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Aconitine is a highly poisonous C 19 -diterpenoid alkaloid identified and isolated from the species of the genus Aconitum. Aconitine is indicated in the treatment of cardiovascular diseases (CVDs) and, due to its neurotoxic effects, is a very effective drug in pain release. A total of 101 relevant scientific papers were manually searched on the Web of Science, Scopus, Science Direct, Google Scholar, PubMed and Dovepress databases and in the books available in the library of the Department of Natural Sciences, the National University of Science and Technology POLITEHNICA Bucharest, Pitesti University Centre, Romania. In combination treatments, aconitine shows antiarrhythmic and anti-inflammatory activity, a synergistic antiproliferative effect and decreased reactive oxygen species (ROS) generation, an improved biodistribution and bioavailability. Additionally, the entrapment of aconitine in engineered nanoparticles represents a promising method for reducing the toxicity of this alkaloid. This review provides, for the first time, a comprehensive picture of the knowledge and research on the synergistic, additive and antagonistic effects of aconitine in combination treatments applied in vivo or in vitro. The summarized studies represent important clues in addressing the multitude of knowledge, which can find their utility in practical and clinical applications.

Background: Radix Aconiti Lateralis (Fuzi), a mono-herbal preparation of Aconitum herbs in the genus Aconitum, is commonly used in traditional Chinese medicine (TCM) to treat critical illnesses. The curative effect of Fuzi is remarkable. However, the toxic effects of Fuzi are still a key clinical focus, and the substances inducing nephrotoxicity are still unclear. Therefore, this study proposes a research model combining "in vitro and in vivo component mining-virtual multi-target screening-active component prediction-literature verification" to screen potential nephrotoxic substances rapidly., Method: The UHPLC-Q-Exactive-Orbitrap MS analysis method was used for the correlation analysis of Fuzi's in vitro-in vivo chemical substance groups. On this basis, the key targets of nephrotoxicity were screened by combining online disease databases and a protein-protein interaction (PPI) network. The computer screening technique was used to verify the binding mode and affinity of Fuzi's components with nephrotoxic targets. Finally, the potential material basis of Fuzi-induced nephrotoxicity was screened., Results: Eighty-one Fuzi components were identified. Among them, 35 components were absorbed into the blood. Based on the network biology method, 21 important chemical components and three potential key targets were screened. Computer virtual screening revealed that mesaconine, benzoylaconine, aconitine, deoxyaconitine, hypaconitine, benzoylhypaconine, benzoylmesaconine, and hypaconitine may be potential nephrotoxic substances of Fuzi., Conclusions: Fuzi may interact with multiple components and targets in the process of inducing nephrotoxicity. In the future, experiments can be designed to explore further. This study provides a reference for screening Fuzi nephrotoxic components and has certain significance for the safe use of Fuzi., (© 2024 John Wiley & Sons Ltd.)

Fuzi, an effective common herb, is often combined with Gancao to treat disease in clinical practice with enhancing its efficacy and alleviating its toxicity. The major toxic and bioactive compounds in Fuzi and Gancao are aconitine (AC) and glycyrrhizic acid (GL), respectively. This study aims to elucidate detoxification mechanism between AC and GL from pharmacokinetic perspective using physiologically based pharmacokinetic (PBPK) model. In vitro experiments exhibited that AC was mainly metabolized by CYP3A1/2 in rat liver microsomes and transported by P-glycoprotein (P-gp) in Caco-2 cells. Kinetics assays showed that the K m and V max of AC towards CYP3A1/2 were 2.38 μM and 57.3 pmol/min/mg, respectively, whereas that of AC towards P-gp was 11.26 μM and 147.1 pmol/min/mg, respectively. GL markedly induced the mRNA expressions of CYP3A1/2 and MDR1a/b in rat primary hepatocytes. In vivo studies suggested that the intragastric and intravenous administration of GL significantly reduced systemic exposure of AC by 27% and 33%, respectively. Drug-drug interaction (DDI) model of PBPK predicted that co-administration of GL would decrease the exposure of AC by 39% and 45% in intragastric and intravenous dosing group, respectively. The consistency between predicted data and observed data confirmed that the upregulation of CYP3A1/2 and P-gp was the crucial detoxification mechanism between AC and GL. Thus, this study provides a demonstration for elucidating the compatibility mechanisms of herbal formula using PBPK modeling and gives support for the clinical co-medication of Fuzi and Gancao., (© 2024 John Wiley & Sons Ltd.)

