by ethylene glycol-perchloric acid-water mixture.

To improve the enzymatic saccharification of lignocellulosic biomass, a mixture of ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) was used for pretreating corn stover in this study. After the optimization in oil-bath system, the optimum pretreatment temperature and time were 130 °C and 30 min, respectively. After the saccharification of 10 g/L pretreated corn stover for 48 h, the saccharification rate was obtained in the yield of 77.4 %. To decrease pretreatment temperature and shorten pretreatment time, ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) media under microwave irradiation was employed to pretreat corn stover effectively at 100 °C and 200 W for 5 min. Finally, the recovered hydrolyzates containing glucose obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. These results would be helpful for developing a cost-effective pretreatment combined with enzymatic saccharification of cellulosic materials for the production of lignocellulosic ethanol.

Change of iodine load and thyroid homeostasis induced by ammonium perchlorate in rats.

Ammonium perchlorate (AP), mainly used as solid propellants, was reported to interfere with homeostasis via competitive inhibition of iodide uptake. However, detailed mechanisms remain to be elucidated. In this study, AP was administered at 0, 130, 260 and 520 mg/kg every day to 24 male SD rats for 13 weeks. The concentrations of iodine in urine, serum thyroid hormones levels, total iodine, relative iodine and total protein, and malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activity in thyroid tissues were measured, respectively. Our results showed that high-dose perchlorate induced a significant increase in urinary iodine and serum thyroid stimulating hormone (TSH), with a decrease of total iodine and relative iodine content. Meanwhile, free thyroxine (FT4) was decreased and CAT activity was remarkably increased. Particularly, the CAT activity was increased in a dose-dependent manner. These results suggested that CAT might be enhanced to promote the synthesis of iodine, resulting in elevated urinary iodine level. Furthermore, these findings suggested that iodine in the urine and CAT activity in the thyroid might be used as biomarkers for exposure to AP, associated with thyroid hormone indicators such as TSH, FT4.

Feasibility of lentiviral­mediated sodium iodide symporter gene delivery for the efficient monitoring of bone marrow­derived mesenchymal stem cell transplantation and survival.

The aim of the present study was to explore the feasibility of lentiviral-mediated sodium iodide symporter (NIS) gene delivery for monitoring bone marrow-derived mesenchymal stem cell (BMSC) transplantation into the infarcted myocardium. For this purpose, we constructed a lentiviral vector (Lv-EF1α-NIS-IRES-EGFP) expressing NIS and enhanced green fluorescent protein (EGFP), and introduced it into BMSCs at different multiplicities of infection (MOI). The expression of EGFP was observed under a fluorescence microscope. Iodine uptake and the inhibition of iodine uptake by sodium perchlorate (NaClO4) in the Lv-EF1α-NIS-IRES-EGFP­treated BMSCs were dynamically monitored in vitro. The Lv-EF1α-NIS-IRES-EGFP­treated BMSCs were transplanted into the infarcted myocardium of Sprague-Dawley rats, and 99mTc99g (Tc, technetium; 99m indicates that technetium is at its excited stage; 99g indicates the atomic weight of technetium) micro-single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging was performed in vivo 1 week following transplantation. The isolated BMSCs successfully differentiated into adipocytes and osteoblasts. The BMSCs were positive for the cell surface markers, CD105, CD29 and CD90, and negative for CD14, CD34 and CD45. Lv-EF1α-NIS-IRES-EGFP was efficiently transfected into the BMSCs. RT-qPCR and western blot analysis confirmed that the BMSCs expressed high protein and mRNA levels of NIS by day 7 following infection, and NIS expression remained at a consistent level from day 14 to 21. In the Lv-EF1α-NIS-IRES-EGFP­treated BMSCs, the accumulation of iodine-125 (125I) was observed in vitro and was successfully monitored by 99mTc99g micro-SPECT/CT imaging at 1 week following transplantation. These results suggest that lentiviral vectors are powerful vehicles for studying gene delivery in BMSCs. It is feasible to use lentiviral vectors to deliver an NIS gene for the non-invasive monitoring of BMSC transplantation and survival in the infarcted myocardium in vivo.

On the dose calculation at the cellular level and its implications for the RBE of (99m)Tc and ¹²³I.

