Corneal regeneration by conditioned medium of human uterine cervical stem cells is mediated by TIMP-1 and TIMP-2.

The aim of the present study was to evaluate the effect and the mechanism of action of the conditioned medium from human uterine cervical stem cells (CM-hUCESC) on corneal wound healing in a rabbit dry eye model. To do this, dry eye and corneal epithelial injuries were induced in rabbits by topical administration of atropine sulfate and NaOH. Hematoxylin-Eosin (H&E) and Ki-67 immunostaining were carried out to evaluate corneal damage and cell proliferation, and real-time PCR was used to evaluate proinflammatory cytokines in the cornea. In addition, in order to investigate possible factors involved in corneal regeneration, primary cultures of rat corneal epithelial cells (rCECs) were used to evaluate cell migration, proliferation, and apoptosis before and after immunoprecipitation of specific factors from the CM-hUCESC. Results showed that CM-hUCESC treatment significantly improved epithelial regeneration in rabbits with dry eye induced by atropine and reduced corneal pro-inflammatory TNF-α, MCP-1, MIP-1α and IL-6 cytokines. In addition, metalloproteinase inhibitors TIMP-1 and TIMP-2, which are present at high levels in CM-hUCESC, mediated corneal regenerative effects by both inducing corneal epithelial cell proliferation and inhibiting apoptosis. In summary, CM-hUCESC induces faster corneal regeneration in a rabbit model of dry eye induced by atropine than conventional treatments, being TIMP-1 and TIMP-2 mediators in this process. The results indicate that an alternative CM-based treatment for some corneal conditions is achievable, although future studies would be necessary to investigate other factors involved in the multiple observed effects of CM-hUCESC.

Atropa belladonna neurotoxicity: Implications to neurological disorders.

Atropa belladonna, commonly known as belladonna or deadly nightshade, ranks among one of the most poisonous plants in Europe and other parts of the world. The plant contains tropane alkaloids including atropine, scopolamine, and hyoscyamine, which are used as anticholinergics in Food and Drug Administration (FDA) approved drugs and homeopathic remedies. These alkaloids can be very toxic at high dose. The FDA has recently reported that Hyland's baby teething tablets contain inconsistent amounts of Atropa belladonna that may have adverse effects on the nervous system and cause death in children, thus recalled the product in 2017. A greater understanding of the neurotoxicity of Atropa belladonna and its modification of genetic polymorphisms in the nervous system is critical in order to develop better treatment strategies, therapies, regulations, education of at-risk populations, and a more cohesive paradigm for future research. This review offers an integrated view of the homeopathy and neurotoxicity of Atropa belladonna in children, adults, and animal models as well as its implications to neurological disorders. Particular attention is dedicated to the pharmaco/toxicodynamics, pharmaco/toxicokinetics, pathophysiology, epidemiological cases, and animal studies associated with the effects of Atropa belladonna on the nervous system. Additionally, we discuss the influence of active tropane alkaloids in Atropa belladonna and other similar plants on FDA-approved therapeutic drugs for treatment of neurological disorders.

Physostigmine Reversal of Dysarthria and Delirium After Iatrogenic Atropine Overdose From a Dental Procedure.

BACKGROUND: Sublingual atropine, dosed at 0.4-0.8 mg, is used by dentists as an antisialogogue to facilitate and increase the speed of procedures. Concentrated ophthalmic atropine drops (10 mg/mL) are commonly used off-label for this purpose. These highly concentrated drops may result in medication errors, atropine toxicity, and the antimuscarinic toxidrome. We report a case of a man who suffered acute delirium and dysarthria (from dry mouth) after an iatrogenic overdose from a dental procedure. His symptoms were initially interpreted as a stroke, but they completely resolved with physostigmine. CASE REPORT: A 57-year-old man presented with acute dysarthria and delirium after a dental procedure; 4 hours earlier he was fitted for a temporary replacement of some premolar/molar teeth. He received sublingual atropine to assist in gingival drying for molding of his prosthesis, but a calculation error resulted in the administration of approximately 113 mg. A stroke evaluation was initially planned; however, 2.5 mg of intravenous physostigmine completely reversed his symptoms. His symptoms reoccurred and were successfully treated twice more with physostigmine; the patient was observed overnight with no additional symptoms and safely discharged the next morning. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Ophthalmic atropine drops are highly concentrated and may cause an overdose after ingestion of small amounts. This novel case highlights the importance of considering antimuscarinic poisoning in cases of acute delirium or dysarthria after dental procedures and stands as a reminder to inquire about the use of atropine drops in such cases. Timely recognition of the antimuscarinic toxidrome and appropriate use of physostigmine may prevent unnecessary testing while providing an effective therapy. This case also highlights the need for observation after resolution of delirium treated with physostigmine.