Hypoaconitine (HA), a major secondary metabolite of aconite (a plant-derived rodenticide), is a highly toxic di-ester alkaloidal constituent. The toxicity of HA is intense with a low LD 50 . However, studies on its toxicity mechanism have mainly focused on cardiotoxicity, with few reports on the mechanism of hepatotoxicity. In this study, we combined metabolomics and network toxicology to investigate the effects of HA on the liver and analyzed the mechanisms by which it causes hepatotoxicity. The results of metabolomics studies indicated diethylphosphate, sphingosine-1-phosphate, glycerophosphorylcholine, 2,8-quinolinediol, guanidinosuccinic acid, and D-proline as differential metabolites after HA exposure. These metabolites are involved in eight metabolic pathways including arginine and proline metabolism, ether lipid metabolism, β-alanine metabolism, sphingolipid metabolism, glutathione metabolism, and glycerophospholipid metabolism. Network toxicology analysis of HA may affect the HIF-1 signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and so on by regulating the targets of ALB, HSP90AA1, MMP9, CASP3, and so on. Integrating the results of metabolomics and network toxicology, it was concluded that HA may induce hepatotoxicity by triggering physiological processes such as oxidative stress, inflammatory response, and inducing apoptosis in hepatocytes., Competing Interests: Declaration of competing interest The authors declared that there are no conflicts of interest in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Yunaconitine (YAC) is a hidden toxin that greatly threatens the life safety of patients who are prescribed herbal medicines containing Aconitum species; however, its underlying mechanism remains unclear., Objective: The objective of this study is to elucidate the functions of P-glycoprotein (P-gp) in regulating the efficacy, toxicity, and pharmacokinetics of YAC., Methods: The efflux function of P-gp on YAC was explored by using Caco-2 monolayers in combination with the P-gp inhibitor verapamil. The impact of P-gp on regulating the analgesic and anti-inflammatory effects, acute toxicity, tissue distribution, and pharmacokinetics of YAC was determined via male Mdr1a gene knocked-out mice and wild-type FVB mice., Results: The presence of verapamil significantly decreased the efflux ratio of YAC from 20.41 to 1.07 in Caco- 2 monolayers (P < 0.05). Moreover, oral administration of 0.07 and 0.14 mg/kg YAC resulted in a notable decrease in writhing times in Mdr1a -/- mice by 23.53% and 49.27%, respectively, compared to wild-type FVB mice (P < 0.05). Additionally, the deficiency of P-gp remarkably decreased the half-lethal dose (LD 50 ) of YAC from 2.13 to 0.24 mg/kg (P < 0.05). Moreover, the concentrations of YAC in the tissues of Mdr1a -/- mice were statistically higher than those in wild-type FVB mice (P < 0.05). Particularly, the brain accumulation of YAC in Mdr1a -/- mice significantly increased by 12- and 19-fold, respectively, after oral administration for 30 and 120 min, when compared to wild-type FVB mice (P < 0.05). There were no significant differences in the pharmacokinetic characteristics of YAC between Mdr1a -/- and wild-type FVB mice., Conclusion: YAC is a sensitive substrate of P-gp. The absence of P-gp enhances the analgesic effect and toxicity of YAC by upregulating its brain accumulation. Co-administration with a P-gp inhibitor may lead to severe YAC poisoning., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aconitine is a crucial toxic component in Chinese herbal medicines such as Aconitum, Aconitum coreanum, and Aconitum soongaricum. The poisoning symptoms of the central nervous system and cardiovascular system caused by it are relatively common in China, and there are many studies on cardiovascular system diseases caused by aconitine. However, the specific mechanism of neurotoxicity induced by aconitine is still unclear. This study explored the effect and mechanism of mitochondrial calcium uniporter on mitochondrial energy metabolism disorder in aconitine poisoning hippocampal neurons. The results showed that after treatment with 400μmol/L aconitine, mitochondrial energy metabolism was abnormal in rat hippocampal neuron cells, the expression of MCU in mitochondria was up-regulated, calcium overload in mitochondria, ATP production decreased, and mitochondrial membrane potential Changes, increased expression of the apoptosis gene Cleaved-Caspase-3. After treatment with the MCU agonist spermine, mitochondrial energy metabolism was significantly abnormal, and cell apoptosis was increased considerably. However, pretreatment with calcium ion channel inhibitor Ruthenium Red (RR) effectively promoted the generation of ATP, thereby improving mitochondrial energy metabolism disorders and reducing cell apoptosis. These results suggest that aconitine induces mitochondrial energy metabolism dysfunction in hippocampal neurons, which may be related to the increased expression of MCU., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Haiying Wu reports financial support was provided by National Natural Science Foundation of China. Haiying Wu reports a relationship with National Natural Science Foundation of China that includes: funding grants., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: The transformations that occur in diterpenoid alkaloids during the process of sand frying for Chinese herbal medicine preparation have yet to be clarified. This study investigated the structural changes that take place in 3-acetylaconitine during a simulation of heat-processing and evaluated the toxicity and biological activity of the pyrolysis products., Methods: The diterpenoid alkaloid 3-acetylaconitine was heated at 180 °C for 15 min to simulate the process of sand frying. The pyrolysis products were separated using column chromatography, and their structures were investigated using high-resolution electrospray ionization mass spectroscopy and nuclear magnetic resonance spectroscopy. Further, in vivo cardiotoxicity and acute toxicity of 3-acetylaconitine and its pyrolysis products were compared, and the aconitine-induced arrhythmia model was employed to evaluate the antiarrhythmic effect of the pyrolysis products., Results: Two new diterpenoid alkaloids, pyroacetylaconitine and 16-epi-pyroacetylaconitine, a pair of epimers at C-16, were isolated. After comparing the structures of these compounds, possible transformation pathways were proposed. Compared with the prototype compound, 3-acetylaconitine, the cardiotoxicity and acute toxicity of the heat-transformed products were significantly decreased. In the biological activity assay, the two pyrolysis products exhibited an effective increase in ventricular premature beat latency, a reduction in the occurrence of ventricular tachycardia, as well as an increase in the rate of arrhythmia inhibition, implying strong antiarrhythmic activity., Conclusion: Compared with 3-acetylaconitine, its pyrolysis products displayed lower toxicity and good antiarrhythmic effects; thus, they have potential for being developed into antiarrhythmic medicines. Please cite this article as: Wang YJ, Wang Y, Tao P. Structural characterization, in vivo toxicity and biological activity of two new pyro-type diterpenoid alkaloids derived from 3-acetylaconitine. J Integr Med. 2023; 21(3): 302-314., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Aconitum species, with a long history of traditional application, were applied to treat rheumatism, arthritis, stroke, and pain in Chinese medical practice. However, misuse of Aconitum species may induce central nervous toxic effects, such as numbness, vomiting, and even coma. Aconitine has been proved to be the main toxic component of Aconitum plants. Neurotoxicity is the main toxic effect of aconitine, while the underlying mechanism of aconitine remains unclear., Aim of the Study: The purpose of the study is to explore the effects and molecular mechanism of ferroptosis caused by aconitine in vivo and in vitro., Materials and Methods: Six-dpf zebrafish larvae and SH-SY5Y cells were treated with different concentrations of aconitine for 24 h. Inhibitors treatment, e.g. pretreatment with Necrostain-1 (Nec-1) and Z-VZD-FMK for 12 h, or with Ferrostain-1 (Fer-1) for 4 h, were involved in the identification of aconitine-induced ferroptosis. Transient transfection experiment was conducted to explore the effects of SLC7A11 in the process of aconitine-induced ferroptosis. The effects of aconitine on morphological changes, lipid peroxidation, ferrous ion, and ferroptosis were detected by transmission electron microscope, flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and western blotting., Results: In SH-SY5Y cells, morphological changes including shrunken mitochondria, increased mitochondrial membranes density and ruptured mitochondrial membranes were captured in aconitine-treated group. The cell viability and GSH content dose-dependently declined, levels of lipid reactive oxygen species (ROS), malondialdehyde (MDA), and ferrous ion significantly increased after aconitine exposure for 24 h. Ferroptosis inhibitor Fer-1 pretreatment effectively increased cell viability, GSH content, and decreased levels of MDA and lipid peroxidation, suggesting that aconitine induced ferroptosis. In addition, the protein expression of SLC7A11 and GPX4 were improved after Fer-1 preincubation, which indicated that aconitine triggered ferroptosis via the inhibition of SLC7A11 and the inactivation of GPX4. Ferroptotic characteristics, including GSH depletion and lipid peroxidation accumulation, were alleviated via overexpression of SLC7A11 to increase protein expression of GPX4. In zebrafish experiment, GSH depletion, lipid peroxidation accumulation, iron overload, and the decreased protein expression of SLC7A11 and GPX4 were also induced in zebrafish larvae after aconitine exposure. Taken together, aconitine triggered ferroptotic cell death via inhibiting SLC7A11/GPX4 signal pathway in vivo and in vitro., Conclusion: All results indicated that aconitine triggered ferroptosis of SH-SY5Y cells and zebrafish larvae nerve cells, which involved the inhibition of SLC7A11/GPX4 signal pathway mediated by lipid peroxidation damage and iron overload., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cardiovascular diseases remain one of the leading causes of death worldwide. Unfortunately, the available pharmacotherapeutic options have limited effectiveness. Therefore, developing new drug candidates remains very important. We selected six novel arylpiperazine alkyl derivatives of salicylamide to investigate their cardiovascular effects. Having in mind the beneficial role of α 1 -adrenergic receptors in restoring sinus rhythm and regulating blood pressure, first, using radioligand binding assays, we evaluated the affinity of the tested compounds for α-adrenergic receptors. Our experiments revealed their high to moderate affinity for α 1 - but not α 2 -adrenoceptors. Next, we aimed to determine the antiarrhythmic potential of novel derivatives in rat models of arrhythmia induced by adrenaline, calcium chloride, or aconitine. All compounds showed potent prophylactic antiarrhythmic activity in the adrenaline-induced arrhythmia model and no effects in calcium chloride- or aconitine-induced arrhythmias. Moreover, the tested compounds demonstrated therapeutic antiarrhythmic activity, restoring a normal sinus rhythm immediately after the administration of the arrhythmogen adrenaline. Notably, none of the tested derivatives affected the normal electrocardiogram (ECG) parameters in rodents, which excludes their proarrhythmic potential. Finally, all tested compounds decreased blood pressure in normotensive rats and reversed the pressor response to methoxamine, suggesting that their hypotensive mechanism of action is connected with the blockade of α 1 -adrenoceptors. Our results confirm the antiarrhythmic and hypotensive activities of novel arylpiperazine derivatives and encourage their further investigation as model structures for potential drugs.

The C 18 -diterpenoid alkaloid lappaconitine (LA) is a non-addictive analgesic used in China. The toxicity (LD 50  = 11.7 mg/kg) limits its application. Two series of LA derivatives, including amides and sulfonamides (1-93), were designed and synthesized by modification on their C4 acetamidobenzoate side chains in this work. In vivo analgesic activity and toxicity of all derivatives were evaluated, and the structure-activity relationship was summarized. Six lead compounds (35, 36, 39, 49, 70, and 89) exhibited approximate analgesic activity to LA but with significantly reduced toxicity. The therapeutic index of these compounds is 14-30 times that of LA. In vivo metabolism study of the lead compounds 39, 49, 70, and 89 were conducted by UPLC-MS E , indicating the reason for the low toxicity of the potential derivatives might be they are difficult to metabolize to toxic metabolite N-deacetyllappaconitine compared to LA. The effects of lead compounds on sodium channels and hERG channels were also studied by ion channel reader (ICR) which further revealed their analgesic and toxicity-attenuating mechanisms. Sodium channel assay revealed that the analgesic mechanism of these lead compounds was inhibiting the Na v  1.7 channels. Taken together, compound 39 was provided as a new analgesic lead compound with significantly low toxicity and comparable activity to LA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Aconitum alkaloids are considered to be the characteristic bioactive ingredients of Aconitum species, which are widely applied to the treatment of diverse diseases, and aconitine (AC) is found in most Aconitum plants. Research evidence shows that low-dose AC has a good therapeutic potential in heart failure, myocardial infarction, neuroinflammatory diseases, rheumatic diseases, and tumors, which has become one of the hotspots in global research in recent years. However, the cardiotoxicity and neurotoxicity of AC have also attracted extensive attention. Excessive use of AC always induces ventricular tachyarrhythmia and heart arrest, even can be potentially lethal. Therefore, AC cannot simply be regarded as a good medicine or a toxicant, but its underlying curative and toxic properties remained chaos. In order to dig the unique pharmacological value of AC while preventing its toxicity, the pharmacological activities and toxic effects of AC were summarized in this paper, providing new insight into the safe and effective use of AC in clinical practice., (© 2022 Société Française de Pharmacologie et de Thérapeutique.)