PURPOSE: Based on the authors' previous findings concerning the radiotoxicity of(99m)Tc, the authors compared the cellular survival under the influence of this nuclide with that following exposure to the Auger electron emitter (123)I. To evaluate the relative biological effectiveness (RBE) of both radionuclides, knowledge of the absorbed dose is essential. Thus, the authors present the dose calculations and discuss the results based on different models of the radionuclide distribution. Both different target volumes and the influence of the uptake kinetics were considered. METHODS: Rat thyroid PC Cl3 cells in culture were incubated with either(99m)Tc or (123)I or were irradiated using 200 kV x-rays in the presence or absence of perchlorate. The clonogenic cell survival was measured via colony formation. In addition, the intracellular radionuclide uptake was quantified. Single-cell dose calculations were based on Monte Carlo simulations performed using Geant4. RESULTS: Compared with external radiation using x-rays (D37 = 2.6 Gy), the radionuclides (99m)Tc (D37 = 3.5 Gy), and (123)I (D37 = 3.8 Gy) were less toxic in the presence of perchlorate. In the absence of perchlorate, the amount of activity a37 that was necessary to reduce the surviving fraction (SF) to 0.37 was 22.8 times lower for (99m)Tc and 12.4 times lower for (123)I because of the dose increase caused by intracellular radionuclide accumulation. When the cell nucleus was considered as the target for the dose calculation, the authors found a RBE of 2.18 for (99m)Tc and RBE = 3.43 for (123)I. Meanwhile, regarding the dose to the entire cell, RBE = 0.75 for (99m)Tc and RBE = 1.87 for (123)I. The dose to the entire cell was chosen as the dose criterion because of the intracellular radionuclide accumulation, which was found to occur solely in the cytoplasm. The calculated number of intracellular decays per cell was (975 ± 109) decays/MBq for (99m)Tc and (221 ± 82) decays/MBq for (123)I. CONCLUSIONS: The authors' data indicate that extra-nuclear targets to Auger electrons exist, which is obvious from our dose calculations. When considering the dose to the cell nucleus, the authors found an enhanced RBE for(99m)Tc and (123)I relative to acute x-ray irradiation and pure extracellular irradiation with both radionuclides. Surprisingly, the authors did not find any radionuclide accumulation in the cell nucleus, indicating that there are additional radiosensitive targets besides the DNA. In addition, the authors demonstrated the necessity of cellular dose calculations in radiobiological experiments using unsealed radionuclides and identified the relevant parameters.

Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

Crosstalk between the thyroid hormone and androgen axes during reproductive development in Silurana tropicalis.

The objectives of this study were to examine the effects of potassium perchlorate (KClO4) treatment on androgen- and thyroid hormone (TH)-related transcript levels during gonadogenesis in the frog Silurana tropicalis. Androgen- and TH-related gene expression was examined in gonad-mesonephros complex (GMC) and liver tissues at stage NF 56 and stage NF 60. These stages of development coincide with the period of sexual differentiation. Real-time RT-PCR analyses revealed that androgen- and TH-related transcript levels in the GMC and liver of stage NF 56 and NF 60 frogs are responsive to KClO4 exposure during prometamorphosis. An increase in srd5α2 mRNA levels in hepatic tissues of KClO4-treated NF 56 tadpoles suggests an important role for hepatic tissues in androgen metabolism. Gene transcript differences highlight possible stage- and tissue-specific sensitivities to KClO4. A greater number of TH- and androgen-related transcriptional changes were discerned in the hepatic tissues compared to the gonads, and overall fewer transcriptional changes were observed in stage NF 60 tadpoles compared to stage NF 56 larvae. Perchlorate suppressed somatic and hind-limb development during the 96-d exposure period. Treatment with KClO4 had no significant effect on sex ratios, however a notable reduction in the percentage of males (33.3% M: 66.7% F) in the highest KClO4 concentration (107 µg/L) was observed. Overall, these findings suggest that KClO4 has secondary androgenic disrupting properties in addition to its known primary thyroid hormone-disrupting role.

Enzyme and acid deconjugation of plasma sulfated metanephrines.

BACKGROUND: Total (i.e. free+sulfated) metanephrines in plasma is a biomarker for the diagnosis of pheochromocytoma/paraganglioma. Sulfated metanephrines must be completely deconjugated by perchloric acid hydrolysis or sulfatase treatment prior to analytical measurement to enable quantification by current techniques. In this report, we compare the yield and efficiency of both methods. METHODS: The deconjugation rate of synthetic sulfated metanephrines (normetanephrine (S-NMN), metanephrine (S-MN) and methoxytyramine (S-MT)) spiked in charcoal-stripped plasma was determined by boiling perchloric acid and compared to sulfatase treatment. Total plasma metanephrines (MN, NMN and MT) were also determined in patient samples by both methods. RESULTS: The complete deconjugation of sulfated metanephrines is achieved after 30 min incubation with 0.1M boiling perchloric acid or upon sulfatase treatment. Ten minutes of acid hydrolysis (gold-standard) leads to a 30% underestimation of metanephrine concentrations. The enzyme hydrolysis is time and amount of sulfatase dependent. The rate of hydrolysis is analyte-dependent (MT>>NMN>MN), although it must contain at least 0.8 U/ml of sample. The Deming regression curves comparing acid versus enzyme hydrolysis on patient samples assessed that both methods gave similar unbiased concentrations. CONCLUSION: Enzyme and acid treatments are equivalent and efficient for removing sulfate from metanephrines as long as the optimal protocol is used for each method. However, the gold standard method for acid hydrolysis at 10 min established more than 20 years ago was not satisfactory regarding the hydrolysis of metanephrines in plasma.