Dexmedetomidine stops benzodiazepine-refractory nerve agent-induced status epilepticus.

Nerve agents are highly toxic chemicals that pose an imminent threat to soldiers and civilians alike. Nerve agent exposure leads to an increase in acetylcholine within the central nervous system, resulting in development of protracted seizures known as status epilepticus (SE). Currently, benzodiazepines are the standard of care for nerve agent-induced SE, but their efficacy quickly wanes as the time to treatment increases. Here, we examine the role of the α2-adrenoceptor in termination of nerve agent-induced SE using the highly specific agonist dexmedetomidine (DEX). Adult male rats were exposed to soman and entered SE as confirmed by electroencephalograph (EEG). We observed that administration of DEX in combination with the benzodiazepine midazolam (MDZ) 20 or 40 min after the onset of SE stopped seizures and returned processed EEG measurements to baseline levels. The protective effect of DEX was blocked by the α2-adrenoceptor antagonist atipamezole (ATI), but ATI failed to restore seizure activity after it was already halted by DEX in most cases, suggesting that α2-adrenoceptors may be involved in initiating SE cessation rather than merely suppressing seizure activity. Histologically, treatment with DEX + MDZ significantly reduced the number of dying neurons as measured by FluoroJade B in the amygdala, thalamus, and piriform cortex, but did not protect the hippocampus or parietal cortex even when SE was successfully halted. We conclude that DEX serves not just as a valuable potential addition to the anticonvulsant regimen for nerve agent exposure, but also as a tool for dissecting the neural circuitry that drives SE.

Antimuscarinic-induced convulsions in fasted mice after food intake: No evidence of spontaneous seizures, behavioral changes or neuronal damage.

Prolonged or repeated seizures have been shown to cause spontaneous recurrent seizures, increased anxiety­related behavior, locomotor hyperactivity, impaired functions of learning and memory, and neuronal damage in the hippocampus and other brain regions in animals. Mice and rats treated with antimuscarinic drugs after fasting for two days or less develop convulsions after being allowed to eat ad libitum. To address whether such behavioral and neuroanatomic changes occur following these convulsions, mice treated i.p. with saline (control) or 2.4 mg/kg atropine and given food after 24 h of fasting were grouped according to seizure scores for behavioral and histological analysis. Following convulsions, the occurrence of spontaneous recurrent seizures was observed for 30 days. Motor activity and grooming behavior were assessed in the open field, and memory was assessed using the novel object recognition test 4 and 7 days after onset of convulsions, respectively. Animals allocated for the histological analysis were decapitated 7 days after onset of convulsions and hippocampal slices were evaluated for the percentage of degenerating neurons stained with Fluoro­Jade C. Spontaneous recurrent seizures, locomotor alterations, anxiety­related behavior, memory impairment, and neuronal loss in the granular layer of the dentate gyrus were not detected in the animals with seizure score 1-2 or 3-5. These results are in accordance with those related to the absence of behavioral changes, cognitive deficits, and hippocampal neuronal damage after single brief seizures in animals and patients with epilepsy.

Alterations in ACE and ABCG2 expression levels in the testes of rats subjected to atropine-induced toxicity.