Ethnopharmacological Relevance: Aconitine, a C 19 -norditerpenoid alkaloid, derives from many medicinal plants such as Aconitum carmichaelii Debx. (Chinese:), Aconitum kusnezoffii Reichb (Chinese:), which were used to rheumatic fever, painful joints and some endocrinal disorders., Aims of the Review: The present paper reviews research progress relating to the pharmacokinetics, physiological and pathological processes of aconitine, while some promising research direction and the detoxification of aconitine are also discussed., Materials and Methods: The accessible literature on aconitine, from 1990 to 2020, obtained from published materials of electronic databases, such as SCI finder, PubMed, Web of Science, Science Direct, Springer and Google Scholar was systematically analyzed., Results: In this review, we address the pharmacokinetics of aconitine, as well as its pharmacological effects including anti-cancer, anti-inflammatory, anti-virus, immunoregulation, analgesic, insecticide and inhibition of androgen synthesis. Further, we summarize the toxicity of aconitine such as cardiotoxicity and neurotoxicity, on which we strikingly focus on the ways to reduce the toxicity of aconitine based., Conclusions: Aconitine plays an vital role in a wide range of physiological and pathological processes and we can reduce the toxicity of aconitine by compatibility and hydrolysis. Although some issues still exist, such as the correlative relationship between the dose and toxicity of aconitine not being clear, our review may provide new ideas for the application of aconitine in the treatment of related diseases., (Copyright © 2022. Published by Elsevier B.V.)

Aconitine is a diterpenoid alkaloid, which mainly exists in the plants of Aconitum. In the last decade, a plethora of studies on the pharmacological activities of aconitine has been conducted and demonstrated that aconitine possessed an extensive range of pharmacological activities such as anti-tumor, anti-inflammatory, analgesic, local anesthesia, and immunomodulatory effects. Pharmacokinetic studies indicated that aconitine may have the characteristics of poor bioavailability, wide distribution, and slow elimination. However, studies have also found that aconitine has toxic effects on the heart, nerves, embryos, etc. Therefore, we believe that aconitine may not be suitable for heart patients and pregnant women to treat related diseases. It is important to note that all of these pharmacological effects require further high-quality studies to determine the clinical efficacy of aconitine. This review aims to summarize the advances in pharmacological, pharmacokinetics, toxicity, and detoxification of aconitine in the last decade with an emphasis on its anti-tumor and anti-inflammatory activities, to provide researchers with the latest information and point out the limitations of relevant research at the current stage and the aspects that should be strengthened in future research., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aconitine is a plant toxin derived from aconitum genus and well known for its neurological and vascular toxicity. However, the mechanism of toxicity on the growth and apoptosis of the neurological cells has not been well investigated. In this study, we used HT22 cell lines derived from hippocampus to explore the mechanism. We began with examination of the viability and DA (dopamine) contents of cells treated with different dose of aconitine. In this study, we investigated the role of apoptosis in AC-induced HT22 cells. Our results showed that aconitine inhibited HT22 cells growth and increased DA contents in a dose dependent manner. Aconitine treatment induced apoptosis in HT22 cells and we found aconitine induced apoptosis by upregulating the expression of Bax, Cyto c, Apaf-1, Caspase9, Fas, Fas-L, Fadd, Caspase8, Caspase3 with concomitant decreasing of Bcl-2 and Bid expression. Collectively, results suggest that aconitine induce apoptosis through mitochondrial-mediated and death receptor signaling pathways in HT22 cells., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Yunaconitine (YAC), crassicauline A (CCA), 8-deacetylyunaconitine (DYA), and 8-deacetylcrassicauline A (DCA), as hidden toxic Aconitum alkaloids, are detected in some products of processed Aconitum carmichaelii lateral root and poisoning cases. The distribution and toxicity of these four components in Aconitum herbs should be further systematically studied for medication safety. This study developed a new UHPLC-QQQ-MS/MS method to determine ten Aconitum alkaloids, including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, YAC, CCA, DYA, and DCA, for Aconitum herbs simultaneously. YAC and CCA were founded in some samples of unprocessed A. carmichaelii lateral root (7.04%), A. carmichaelii root (9.43%), A. brachypodum root (6.00%), and A. ouvrardianum root (100%). Four hidden toxic Aconitum alkaloids were detected in processed A. carmichaelii lateral root (2.56%) and A. vilmorinianum root (100%). Four hidden toxic Aconitum alkaloids played significant roles in the classification of Aconitum herbs by OPLS-DA analysis. The acute toxicity test was performed by up-and-down procedure (UDP). The oral administration of the half lethal dose (LD 50 ) of YAC, CCA, DYA, and DCA to female ICR mice was 2.37 mg/kg, 5.60 mg/kg, 60.0 mg/kg, and 753 mg/kg, respectively. The LD 50 by intravenous injection was 0.200 mg/kg, 0.980 mg/kg, 7.60 mg/kg, and 34.0 mg/kg, respectively. The LD 50 of unprocessed A. carmichaelii lateral root, A. vilmorinianum root, and A. brachypodum root to mice orally was 1.89 g/kg, 0.950 g/kg, and 0.380 g/kg, respectively. Symptoms of Aconitum alkaloid poisoning in mice were decreased activity, fur erect, palpebral edema, vomiting, polypnea, and convulsions. The main change of organs was flatulence. No poisoning or death occurred in mice at the maximum dosage (27.0 g/kg) of A. ouvrardianum root orally. To better control the quality and safety of Aconitum herbs, this study provides favorable support for improving the existing standards to strengthen the supervision of the four hidden toxic Aconitum alkaloids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aconitine linoleate (1) is a lipo-diterpenoid alkaloid, isolated from Aconitum sinchiangense W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of 1 against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation in vivo. The cytotoxic activities of 1 were measured in vitro. Also, we investigated the latent mechanism of 1 by cell cycle analysis in MCF-7/ADR cells and topo I and topo IIα inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of 1 was detected on mice. 1 exhibited significant antitumor activity against both MCF-7 and MCF-7/ADR cells, with IC 50 values of 7.58 and 7.02 μM, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than Adriamycin in MCF-7/ADR cell lines. The molecular docking and the topo inhibition test found that it is a selective inhibitor of topoisomerase IIα. Moreover, activation of the damage response pathway of the DNA leads to cell cycle arrest at the G 0 G 1 phase. Furthermore, the in vivo acute toxicity of 1 in mice displayed lower toxicity than aconitine, with LD 50 of 2.2 × 10 5  nmol/kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, 1 has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells and greatly reduces in vivo toxicity, which suggests this kind of lipo-alkaloids is powerful and promising antitumor compounds for breast cancer., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aconitine, a highly toxic alkaloid derived from Aconitum L., affects the central nervous system and peripheral nervous system. However, the underlying mechanism of aconitine-induced neurotoxicity remains unclear. This study investigates the effects and mechanism of aconitine on mitochondrial energy metabolism in SH-SY5Y cells. Results demonstrated that aconitine exposure suppressed cell proliferation and led to an increase in reactive oxygen species (ROS) and excessive lactate dehydrogenase (LDH) release. Aconitine (400 μmol/L) induced abnormal mitochondrial energy metabolism that quantified by the significant decrease in ATP production, basal respiration, proton leak, maximal respiration, and succinate dehydrogenase (SDH) activity. Phosphorylation of AMPK was significantly reduced in aconitine-treated SH-SY5Y cells. The AMPK activator AIACR pretreatment effectively promoted ATP production to ameliorate mitochondrial energy metabolism disorder caused by aconitine. Mitochondrial biosynthesis was inhibited after treatment with 400 μmol/L aconitine, which was characterized by mitochondria number, TFAM expression, and mtDNA copy number. Moreover, aconitine prompted the down-regulation of mitochondrial fusion proteins OPA1, Mfn1 and Mfn2, and the up-regulation of mitochondrial fission proteins p-Drp1 and p-Mff. These results suggest that aconitine induces mitochondrial energy metabolism dysfunction in SH-SY5Y cells, which may involve the inhibition of AMPK signaling and abnormal mitochondrial dynamics., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aconitine (AC) is well-known as the main toxic ingredient and active compound of Aconitum species, of which several aconites are essential herbal medicines of Traditional Chinese Medicine (TCM) and widely applied to treat diverse diseases for their excellent anti-inflammatory, analgesic, and cardiotonic effects. However, the cardiotoxicity and neurotoxicity of AC attracted a lot of attention and made it a favorite botanic poison in history. Nowadays, the narrow therapeutic window of AC limits the clinical application of AC-containing herbal medicines; overdosing on AC always induces ventricular tachyarrhythmia and heart arrest, both of which are potentially lethal. But the underlying cardiotoxic mechanisms remained chaos. Recently, beyond its cardiotoxic effects, emerging evidence shows that low doses of AC or its metabolites could generate cardioprotective effects and are necessary to aconite's clinical efficacy. Consistent with TCM's theory that even toxic substances are powerful medicines, AC thus could not be simply identified as a toxicant or a drug. To prevent cardiotoxicity while digging the unique value of AC in cardiac pharmacology, there exists a huge urge to better know the characteristic of AC being a cardiotoxic agent or a potential heart drug. Here, this article reviews the advances of AC metabolism and focuses on the latest mechanistic findings of cardiac efficacy and toxicity of this aconite alkaloid or its metabolites. We also discuss how to prevent AC-related cardiotoxicity, as well as the issues before the development of AC-based medicines that should be solved, to provide new insight into the paradoxical nature of this ancient poison., (© 2021 Wiley Periodicals LLC.)

