Exposure of human glioblastoma cells to thimerosal inhibits the thioredoxin system and decreases tumor growth-related factors.

Glioblastoma multiforme (GBM) is the most common, aggressive, and fatal primary malignant brain tumor in adults. The therapeutic efficacy of temozolomide (TMZ) is limited owing to frequent treatment resistance. The latter is in part related to the overexpression of redox systems such as the thioredoxin system. This system is fundamental for cell survival and proliferation, regulating hypoxia inducible factor-1alpha (HIF-1α) activity, in turn controlling vascular endothelial growth factor (VEGF), which is indispensable for tumor invasiveness, angiogenesis and microenvironment maintenance. HIF-1α can also be regulated by the signal transducer and activator of transcription 3 (STAT3), an oncogene stimulated by pro-inflammatory cytokines and growth factors. The thioredoxin system has several known inhibitors including mercury compounds such as Thimerosal (TmHg) which readily crosses the blood-brain barrier (BBB) and accumulates in the brain. Though previously used in various applications epidemiological evidence on TmHg's neurotoxicity is lacking. The objective of this study was to verify whether thimerosal is a suitable candidate for hard repurposing to control glioblastoma; therefore, the effects of this molecule were evaluated in human GBM (U87) cells. Our novel results show that TmHg decreased cellular viability (>50%) and migration (up to 90% decrease in wound closure), reduced thioredoxin reductase (TrxR/TXNRD1) and thioredoxin (Trx) activity, and increased reactive oxygen species (ROS) generation. Moreover, TmHg reduced HIF-1α expression (35%) as observed by immunofluorescence. Co-exposure of U87 cells to TmHg and TMZ reduced HIF-1α, VEGF, and phosphorylated STAT3. Consequently, TmHg alone or combined with chemotherapeutic drugs can reduce neoangiogenesis and ameliorate glioblastoma progression and treatment.

Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.

Errata: Effect of Thimerosal on Arrhythmia Induced by Coronary Ligation: The Involvement of ATP-dependent Potassium Channels.

Several errors (shown with underlines) in the following list appeared in the article "Effect of Thimerosal on Arrhythmia Induced by Coronary Ligation: The Involvement of ATP-dependent Potassium Channels" by Ömer Bozdogan, Ersöz Gonca, Melih Nebigil, Eylem Suveren Tiryaki (Vol. 46 No.4, 711-721, 2005).

Comparative characteristics between calyculin A-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa.

This study aimed to characterize calyculin A (CL-A)-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa. Hyperactivation was effectively induced by treating with 10 nM CL-A for 60 min in the presence of cyclic AMP analogs, extracellular Ca2+, and albumin or with 12.5 µM thimerosal briefly in the absence of these capacitation-supporting factors. Majority of the spermatozoa exhibiting CL-A-induced hyperactivation were characterized by the 3-dimensional helical movement with head rotation, higher degree of flagellar curvature, and faster beating of the flagella than those exhibiting thimerosal-induced hyperactivation of the 2-dimensional planar movement without head rotation. The CL-A-induced hyperactivation was linked to the activation of cAMP/protein phosphorylation-dependent signaling cascades and to the decreased activity of glycogen synthase kinase-3α (GSK-3α). In contrast, the thimerosal-induced hyperactivation was suppressed by pretreatment with CL-A and cyclic AMP analogs in the absence of CaCl2 to activate cAMP/protein phosphorylation-dependent signaling cascades. Additionally, the intracellular Ca2+ level in live sperm flagella was significantly higher in the CL-A-treated samples than in the thimerosal-treated samples. These results indicate that CL-A-induced hyperactivation of cryopreserved bovine spermatozoa is an extracellular Ca2+-dependent type with the 3-dimensional helical movement, which can be regulated not only by the activation of cAMP/protein phosphorylation-dependent signaling cascades, leading to a large enhancement of the intracellular Ca2+ level, but also by the reduction in GSK-3α activity. Considering the different characteristics of thimerosal-induced hyperactivation, our results suggest that the diversity of sperm hyperactivation arises from different combinations of flagellar bending and head rotation.

Caffeine induces sperm detachment from sperm head-to-head agglutination in bull.