Atropine-induced damage is associated with enzyme and protein alterations. The aim of the present study was to investigate atropine­induced alterations in testicular expression levels of angiotensin­converting enzyme (ACE) and adenosine 5'-triphosphate binding cassette sub­family G member 2 (ABCG2) following atropine treatment. Male Wistar rats received 15 mg/kg/day atropine for 7 days; control rats received an identical volume of saline, Following treatment, the testes were harvested for immunohistochemistry and in situ hybridization to examine the protein and gene expression levels of ACE and ABCG2 by digital image analysis. ACE gene and protein expression levels were significantly reduced in the testes of atropine­treated rats, compared with control rats (P=0.0001 and P<0.001, respectively). In addition, ABCG2 gene and protein expression levels were significantly increased in the testes of atropine­treated rats, compared with control rats (P=0.0017 and P<0.001, respectively). Thus, the results of the present study demonstrate that testicular protein and gene expression levels of ACE and ABCG2 were altered as a result of atropine­induced toxicity in the rats. These alterations may result in abnormal testicular function, and the proteins and genes identified in the present study may be useful to elucidate the mechanisms underlying atropine­induced toxicity and provide a direction for further studies.

Evaluation of the benefit of the bispyridinium compound MB327 for the antidotal treatment of nerve agent-poisoned mice.

The potency of the bispyridinium non-oxime compound MB327 [1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) diiodide] to increase the therapeutic efficacy of the standard antidotal treatment (atropine in combination with an oxime) of acute poisoning with organophosphorus nerve agents was studied in vivo. The therapeutic efficacy of atropine alone - or atropine in combination with an oxime, MB327, or both an oxime and MB237 - was evaluated by the determination of LD50 values of several nerve agents (tabun, sarin and soman) in mice with and without treatment. The addition of MB327 increased the therapeutic efficacy of atropine alone, and atropine in combination with an oxime, against all three nerve agents, although differences in the LD50 values only reached statistical significance for sarin. In conclusion, the addition of the compound MB327 to the standard antidotal treatment of acute poisonings with nerve agents was beneficial regardless of the chemical structure of the nerve agent, although at the dose employed, MB327 in combination with atropine, or atropine and an oxime, provided only a modest increase in protection ratio. These results from mice, and previous ones from guinea-pigs, provide consistent evidence for additional, albeit modest, efficacy resulting from the inclusion of the antinicotinic compound MB327 in standard antidotal therapy. Given the typically steep probit slope for the dose-lethality relationship for nerve agents, such modest increases in protection ratio could provide significant survival benefit.

Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis.

Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic.

Cytotoxicity of atropine to human corneal epithelial cells by inducing cell cycle arrest and mitochondrion-dependent apoptosis.

Atropine is an anticholinergic drug for mydriasis in eye clinic, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of atropine to the cornea and its cellular and molecular mechanisms remain unknown. In this study, we investigated the cytotoxicity of atropine to corneal epithelium and its underlying mechanisms using an in vitro model of non-transfected human corneal epithelial (HCEP) cells. Our results showed that atropine, above the concentration of 0.3125 g/l (1/32 of its therapeutic dosage in eye clinic), had a dose- and time-dependent toxicity to HCEP cells by inducing morphological abnormality, cytopathic effect, viability decline, and proliferation retardation. Moreover, the proliferation-retarding effect of atropine on the cells was achieved by inducing G1/S phase arrest and downregulation of E-cadherin and ß-catenin. Besides, atropine also had an apoptosis-inducing effect on the cells by inducing phosphatidylserine externalization, plasma membrane permeability elevation, DNA fragmentation and apoptotic body formation. Furthermore, atropine could also induce activations of caspase-2, -3 and -9, disruption of mitochondrial transmembrane potential, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor, implying a death receptor-mediated mitochondrion-dependent pathway is most probably involved in the apoptosis of HCEP cells induced by atropine. Taken together, our results suggest that atropine has remarkable cytotoxicity to HCEP cells by inducing cell cycle arrest and death receptor-mediated mitochondrion-dependent apoptosis.

The effects of taurine on vigabatrin, high light intensity and mydriasis induced retinal toxicity in the pigmented rat.