Medicinal plants of the genus Aconitum are one of the most commonly used herbs in traditional medicine in East Asia to treat conditions related to the heart, pain, or inflammation. However, these herbs are also dangerous as accidental poisoning due to misuse is a recurring issue. These plants contain a number of diester-diterpenoid alkaloid compounds and aconitine is the most abundant and active one. This study investigated neurotoxicity of aconitine to zebrafish embryos in early development in relation to serotonin regulation. Experimental results showed that aconitine exposure (1, 10, and 100 μM) increased frequency of coiling behavior in zebrafish embryos in a dose-dependent manner and this effect can be triggered by either exposure to 5-hydroxytryptamine 1A (5-HT1A) receptor agonist (±)-8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) or overexpression of serotonin receptor 5-htr1ab. At the same time, coiling behavior caused by aconitine exposure could be rescued by co-exposure to 5-HT1A receptor antagonist WAY-100635 Maleate (WAY100635) and knockdown of 5-htr1ab using morpholino. Exposure to aconitine also significantly increased serotonin receptor 5-htr1ab and 5-htr1bd gene expression at 24 h post fertilization (hpf), but decreased their expression and protein expression of the serotonin receptor at 96 hpf with the high dose. These results suggest that neurotoxicity caused by aconitine is mediated through the 5-HT receptor., (© 2020 John Wiley & Sons, Ltd.)

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 μg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 μM) or MACO (0.1, 0.3 μM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an I Na blocker tetrodotoxin (2 μM) and partly abolished by a specific Na + /K + pump (NKP) blocker ouabain (0.1 μM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (I Na/K ) and increased peak I Na by accelerating the sodium channel activation with the EC 50 of 8.36 ± 1.89 and 1.33 ± 0.16 μM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na + and Ca 2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak I Na via accelerating sodium channel activation and inhibiting the I Na/K . These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

BACKGROUND Accumulating evidence suggests that cardiotoxicity is one of the main manifestations of aconitine (AC) poisoning. However, the molecular mechanism of AC-induced cardiotoxicity remains unclear, there is little direct evidence for therapeutic targets and drugs of AC-induced cardiotoxicity. MATERIAL AND METHODS Zebrafish were exposed to AC to evaluate cardiotoxicity by calculating the heart rates and observing the changes of cardiac and vascular structure. RNA-seq (RNA sequencing) and bioinformatics analysis were used to obtain differentially expressed genes (DEGs). The anti-AC cardiotoxicity compound was identified via connectivity map (CMAP) analysis and molecular docking. RESULTS AC-induced cardiotoxicity in zebrafish predominantly included arrhythmias, extended sinus venous and bulbus arteriosus (SV-BA) distance, and larger pericardial edema aera. A total of 1380 DEGs were identified by RNA-seq and bioinformatics analysis. cyclin-dependent kinase-1 (CDK1) was screened as the hub gene and the most potential therapeutic target due to its significant downregulation in cardiotoxicity based on protein-protein interaction (PPI) and drug-gene interaction (DGIdb) network analysis. Cell cycle signal pathway was the most significant pathways identified in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, the expression of CDK1 was validated in the Gene Expression Omnibus (GEO) database GSE71906, GSE65705, and GSE95140. Finally, heptaminol was identified as a novel anti-AC cardiotoxicity compound via CMAP analysis and molecular docking. CONCLUSIONS Totally, hub genes and key pathways identified in this study can aid in the understanding of the molecular changes in AC-induced cardiotoxicity. Meanwhile, we provide a systematic method to explore drug toxicity prevention and treatment.

Fuzi, the processed lateral roots of Aconitum carmichaelii Debx., is a traditional herbal medicine that is well known for its excellent pharmacological effects and acute toxicity. Aconitine is one of the diester-diterpene alkaloids and well-known for its arrhythmogenic effects. However, the effects of aconitine in zebrafish have rarely been studied. Therefore, we investigated the effects of aconitine on zebrafish embryos and H9c2 cells. Zebrafish embryos at 48 hours postfertilization were exposed to aconitine, and then, cardiac function and apoptosis were measured. Through transcriptomic analysis, the cardiotoxicity of aconitine in zebrafish embryos was involved in regulating Ca 2+ signal pathways. A reverse transcription-polymerase chain reaction was performed to verify the expression of Ca 2+ pathway-related genes after 12, 24, 36 and 48 hours of treatment. Meanwhile, intracellular Ca 2+ concentrations and cell apoptosis were observed in H9c2 cells treated with half-maximal inhibitory concentration values of aconitine for 30 minutes. The protein levels of troponin T (TnT), caspase 3, Bcl-2 and Bax were detected by western blot analysis. In vivo, 2.0 and 8.0 μm aconitine decreased the heart rate and inhibited the contraction of ventricles and atria in a dose- and time-dependent manner. Furthermore, aconitine increased expression of cacna1c, RYR2, atp2a2b, Myh6, troponin C, p38, caspase 3, Bcl-2 and Bax for 12 hours. In vitro, 1.5 and 4.5 mm aconitine caused intracellular Ca 2+ ion oscillation, increased rates of apoptosis, inhibited TnT and Bcl-2 protein expression, and promoted caspase 3 and Bax protein expression. These data confirmed that aconitine at various concentrations induced cardiac dysfunction and apoptosis were related to the Ca 2+ signaling pathway., (© 2020 John Wiley & Sons, Ltd.)