Sperm head-to-head agglutination is a well-known known phenomenon in mammalian and non-mammalian species. Although several factors have been reported to induce sperm agglutination, information on the trigger and process of sperm detachment from the agglutination is scarce. Since hyperactivated motility is involved in bovine sperm detachment from the oviduct, we focused on caffeine, a well-known hyperactivation inducer, and aimed to determine the role of caffeine in sperm detachment from agglutination. Agglutination rate of bovine sperm was significantly decreased upon incubation with caffeine following pre-incubation without caffeine. Additionally, we observed that bovine sperm were detached from agglutination only when the medium contained caffeine. The detached sperm showed more asymmetrical flagellar beating compared to the undetached motile sperm, regardless of whether before or after the detachment. Intriguingly, some sperm that detached from agglutination re-agglutinated with different sperm agglutination. These findings indicated caffeine as a trigger for sperm detachment from the agglutination in bull. Furthermore, another well-known hyperactivation inducer, thimerosal, also significantly reduced the sperm agglutination rate. Overall, the study demonstrated the complete process of sperm detachment from sperm head-to-head agglutination and proposed that hyperactivated motility facilitates sperm detachment from another sperm. These findings would provide a better understanding of sperm physiology and fertilization process in mammals.

SNAP23 is required for constitutive and regulated exocytosis in mouse oocytes†.

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using Trim-away, a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits regulated exocytosis in response to a Ca2+ stimulus (cortical granule exocytosis), as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs.

Alterations of neuroimmune cell density and pro-inflammatory cytokines in response to thimerosal in prefrontal lobe of male rats.

Evidence suggests that the effect of heavy metals on neuroimmune cells lead to neurogenic inflammatory responses. In this study, immune cells [mast cells (MCs) and microglia] and pro-neuroinflammation cytokines (interleukin-1b and tumor necrosis factor-α) were assessed in the prefrontal lobe of rat brains exposed to thimerosal in different timeframes. A total of 108 neonatal Wistar rats were divided into three groups having three subgroups. The experimental groups received a single dose of thimerosal (300 µg/kg) postnatally at 7, 9, 11, and 15 days. The vehicle groups received similar injections of phosphate-buffered saline in a similar manner. The control groups received nothing. Samples of the prefrontal cortex were collected and prepared for stereological, immunohistochemical, and molecular studies at timeframes of 12 or 48 h (acute phase) and 8 days (subchronic phase) after the last injection. The average density of the microglia and MCs increased significantly in the experimental groups. This increase was more evident in the 48 h group. At 8 days after the last injection, there was a significant decrease in the density of the MCs compared to the 12 and 48 h groups. Alterations in pro-inflammatory cytokines were significant for all timeframes. This increase was more evident in the 48 h group after the last injection. There was a significant decrease in both neuroinflammatory cytokines at 8 days after the last injection. It was found that ethylmercury caused abnormal neurogenic inflammatory reactions and alterations in the neuroimmune cells that remained for a longer period in the brain than in the blood.

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study.

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200 µg Hg during the first 6 months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.

Regulation of endoplasmic reticulum Ca(2+) oscillations in mammalian eggs.

Changes in the intracellular concentration of free calcium ([Ca(2+)]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca(2+)]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca(2+)]i oscillations. The source of Ca(2+) during oscillations is the endoplasmic reticulum ([Ca(2+)]ER), but it is presently unknown how [Ca(2+)]ER is regulated. Here, we show using mouse eggs that [Ca(2+)]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca(2+)]ER and cause differential effects on the refilling and overall load of [Ca(2+)]ER. We also found that Ca(2+) influx is required to refill [Ca(2+)]ER, because the loss of [Ca(2+)]ER was accelerated in medium devoid of Ca(2+). Pharmacological inactivation of the function of the mitochondria and of the Ca(2+)-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca(2+)]ER, especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [Ca(2+)]i rises, promotes initiation of oscillations and enhances refilling of [Ca(2+)]ER. Collectively, our results provide novel insights on the regulation of [Ca(2+)]ER oscillations, which underlie the unique Ca(2+)-signalling system that activates the developmental program in mammalian eggs.

Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 µm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 µM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Effects of contact lens solution disinfectants against filamentous fungi.

PURPOSE: To assess and compare the antifungal activity of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are disinfectants used in multipurpose disinfectant solutions (MPDSs) against ocular pathogenic Fusarium solani and Aspergillus flavus isolates in vitro. METHODS: The in vitro activity of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine was assessed against 40 isolates of ocular pathogenic fungi that included 24 F. solani and 16 A. flavus isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the CLSI (Clinical and Laboratory Standards Institute) M38-A document. RESULTS: MIC90 (minimum inhibitory concentration for 90% of the organisms) values of PHMB were 4 and 16 µg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 µg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 µg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 µg/mL for F. solani and A. flavus, respectively. CONCLUSIONS: As a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations.

The association of cysteine to thiomersal attenuates its apoptosis-mediated cytotoxicity in zebrafish.