The overall purpose of this study was to establish a model that may be used for examining the effect of Vigabatrin-induced retinal toxicity in pigmented rats, and subsequently examine the possible effects of taurine on the retinal toxicity. In the first part of the study, pigmented Long Evans rats were subjected to combinations of induced mydriasis, low/high light intensities (40/2000 lx) and oral administration of near-MTD (Maximum Tolerated Dose) doses (200 mg/kg/day) of Vigabatrin for up to 6 weeks. The combination of mydriasis and high light intensity applied to Long Evans rats resulted in retinal damage that was increased by the administration of Vigabatrin. In the second part of the study Long Evans rats were subjected to combinations of induced mydriasis and high/low light intensity (40/2000 lx) while being orally administered low (30 mg/kg/day) or high (200 mg/kg/day) doses of Vigabatrin for up to 6 weeks. In addition, selected groups of animals were administered taurine via the drinking water (20 mg/ml), resulting in systemic taurine concentrations of approximately threefold the endogenous concentration. The combined results of the studies demonstrate that retinal damage can be induced in pigmented animals when combining mydriasis and high light intensity. Retinal damage was functionally evaluated by electroretinography (ERG), then confirmed by histopathology. While depending on mydriasis and high light intensity, administration of Vigabatrin increased the retinal toxicity and resulted in the formation of rosette-like structures in the retina in a dose-related manner. Administration of taurine did not alleviate the Vigabatrin-induced retinal toxicity, as demonstrated either functionally by ERG or morphologically, although systemic concentrations of 3-fold the endogenous levels were reached, and it was thus not possible to demonstrate a protective effect of taurine in these pigmented animals.

Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma.

While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

Simple validated LC-MS/MS method for the determination of atropine and scopolamine in plasma for clinical and forensic toxicological purposes.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of atropine and scopolamine in 100µL human plasma was developed and validated. Sample pretreatment consisted of protein precipitation with acetonitrile followed by a concentration step. Analytes and levobupivacaine (internal standard) were separated on a Zorbax XDB-CN column (75mm×4.6mm i.d., 3.5µm) with gradient elution (purified water, acetonitrile, formic acid). The triple quadrupole MS was operated in ESI positive mode. Matrix effect was estimated for deproteinised plasma samples. Selected reaction monitoring (SRM) was used for quantification in the range of 0.10-50.00ng/mL. Interday precision for both tropanes and intraday precision for atropine was <10%, intraday precision for scopolamine was <14% and <18% at lower limit of quantification (LLOQ). Mean interday and intraday accuracies for atropine were within ±7% and for scopolamine within ±11%. The method can be used for determination of therapeutic and toxic levels of both compounds and has been successfully applied to a study of pharmacodynamic and pharmacokinetic properties of tropanes, where plasma samples of volunteers were collected at fixed time intervals after ingestion of a buckwheat meal, spiked with five low doses of tropanes.

Acute effects of a low-dose atropine/scopolamine mixture as a food contaminant in human volunteers.

To verify the assumptions in our previous risk assessment of an atropine/scopolamine mixture in buckwheat flour, we performed a randomized, double-blind, placebo-controlled cross-over study in 20 healthy, adult volunteers. The volunteers ingested a traditional Slovenian buckwheat meal, made of boiled buckwheat flour to which alkaloids were added. In addition to the placebo they ingested 0.12/0.10, 0.37/0.29, 1.22/0.95, 3.58/2.81 and 12.10/9.50 µg kg(-1) body mass (BM) of the atropine/scopolamine mixture. The changes in body temperature, heart rate, salivary and sweat secretion, pupil size, near-point vision and subjective symptoms were recorded regularly for 4 h after the ingestion. Decreased salivary and sweat secretion, increased heart rate and pupil size and reduced near-point vision accompanied by characteristic subjective symptoms were observed at 12.10/9.50 µg kg(-1) BM. At doses of 0.37/0.29 and 1.22/0.95 µg kg(-1) BM, a significant decrease in the heart rate was noted, which we consider to be a critical effect of a low-dose exposure to the atropine/scopolamine mixture. Although this did not have any clinical relevance in our subjects, it may have serious implications if it occurred in people with pre-existent cardiac conditions or those on medications that may cause bradycardia. No significant changes in the observed end points were noted at 0.12/0.10 µg kg(-1) BM. We estimate that the NOAEL (No Observed Adverse Effect Level) for the atropine/scopolamine mixture lies between the lower two administered doses. Applying the uncertainty factor of 10, we propose a new provisional Acute Reference Doses (ARfDs) of the mixture, i.e. 0.01 µg kg(-1) BM for each alkaloid, and a further refinement using higher-tier approaches.