Despite the high profile of aconine in WuTou injection, there has been no preparative technology or structural studies of its salt as the pharmaceutical product. The lack of any halide salt forms is surprising as aconine contains a tertiary nitrogen atom. In this work, aconine was prepared from the degradation of aconitine in Aconiti kusnezoffii radix (CaoWu). A green chemistry technique was applied to enrich the lipophilic-poor aconine. Reaction of aconine with hydrochloride acid resulted in protonation of the nitrogen atom and gave a novel salt form (C 25 H 42 NO 9 + ·Cl - ·H 2 O; aconine hydrochloride monohydrate, AHM), whose cation in the crystal structure was elucidated based on extensive spectroscopic and X-ray crystallographic analyses. The AHM crystal had a Z' = 3 structure with three independent cation-anion pairs, with profound conformational differences among the aconine cations. The central framework of each aconine cation was compared with that of previously reported aconitine, proving that protonation of the nitrogen atom induced the structure rearrangement. In the crystal of AHM, aconine cations, chloride anions and water molecules interacted through inter-species O-H...Cl and O-H...O hydrogen bonds; this complex hydrogen-bonding network stabilizes the supramolecular structure. The seriously disordered solvent molecules were treated using the PLATON SQUEEZE procedure [Spek (2015). Acta Cryst. C71, 9-18] and their atoms were therefore omitted from the refinement. Bioactivity studies indicated that AHM promoted in vitro proliferative activities of RAW264.7 cells. Molecular docking suggested AHM could target cardiotoxic protein through the hydrogen-bonding interactions. The structural confirmation of AHM offers a rational approach for improving the pharmaceutical technology of WuTou injection.

Objectives: Aconitine, the natural product extracted from Aconitum species, is widely used for the treatment of various diseases, including rheumatism, arthritis, bruises, fractures and pains. However, many studies have reported cardiotoxicity and neurotoxicity caused by aconitine, but the detailed mechanism underlying aconitine's effect on these processes remains unclear., Materials and Methods: The effects of aconitine on the inflammation, apoptosis and viability of H9c2 rat cardiomyocytes were evaluated by flow cytometry, Western blot, RNA sequencing and bioinformatics analysis., Results: Aconitine suppressed cardiomyocyte proliferation and induced inflammation and apoptosis in a dose- and time-dependent manner. These inflammatory damages could be reversed by a TNFα inhibitor and BNIP3-mediated mitophagy. Consistent with the in vitro results, overexpression of BNIP3 in heart tissue partially suppressed the cardiotoxicity of aconitine by inhibiting apoptosis and the NLRP3 inflammasome., Conclusions: Our findings lay a foundation for the application of a TNFα inhibitor and BNIP3 to aconitine-induced cardiac toxicity prevention and therapy, thereby demonstrating potential for further investigation., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Ethnopharmacological Relevance: CaoWu (Aconiti Kusnezoffii Radix), well known for its high toxicity leading to fatal ventricular arrhythmias, is detoxified by HeZi (Terminalia Chebula Retz) decoction to prepare ZhiCaoWu (Aconiti Kusnezoffii Radix Preparata) as one part of ingredients of NaRu-3 pill which is used for the treatment of rheumatoid arthritis (RA). Aconitine (AC) is a highly toxic alkaloid of CaoWu and it is used as toxic target marker for the quality control (QC) of ZhiCaoWu. In the traditional processing method, the vanish of astringent or spicy feeling in tongue is the important detoxification indicator of ZhiCaoWu. However, how CaoWu is detoxified to ZhiCaoWu and whether the appropriate content of AC in ZhiCaoWu can be efficiently perceived after the empirical detoxification still lack factual basis., Aim of the Study: The present study aimed to optimize the traditional processing method for precision detoxification of CaoWu through biomimetic linking kinetics and human toxicokinetics (TK) of AC, with a view of providing insights into the changes of toxic target marker., Materials and Methods: CaoWu medicinal slices (Mes) and coarse powder (Cop) were processed by blank HeZi decoction through the soaking method for 7 days. High-performance liquid chromatography (HPLC) was used for the analysis of the samples. The acidity of blank HeZi decoction and HeZi processing decoction was directly determined by pH meter. The non-compartment analysis (NCA) was used to have an intuitive appreciation for AC and pH changes in HeZi processing decoction while the compartment model method was used to build the biomimetic linking kinetics model with the covariate. The inter-species scaling of animal TK parameters was conducted to predict human AC TK profiles. The possible uptake ways of AC (rapid-release or extended-release) for humans were attempted to assess the poisoning risk of AC in NaRu-3 pill. Based on the target content of AC in ZhiCaoWu, the biomimetic linking kinetics model was explored to optimize the traditional processing detoxification method of CaoWu. The assays of determining inflammatory cytokines in lipopolysaccharides (LPS)-induced RAW264.7 cells were performed to investigate the inflammatory modulation effects of AC in vitro., Results: ZhiCaoWu was prepared by eliminating redundant AC in CaoWu through the repeatable replacement of HeZi processing decoction in which its acidity (pH) was affected. AC-pH changes in HeZi processing decoction were adequately depicted by a biomimetic linking kinetics model whose predictive power was determined by comparing the predictions of AC in ZhiCaoWu with the reported data. Rapid-release AC at the converted dose of 111.1 and 417.6 μg (0.011 and 0.042% of AC in NaRu-3 pill) reached maximum blood concentrations of 26.1 and 98.1 ng/mL at 0.3 h, in comparison with minimum human lethal concentration (100 ng/mL). Achieving the target content of AC (0.04%) in ZhiCaoWu or AC (0.011%) in NaRu-3 pill to precisely control the poisoning risk, the potential optimized protocols were that the processing time at 0.2-0.8% of AC in CaoWu was 2.0-4.4 days for Cop and 2.7-6.2 days for Mes. Correspondingly, pH values in HeZi processing decoction were 3.95 and 3.77 for Cop and Mes, respectively. Meanwhile, Lipopolysaccharides (LPS)-induced RAW264.7 cells were exposed to 0, 20, and 200 μM of AC for 12 h and AC at 20 μM enhanced the levels of IL-6, IL-10 and TNF-α., Conclusions: Thus, for the first time, a biomimetic linking kinetics model was built to optimize the traditional detoxification method. Moreover, pH changes could be developed as surrogate endpoint for guiding the processing detoxification of CaoWu. Notably, setting the content limit of AC (0.011%) was very rational to control the poisoning risk of NaRu-3 pill. In addition, it was possible that there existed the more complex mechanisms of AC for inflammatory modulation in vitro., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The brown planthopper, Nilaparvata lugens (Stål), severely damages rice production and develops high level resistance to several classes of insecticides. To find potential insecticidal resources is always important. As an environmentally friendly compound, aconitine exhibits potential pesticide features. In the present study, the pesticide and knockdown effects of aconitine were first tested on the brown planthopper. The results showed that the knockdown rates for an aconitine concentration of 200 ppm was 83.6%. The insecticidal LD50 was 22.68 ng/pest (95% CI, 17.75-28.99). The molecular mechanisms responding to aconitine application were analyzed through transcriptional sequencing. Compared to that of the knockdown nymphs of the brown planthoppers, the enzymes CYP3A4, UDP-glucuronosyltransferase (UGT), GST, carboxylesterase (EC3.1.1.1), and GABAergic synapse were up-regulated. We inferred that aconitine might be neurotoxic to the brown planthoppers, and the conscious nymphs resist the drug neurotoxicity through the upregulation of CYP3A4, UGT, and GABA receptor mutation. Although aconitine is not safe for mammals, it may be a leading compound to develop novel insecticides., Competing Interests: The authors have declared that no competing interests exist.