Thiomersal (TM) is an excellent preservative that is used in a wide variety of products, like pharmaceuticals, cosmetics, and vaccines, etc. Its usage has been in decline because of safety concerns. Since vaccine production is on the rise, its use may increase further in low-income and developing countries, as a cost-effective vaccine preservative. Further, Thiomersal is still being used as an essential component in various pharmaceutical preparations. In this light, the present study addresses its mechanism of toxicity in zebrafish and unveils a novel strategy for lessening its negative effects by conjugating cysteine to it, while retaining its antibacterial efficacy. We show that the mitochondrial membrane potential is destabilised by TM, leading to the induction of apoptosis. Interestingly, TM-cysteine conjugate (at a ratio of 1:1) showed no toxicity in zebrafish, whereas TM alone was highly toxic. Importantly, assaying for the bactericidal activity, tested using Escherichia coli (E. coli) and Methicillin-resistant Staphylococcus aureus (MRSA), revealed that the conjugate retains the antibacterial activity, demonstrating that the TM-cysteine conjugate is a safer alternative to TM as a vaccine preservative, and in all the other products that still use TM.

Does acute exposure to thimerosal, an organic mercury compound, affect the mitochondrial function of an infant model?

BACKGROUND: Thimerosal (TM) is a toxic, organometallic mercury compound (which releases ethyl-mercury-containing compounds in aqueous solutions) used as a preservative in vaccines. Mitochondria are organelle which are highly vulnerable to many chemical compounds, including mercury (Hg) and its derivatives. METHOD: Wistar rats (at 21 days of age) were used to model a child's TM exposure following childhood vaccination, divided in two groups: TM exposed (20 µg/kg/day) and unexposed controls (saline solution), both for 24 h. Atomic Fluorescence Spectrometry was used to quantify the amounts of mercury in tissues. The electron transport chain (ETC) from isolated mitochondria was evaluated using an oxygen electrode. The mitochondrial membrane potential and H2O2 production were analyzed using selective fluorescence probes. The activity of some enzymes (SOD, CAT, GPx, and AChE) and secondary markers of oxidative stress (GSH, GSSG, total free thiol) were also examined in tissues. RESULTS: Hg accumulation in the brain and liver was higher in exposed animals when compared to the control. Liver-isolated mitochondria showed that TM improved respiratory control by 23%; however, states 3 and 4 of the ETC presented a decrease of 16% and 37%, respectively. Furthermore, brain-isolated mitochondria presented an improvement of 61% in respiratory control. Brain enzyme activities were significantly impacted in TM-exposed rats compared to unexposed rats as follows: decreases in SOD (32%) and AChE (42%) and increases in GPx (79%) and CAT (100%). GPx enzyme activity in the liver was significantly increased (37%). Among secondary oxidative stress markers, the brain's total reduced thiol (SH) concentration was significantly increased (41%). CONCLUSION: Acute TM treatment exposure in a Wistar rat model mimicking TM exposure in an infant following childhood vaccination significantly damaged brain bioenergetic pathways. This study supports the ability of TM exposure to preferentially damage the nervous system.

MHC class I dimer formation by alteration of the cellular redox environment and induction of apoptosis.

Many MHC class I molecules contain unpaired cysteine residues in their cytoplasmic tail domains, the function of which remains relatively uncharacterized. Recently, it has been shown that in the small secretory vesicles known as exosomes, fully folded MHC class I dimers can form through a disulphide bond between the cytoplasmic tail domain cysteines, induced by the low levels of glutathione in these extracellular vesicles. Here we address whether similar MHC class I dimers form in whole cells by alteration of the redox environment. Treatment of the HLA-B27-expressing Epstein-Barr virus-transformed B-cell line Jesthom, and the leukaemic T-cell line CEM transfected with HLA-B27 with the strong oxidant diamide, and the apoptosis-inducing and glutathione-depleting agents hydrogen peroxide and thimerosal, induced MHC class I dimers. Furthermore, induction of apoptosis by cross-linking FasR/CD95 on CEM cells with monoclonal antibody CH-11 also induced MHC class I dimers. As with exosomal MHC class I dimers, the formation of these structures on cells is controlled by the cysteine at position 325 in the cytoplasmic tail domain of HLA-B27. Therefore, the redox environment of cells intimately controls induction of MHC class I dimers, the formation of which may provide novel structures for recognition by the immune system.

Unexpected flagellar movement patterns and epithelial binding behavior of mouse sperm in the oviduct.

In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.

Administration of thimerosal to infant rats increases overflow of glutamate and aspartate in the prefrontal cortex: protective role of dehydroepiandrosterone sulfate.