A new biological test utilising the yeast Saccharomyces cerevisiae for the rapid detection of toxic substances in water.

This study evaluates the toxic effects of five substances (atropine, fenitrothion, potassium cyanide, mercuric chloride and lead nitrate) on the yeast Saccharomyces cerevisiae. It describes a new biological toxicity test based on inhibition of S. cerevisiae viability and compares it with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminiscence inhibition (EN ISO 11348-2). The new biological test -S. cerevisiae lethal test - is cheaper and 24 times faster than the D. magna test. The test speed is comparable with the V. fischeri test but the new test is more sensitive for some substances. The test indicates reliably the presence of all used toxicants in water in concentrations which are significantly lower than the concentration in toxic or lethal doses for man. Therefore, this new toxicity test could be proposed for rapid detection of toxic substances in water.

E-cadherin and 5-HT alterations in the heart of rats having undergone atropine-induced toxicity.

Atropine-induced heart damage is associated with changes in the expression of various enzymes and proteins. The purpose of this study was to investigate atropine-induced alterations in cardiac E-cadherin and 5-hydroxytryptamine (5-HT) after atropine administration. Male Wistar rats were randomly divided into two groups: a control group and an atropine group. The atropine group intraperitoneally received atropine at a single dose of 15 mg/kg for 7 days; the controls received the same amount of saline via the same route. On Day 8, the rats were anesthetized, and a thoracotomy was performed in all animals. Immunohistochemical analysis was performed to evaluate protein expression of E-cadherin and 5-HT. Sections were analyzed by digital image analysis. Cardiac protein expression of E-cadherin and 5-HT was altered after atropine­induced toxicity in the rat. The expression levels of E-cadherin and 5-HT were significantly decreased after atropine treatment, supported by IOD analysis, when compared with the control (P<0.05). The current findings indicate that such changes would be reflected in abnormal cardiac function, and these proteins may be useful for revealing the mechanisms underlying atropine-induced toxicity and may also provide various clues for further research.

Intoxicación aguda por atropina de causa yatrógena / A case of acute iatrogenic atropine poisoning

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Development of an in vitro model for the simultaneous study of the efficacy and hematotoxicity of antileukemic compounds.

Hematopoietic system displays a wide spectrum of cell populations hierarchically organized in the bone marrow. Homeostasis in this system requires equilibrium between the self-renewal of the stem cells and their capacity of differentiation. Any failure on this equilibrium could lead to fatal consequences, such as the development of leukemia. Due to its rapid rate of renewal, hematopoietic tissue is a major target for antitumoral compounds and often becomes a dose limiting factor in the development of antineoplastics. Our aim was to develop an in vitro model for predicting the efficacy of antitumoral compounds on leukemic cells and their toxic effects on the healthy hematopoietic cells. The mouse myelomonocytic leukemia WEHI-3b was transduced with a lentiviral vector for expressing the green fluorescence protein. Mixed semisolid clonogenic cultures of transduced WEHI-3b and murine bone marrow cells were exposed to five pharmaceuticals: daunorubicin (positive control), atropine sulphate (negative control) and three in different stages of clinical development (trabectedin, Zalypsis(®) and PM01183). Colonies of leukemic cells were distinguishable from healthy CFU-GM under fluorescence microscope. The sensitivity of leukemic cells to daunorubicin, trabectedin, Zalypsis(®) and PM01183 was higher compared to healthy cells. The effect of a non-antitumoral compound, atropine sulphate, was the same on both populations. Our results show that this in vitro model is a valuable tool for studying the effect of antitumoral compounds in both tumoral and normal hematopoietic cells under the same toxic microenvironment and could safe time and facilitate the reduction of the number of animals used in preclinical development of pharmaceuticals.