Hypaconitine is an active and highly toxic constituent derived from Aconitum species. Here we aimed to determine the chronotoxicity of hypaconitine in mice, and to investigate a potential role of metabolism in hypaconitine chronotoxicity. Cardiac toxicity was assessed by measuring CK (creatine kinase) and LDH (lactate dehydrogenase) levels after hypaconitine administration to wild-type and Bmal1 -/- (a clock disrupted model) mice at different times of day. The mRNA and protein levels of Cyp3a11 in mouse livers were determined by qPCR and western blotting, respectively. In vitro metabolism was assessed using liver microsomes. Pharmacokinetic study of hypaconitine was performed with wild-type mice. We observed injection time-dependent toxicity (i.e., a more severe toxicity during the light phase than the dark phase) for hypaconitine in mice. The chronotoxicity was attributed to a difference in systemic exposure of hypaconitine caused by time of day-dependent metabolism. Furthermore, circadian metabolism of hypaconitine was accounted for by the diurnal expression of Cyp3a11, a major enzyme for hypaconitine detoxification in the liver. Moreover, Bmal1 ablation in mice abolished the daily rhythm of Cyp3a11 expression and abrogated the time-dependency of hypaconitine toxicity. In conclusion, circadian Cyp3a11 metabolism contributed to chronotoxicity of hypaconitine in mice. This metabolism-based chronotoxicity would facilitate the formulation of best timing for drug administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aconitine, a major aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias and neurological symptoms. One mechanism of aconitine-induced toxic responses is the induction of apoptosis. Apoptosis and autophagy are interconnected processes and the two pathways share critical components. In this study, we investigated the role of autophagy in aconitine-induced toxicity using mouse model. 120 mice were randomly divided into 4 experimental groups (normal saline), low dose group (0.14 μmol/L), medium dose group (0.28 μmol/L) and high dose group (0.56 μmol/ L). 30 mice in each group were administered with aconitine (lavage) for 30 days. The livers were collected for analysis of autophagy-related proteins by Western blotting. The expression of LC3II/LC3I ratio and Beclin 1 were found to increase and then decrease with the highest expression at 10 days and the p62 showed a time-dependent decreases. Autophagy is regulated by the mTOR pathway, we further analyzed the effects of aconitine on this pathway and found aconitine inhibited, phosphorylation of p-PI3K, p-Akt and p-mTOR. The p-p70s6k and p-4EBP1 which are downstream of mTOR were concomitantly decreased. These results suggest that aconitine induce autophagy in mouse liver. The PI3K/Akt/mTOR signaling pathway is involved in the regulation of aconitine-induced autophagy in the liver of mice., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aconitine (AC) and mesaconitine (MA) are major bioactive diterpenoid alkaloids derived from herbal aconitum plants. Emerging evidence indicates that AC plays a pivotal role in the cardiotoxicity for aconite poisoning. However, the cardiotoxicity data of MA, especially those on the difference between AC and MA are quite limited. Zebrafish embryos were used in this study for toxicological screening, and the cardiac morphology and function were observed. Embryos were analyzed by means of high-performance liquid chromatography (HPLC) after exposure and pharmacokinetic behaviors were also investigated. Results showed that 1.5% of the aconitum alkaloids penetrated into the zebrafish embryos. 2.5 μg/L AC and 20 μg/L MA caused a deficient cardiovascular system with yolk sac hemorrhage and early cardiac dysfunctions were observed in 96 h post-fertilization. AC showed greater cardiotoxicity than MA by comparing the EC 50 of pericardium edema. Aconitum alkaloids exposure also resulted in a significant decrease in the expression of cardiac genes (Tbx5, Gata4, and Nkx2.5) from an early stage (12-24 hpf), which may partly explained that the death caused by aconitum is most likely to occur within the first 24 h. In addition, a high percentage of apoptotic cells was observed in the brain region, which identified another potential target of the DDA action in zebrafish embryos., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Larkspurs (Delphinium spp.) are native perennial plants that have a serious toxic potential to cattle on foothill and mountain rangelands in the western United States. Livestock death due to larkspur toxicity is attributed to norditerpenoid alkaloids. Diagnosing plant poisonings in livestock is often challenging. The objective of this study was to evaluate the use of three matrices; earwax, oral fluid, and nasal mucus, as noninvasive specimens to determine livestock exposure to larkspurs. Reversed phase high performance liquid chromatography - high resolution mass spectrometry was used to analyze for norditerpene alkaloids, in all three matrices, in cattle administered a single dose of larkspur. Earwax, oral fluid, and nasal mucus were collected over 6 days post-dosing. Methyllycaconitine (MLA) and deltaline concentrations in earwax ranged from 0.4 ± 0.1 to 0.2 ± 0.06 and 0.6 ± 0.5 to 0.11 ± 0.08 ng/mg, respectively. MLA and deltaline concentrations in oral fluid ranged from 0.08 ± 0.03 to 0.01 ± 0.002 ng/mg and 0.07 ± 0.03 ng/mg to not detected (ND), respectively. MLA and deltaline concentrations in nasal mucus ranged from 0.2 ± 0.13 to 0.03 ± 0.01 ng/mg and 0.2 ± 0.12 ng/mg to ND, respectively. The ability to detect differing norditerpene alkaloid chemotypes from two different Delphinium spp. was also possible in the three matrices. This study demonstrates the potential of earwax, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by larkspur plants., (Published by Elsevier Ltd.)

Through the comprehensive and systematic research of domestic and overseas literature and information, we studied ancient original records on Aconiti Kusnezoffii Radix and its toxicity, analyzed related adverse cases and the animal toxicity experiments in recent years, then systematically analyzed the safety of Aconitum and its preparations, and finally we summarized the clinical characteristics and potential risk factors related to the safety of Aconitum. A report on adverse events of Aconitum in 76 patients with myocardial damage and renal damage accounting for 53.9% and 42.1% respectively, indicated that the safety problems of Aconitum may be related to heart toxicity and liver-kidney toxicity. Aconitum had complex compositions, and based on the animal experiments, Aconitum decoction had the highest toxicity at 2 h, and it reduced significantly at 4 h, which showed that the toxic components mainly depend on the hydrolysis or the decomposition degree of diester diterpenoid alkaloids. According to the toxicity study, Aconitum toxicity might occur in cardiovascular system, nervous system, kidney, embryo, reproductive system, and it was contraindicated in pregnant women. So far, specific antidote for aconitine poisoning is still a blank. The key for treatment is to correct arrhythmia timely and effectively, maintain stable vital signs, and meanwhile, give gastric lavage, intravenous fluid infusion and other therapies. So we suggest that the basic study for Aconitum toxicology should be strengthened, and the pharmacology and mechanism of toxicity, as well as the mechanism of processing for raising efficiency and reducing toxicity, should be further clarified to determine the quantity-effect relationship and eliminate safety hazards in using Aconitum., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Poisoning of cattle by larkspur plants (Delphinium spp.) is a concern for cattle ranchers in western North America. Previous research studies have evaluated the toxicokinetic profile of multiple larkspur toxins in several livestock species. However, those studies were all performed by orally dosing plant material. Consequently some toxicokinetic parameters could not be definitively determined. In this study, we compared the serum toxicokinetic profile of the larkspur alkaloids methyllycaconitine (MLA) and deltaline in goats dosed both IV and via oral gavage. The results from this study indicate that the toxic alkaloids in larkspurs undergo flip-flop kinetics, meaning the rate of absorption of the alkaloids is slower than the rate of elimination. The implications of flip-flop kinetics in treating animals poisoned by larkspur is discussed., (Published by Elsevier Ltd.)

Aconitine (AC) is the primary bioactive/toxic alkaloid in plants of the Aconitum species. Our previous study demonstrated that Mdr1 was involved in efflux of AC. However, the mechanism by which Mdr1 regulates the efficacy/toxicity of AC in vivo remains unclear. The present study aimed to determine the effects of Mdr1a on the efficacy/toxicity and pharmacokinetics of AC in wild-type and Mdr1a -/- FVB mice. After oral administration of AC, significantly higher analgesic effect was observed in Mdr1a -/- mice (49% to 105%) compared to wild-type mice (P<0.05). The levels of s100-β protein and creatine kinase, which indicate cerebral and myocardial damage, respectively, were also significantly increased (P<0.05) in Mdr1a -/- mice. Histopathological examination revealed that the Mdr1a -/- mice suffered from evident cerebral and myocardial damages, but the wild-type mice did not. These findings suggested that Mdr1a deficiency significantly promoted the analgesic effect of AC and exacerbated its toxicity. Pharmacokinetic experiments showed that T 1/2 of AC in the Mdr1a -/- mice was significantly higher (from 87% to 300%) than that in wild-type mice (P<0.05). The distribution of AC in the brain of Mdr1a -/- mice was 2- to 32-fold higher than that in the brains of wild-type mice (P<0.05). Toxic reactions were more severe in Mdr1a -/- mice compared to wild-type mice. In conclusion, Mdr1a deficiency significantly enhanced the analgesic effect of AC and exacerbated its toxicity by upregulating its distribution to the brain and decreasing its plasma elimination rate. Thus, Mdr1a dysfunction may cause severe AC poisoning., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aconitine (ACO) is well-known for causing lethal ventricular tachyarrhythmias. While cardiac Na+ channel opening during repolarization has long been documented in animal cardiac myocytes, the cellular effects and mechanism of ACO in human remain unexplored. This study aimed to assess the proarrhythmic effects of ACO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACO concentration-dependently (0.3 ~ 3.0 μM) shortened the action potentials (AP) durations (APD) in ventricular-like hiPSC-CMs by > 40% and induced delayed after-depolarization. Laser-scanning confocal calcium imaging analysis showed that ACO decreased the duration and amplitude of [Ca2+]i transients and increased in the beating frequencies by over 60%. Moreover, ACO was found to markedly reduce the L-type calcium channel (LTCC) currents (ICa,L) in hiPSC-CMs associated with a positive-shift of activation and a negative shift of inactivation. ACO failed to alter the peak and late Na+ currents (INa) in hiPSC-CMs while it drastically increased the late INa in Guinea-pig ventricular myocytes associated with enhanced activation/delayed inactivation of INa at -55 mV~ -85 mV. Further, the effects of ACO on ICa,L, INa and the rapid delayed rectifier potassium current (Ikr) were validated in heterologous expression systems by automated voltage-clamping assays and a moderate suppression of Ikr was observed in addition to concentration-dependent ICa,L inhibition. Lastly, increased beating frequency, decreased Ca2+ wave and shortened field potential duration were recorded from hiPSC-CMs by microelectrode arrays assay. In summary, our data demonstrated that LTCC inhibition could play a main role in the proarrhythmic action of ACO in human cardiomyocytes., Competing Interests: The authors have declared that no competing interests exist.