Thimerosal, a mercury-containing vaccine preservative, is a suspected factor in the etiology of neurodevelopmental disorders. We previously showed that its administration to infant rats causes behavioral, neurochemical and neuropathological abnormalities similar to those present in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration (4 injections, i.m., 240 µg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine and alanine; measured 10-14 weeks after the injections. Four injections of thimerosal at a dose of 12.5 µg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time (but based on thimerosal pharmacokinetics, could have been effective soon after its injection). Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our data imply that neonatal exposure to thimerosal-containing vaccines might induce excitotoxic brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against mercurials-induced neurotoxicity.

Metronidazole thiosalicylate conjugates: synthesis, crystal structure, docking studies and antiamoebic activity.

Metronidazole thiosalicylate conjugates were synthesized and crystallised in order to discover new molecules having better efficacy than therapeutically administered drug metronidazole, used against Entamoeba histolytica. The three compounds (4-6) showed lower IC(50) values than metronidazole on HM1:IMSS strain of E. histolytica and displayed low cytotoxicity on MCF-7 cell line. In order to get an insight into the mechanisms of action of these compounds, a homology model of E. histolytica thioredoxin reductase (EhTHRase) was constructed and molecular docking was performed into the binding pocket to identify the nature of interactions. The docking studies suggest that the improved inhibitory activity of the newly synthesised metronidazole analogues could be due to involvement of the additional hydrophobic interactions in the binding mode. The result of the present study indicates the molecular fragments that play an essential role in improving the antiamoebic activity.

Consequences of thimerosal on human erythrocyte hemoglobin: Assessing functional and structural protein changes induced by an organic mercury compound.

BACKGROUND: Thimerosal (TM) is an organic mercury compound used as a preservative in many pharmacological inputs. Mercury toxicity is related to structural and functional changes in macromolecules such as hemoglobin (Hb) in erythrocytes (Ery). METHOD: Human Hb and Ery were used to evaluate O2 uptake based on the TM concentration, incubation time, and temperature. The influence of TM on the sulfhydryl content, production of reactive oxygen species (ROS), and membrane fragility was also evaluated. Raman spectra and atomic force microscopy (AFM) profiles for Ery in the presence and absence of TM were calculated, and docking studies were performed. RESULTS: At 37 °C, with 2.50 µM TM (higher concentration) and after 5 min of incubation in Hb and Ery, we observed a reduction in O2 uptake of up to 50 %, while HgCl2, which was used as a positive control, showed a reduction of at least 62 %. Total thiol assays in the presence of NEM (thiol blocker) quantified the preservation of almost 60 % of free SH in Ery. Based on the Raman spectrum profile from Ery-TM, structural differences in the porphyrinic ring and the membrane lipid content were confirmed. Finally, studies using AFM showed changes in the morphology and biomechanical properties of Ery. Theoretical studies confirmed these experimental results and showed that the cysteine (Cys) residues present in Hb are involved in the binding of TM. CONCLUSION: Our results show that TM binds to human Hb via free Cys residues, causing conformation changes and leading to harmful effects associated with O2 transport.

Effects of Treadmill Exercise on Social Behavior in Rats Exposed to Thimerosal with Respect to the Hippocampal Level of GluN1, GluN2A, and GluN2B.

Thimerosal (THIM) kills brain neurons via induction of apoptosis and necrosis and induces the pathological features of autism spectrum disorder (ASD) in rats. THIM also affects the function of glutamatergic receptors. On the other hand, exercise induces both improvement and impairment effects on memory, depending on intensity, type, and duration. Treadmill exercise can also alter the expression of glutamatergic receptors. In this study, we aimed to investigate the effect of THIM and three protocols of treadmill exercise on social interaction memory and hippocampal expression of GluN1, GluN2A, and GluN2B in rats. THIM was injected intramuscularly at the dose of 300 µg/kg. The three-chamber apparatus was used to evaluate social interaction memory, and western blotting was used to assess protein expression. The results showed that THIM impaired social memory. Exercise 1 impaired social affiliation in controls. Social memory was impaired in all exercise groups of controls. Exercise 1 + 2 impaired social affiliation in THIM rats. Social memory was impaired in all groups of THIM rats. Exercises 2 and 1 + 2 decreased the expression of GluN1, and exercise 1 increased the expression of GluN2A and GluN2B in controls. THIM increased the expression of GluN2B, while exercise 1 reversed this effect. All exercise protocols increased the expression of GluN2A, and exercises 2 and 1 + 2 increased the expression of GluN1 in THIM rats. In conclusion, both THIM and exercise impaired social memory. Of note, the results did not show a separate and influential role for glutamatergic subunits in modulating memory processes following THIM injection or exercise.