The relationship between atrial fibrillation (AF) and flap survival has not been fully characterized. Therefore, the goal of this study was to investigate the effect of AF on survival areas of pedicled flap and survival rates of free flap in an experimental rat AF model. An aconitine-induced rat AF model was established without intubation anesthesia. Survival areas of the pedicled rectangular epigastric flap were compared between AF rats (n = 7) and control rats (n = 7), and survival rates of the free epigastric flap were compared between AF rats (n = 10) and control rats (n = 10). Animals that died during the study or in which AF was not induced were excluded from study. A total of 64 rats were assessed in this study. Atrial fibrillation was induced with a success rate of 77.8% (21/27) throughout the study. Pedicled flap survival area was significantly higher in controls (75.1 ± 9.0%; n = 7) than that in AF animals (55.7 ± 13.0%; n = 7) (P < 0.01, nonpaired Student t test). Free flap survival rates were 80% in controls and 40% in AF animals (P = 0.07, χ² test). This is the first study to develop an aconitine-induced model of AF in rats. Atrial fibrillation has a detrimental effect on survival areas of the pedicled flap and survival rates of the free flap.

It is well known that various traditional Chinese medicines produce antiarrhythmic actions. The aims of this study were to examine whether total flavones derived from Choerospondias axillaris folium (TFCF) also produced antiarrhythmic effects using a rat model of aconitine-induced arrhythmia and to compare these observations with the effects of total flavones of Choerospondias axillaris fructus (TFC). Wistar rats were orally administered TFC (0.2 g/kg) or TFCF (0.1, 0.2, or 0.4 g/kg) daily for 7 d. Subsequently, aconitine iv at 25 µg/kg was used to induce arrhythmia in these animals. Control (C) physiological saline and positive verapamil rats were also administered orally. The starting times of ventricular ectopic beats (VE), ventricular tachycardia (VT), ventricular fibrillation (VF), and heart arrest (HA) were recorded. In comparison to C, TFCF and TFC significantly prolonged the starting time of VE, VT, VF, and HA induced by aconitine. With respect to hemodynamics, TFC and high-dose TFCF were effective in reducing HR without associated changes in BP in all groups. TFC and TFCF decreased left ventricular systolic pressure (LVSP) and maximal velocity rate of ventricular pressure (+dp/dt max and -dp/dt min) with no marked effect on left ventricular end diastolic pressure (LVEDP) and -dp/dtmin. Data demonstrated that TFCF and TFC were equally effective in diminishing the aconitine-mediated arrhythmias. In addition, TFCF and TFC produced a similar reduction in HR with no accompanying change in BP. These findings indicate that the TFCF- and TFC-induced alterations may be attributed to inhibition of ventricular contraction without altering ventricular diastolic function.

Seizures are associated with altered autonomic activity, which has been implicated in the development of cardiac dysfunction and structural damage. This study aimed to investigate the involvement of the autonomic nervous system in seizure-induced cardiomyopathy. Male Sprague-Dawley rats (320-350 g) were implanted with EEG/ECG electrodes to allow simultaneous telemetric recordings during seizures induced by intrahippocampal (2 nmol, 1 μl/min) kainic acid and monitored for 7 days. Seizure activity occurred in conjunction with increased heart rate (20%), blood pressure (25%), and QTc prolongation (15%). This increased sympathetic activity was confirmed by the presence of raised plasma noradrenaline levels at 3 h post-seizure induction. By 48 h post-seizure induction, sympathovagal balance was shifted in favor of sympathetic dominance, as indicated by both heart rate variability (LF/HF ratio of 3.5 ± 1.0) and pharmacological autonomic blockade. Functional cardiac deficits were evident at 7 and 28 days, as demonstrated by echocardiography showing a decreased ejection fraction (14% compared with control, P < 0.05) and dilated cardiomyopathy present at 28 days following seizure induction. Histological changes, including cardiomyocyte vacuolization, cardiac fibrosis, and inflammatory cell infiltration, were evident within 48 h of seizure induction and remained present for up to 28 days. These structural changes most probably contributed to an increased susceptibility to aconitine-induced arrhythmias. This study confirms that prolonged seizure activity results in acute and chronic alterations in cardiovascular control, leading to a deterioration in cardiac structure and function. This study further supports the need for modulation of sympathetic activity as a promising therapeutic approach in seizure-induced cardiomyopathy., (Copyright © 2015 the American Physiological Society.)

Sodium channels, found ubiquitously in animal muscle cells and neurons, are one of the main target sites of many naturally-occurring, insecticidal plant compounds and agricultural pesticides. Pyrethroids, derived from compounds found only in the Asteraceae, are particularly toxic to insects and have been successfully used as pesticides including on flowering crops that are visited by pollinators. Pyrethrins, from which they were derived, occur naturally in the nectar of some flowering plant species. We know relatively little about how such compounds--i.e., compounds that target sodium channels--influence pollinators at low or sub-lethal doses. Here, we exposed individual adult forager honeybees to several compounds that bind to sodium channels to identify whether these compounds affect motor function. Using an assay previously developed to identify the effect of drugs and toxins on individual bees, we investigated how acute exposure to 10 ng doses (1 ppm) of the pyrethroid insecticides (cyfluthrin, tau-fluvalinate, allethrin and permethrin) and the nectar toxins (aconitine and grayanotoxin I) affected honeybee locomotion, grooming and wing fanning behaviour. Bees exposed to these compounds spent more time upside down and fanning their wings. They also had longer bouts of standing still. Bees exposed to the nectar toxin, aconitine, and the pyrethroid, allethrin, also spent less time grooming their antennae. We also found that the concentration of the nectar toxin, grayanotoxin I (GTX), fed to bees affected the time spent upside down (i.e., failure to perform the righting reflex). Our data show that low doses of pyrethroids and other nectar toxins that target sodium channels mainly influence motor function through their effect on the righting reflex of adult worker honeybees.

Aconite species have played an important role in human history. Aconitum species have been used worldwide as poisons as well as remedies. Their potential in targeting several ailments such as pain, rheumatism, and lethargy has been recognized by Western, Chinese, and Indian health care practitioners. Aconite use in herbal preparations has declined, especially in Europe and the United States, in the first half of the twentieth century due to several reported toxicity cases. The situation has changed with the application of new technologies for the accurate analysis of its toxic components and the development of efficient detoxification protocols. Some Asian countries started small clinical trials to evaluate the potency and safety of different marketed aconite preparations. The current review summarizes therapeutic uses of aconite preparations in China, Taiwan, India, and Japan. It also highlights clinical trial results with special emphasis on their limitations. Modern drugs and pharmacopoeial preparations derived from aconite are also discussed., (Georg Thieme Verlag KG Stuttgart · New York.)

Larkspurs ( spp.) are one of the most serious toxic plant problems on foothill and mountain rangelands in the western United States. A considerable amount of research has been conducted over the years in both field and pen settings. The results of these research efforts have significantly increased our understanding of the poisoning of cattle by larkspurs. However, most of the pen studies conducted thus far have used a dosing regimen of a single bolus dose, which does not accurately mimic the manner by which cattle are poisoned by larkspur while grazing. Consequently, the objective of this study was to evaluate the acute toxicity of tall larkspur ( collected near Manti, UT) when administered in multiple doses, with the intent to identify a no observable adverse effect level (NOAEL). The adverse effect selected for this study was muscle weakness to the point the cattle could no longer remain ambulatory as would be required in a grazing environment, thus becoming sternally recumbent when exercised. Hereford steers were administered various doses of tall larkspur at 12-h intervals for 4 d or until they showed marked signs of muscle weakness. The results suggest that a dose of 2 mg kg∙d -(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids is the NOAEL for a tall larkspur population with a norditerpenoid alkaloid profile containing 4 mg MSAL-type alkaloids/g plant material and 12 mg non-MSAL-type alkaloids/g plant material. Additionally, a computer model was generated to simulate multiple-dosing regimens at the various doses as well as different dosing regimens. The results from this study suggest that a 500-kg steer can consume a daily dose of 1.25 kg of fresh tall larkspur (with a similar alkaloid profile) without becoming severely poisoned (suffering from muscle weakness to the point of recumbency). Additionally, these results indicate that a serum concentration of approximately 355 ng methyllycaconitine/mL may represent a toxic threshold.

Context: Diesel exhaust (DE) has been shown to increase the risk of cardiac arrhythmias. Although biodiesel has been proposed as a "safer" alternative to diesel, it is still uncertain whether it actually poses less threat., Objective: We hypothesized that exposure to pure or 20% soy biodiesel exhaust (BDE) would cause less sensitivity to aconitine-induced arrhythmia than DE in rats., Methods: Spontaneously hypertensive (SH) rats implanted with radiotelemeters were exposed once or for 5 d (4 h) to either 50 mg/m(3) (low), 150 mg/m(3) (medium), or 500 mg/m(3) (high) of DE (B0), 20% (B20) or 100% (B100) soy biodiesel exhaust. Arrhythmogenesis was assessed 24 h later by continuous infusion of aconitine, an arrhythmogenic drug, while heart rate (HR), and electrocardiogram (ECG) were monitored., Results: Rats exposed once or for 5 d to low, medium, or high B0 developed arrhythmia at significantly lower doses of aconitine than controls, whereas rats exposed to B20 were only consistently sensitive after 5 d of the high concentration. B100 caused mild arrhythmia sensitivity at the low concentration, only after 5 d of exposure at the medium concentration and after either a single or 5 d at the high concentration., Discussion and Conclusions: These data demonstrate that exposure to B20 causes less sensitivity to arrhythmia than B0 and B100. This diminished effect may be due to lower irritant components such as acrolein and nitrogen oxides. Thus, in terms of cardiac health, B20 may be a safer option than both of the pure forms.

This study compared transdermal aconitine delivery using solid lipid nanoparticles (SLN) and microemulsion (ME) vehicles. Aconitine-loaded SLN and ME were formulated with the same surfactant, cosurfactant, and water content, with an equal amount of oil matrix (ATO 888 for SLN and ethyl oleate for ME). These nanosized formulations (70-90 nm) showed suitable pH values and satisfactory skin tissue biocompatibility. SLN contained a higher concentration of smaller nanoparticles, compared with that in ME. Neither of the nanocarriers penetrated across excised skin in their intact form. In vitro transdermal delivery studies found that transdermal aconitine flux was lower from SLN than from ME (p < 0.05), but skin aconitine deposition was higher using SLN (p < 0.05). Fluorescence-activated cell sorting indicated that in vitro uptake of fluorescently labeled SLN by human immortalized keratinocyte (HaCaT) cells was greater than that of ME, indicating that a transcellular pathway may contribute to cutaneous drug absorption more effectively from SLN. In vivo studies found that these formulations could loosen stratum corneum layers and increase skin surface crannies, which may also enhance transdermal aconitine delivery. SLN produced a more sustained aconitine release, indicating that compared with ME, this transdermal delivery vehicle may reduce the toxicity of this drug., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Aconitine is a major bioactive diterpenoid alkaloid with high content derived from herbal aconitum plants. Emerging evidence indicates that voltage-dependent Na(+) channels have pivotal roles in the cardiotoxicity of aconitine. However, no reports are available on the role of Ca(2+) in aconitine poisoning. In this study, we explored the importance of pathological Ca(2+) signaling in aconitine poisoning in vitro and in vivo. We found that Ca(2+) overload lead to accelerated beating rhythm in adult rat ventricular myocytes and caused arrhythmia in conscious freely moving rats. To investigate effects of aconitine on myocardial injury, we performed cytotoxicity assay in neonatal rat ventricular myocytes (NRVMs), as well as measured lactate dehydrogenase level in the culture medium of NRVMs and activities of serum cardiac enzymes in rats. The results showed that aconitine resulted in myocardial injury and reduced NRVMs viability dose-dependently. To confirm the pro-apoptotic effects, we performed flow cytometric detection, cardiac histology, transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The results showed that aconitine stimulated apoptosis time-dependently. The expression analysis of Ca(2+) handling proteins demonstrated that aconitine promoted Ca(2+) overload through the expression regulation of Ca(2+) handling proteins. The expression analysis of apoptosis-related proteins revealed that pro-apoptotic protein expression was upregulated, and anti-apoptotic protein BCL-2 expression was downregulated. Furthermore, increased phosphorylation of MAPK family members, especially the P-P38/P38 ratio was found in cardiac tissues. Hence, our results suggest that aconitine significantly aggravates Ca(2+) overload and causes arrhythmia and finally promotes apoptotic development via phosphorylation of P38 mitogen-activated protein kinase., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Aconitum carmichaelii Debx. (Fuzi), a commonly use traditional Chinese medicine (TCM), has often been used in combination with Rhizoma Glycyrrhizae (Gancao) to reduce its toxicity due to diester diterpenoid alkaloids aconitine, mesaconitine, and hypaconitine. However, the mechanism of detoxication is still unclear. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizinic acid (GL), the major component of Gancao. In present study, the effect of GA on the changes of metabolic profiles induced by mesaconitine was investigated using NMR-based metabolomic approaches., Materials and Methods: Fifteen male Wistar rats were divided into a control group, a group administered mesaconitine alone, and a group administered mesaconitine with one pretreatment with GA. Their urine samples were used for NMR spectroscopic metabolic profiling. Statistical analyses such as orthogonal projections to latent structures-discriminant analysis (OPLS-DA), t-test, hierarchical cluster, and pathway analysis were used to detect the effects of pretreatment with GA on mesaconitine-induced toxicity., Results: The OPLS-DA score plots showed the metabolic profiles of GA-pretreated rats apparently approach to those of normal rats compared to mesaconitine-induced rats. From the t-test and boxplot results, the concentrations of leucine/isoleucine, lactate, acetate, succinate, trimethylamine (TMA), dimethylglycine (DMG), 2-oxo-glutarate, creatinine/creatine, glycine, hippurate, tyrosine and benzoate were significantly changed in metabolic profiles of mesaconitine-induced rats. The disturbed metabolic pathways include amino acid biosynthesis and metabolism., Conclusions: GA-pretreatment can mitigate the metabolic changes caused by mesaconitine-treatment on rats, indicating that prophylaxis with GA could reduce the toxicity of mesaconitine at the metabolic level., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Aconitine (AC) is a highly toxic alkaloid from bioactive plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. In this study, we systematically evaluated the potential role of P-glycoprotein (P-gp) in the mechanisms underlying the low and variable bioavailability of oral AC. First, the bidirectional transport of AC across Caco-2 and MDCKII-MDR1 cells was investigated. The efflux of AC across monolayers of these two cell lines was greater than its influx. Additionally, the P-gp inhibitors, verapamil and cyclosporin A, significantly decreased the efflux of AC. An in situ intestinal perfusion study in rats showed that verapamil co-perfusion caused a significant increase in the intestinal permeability of AC, from 0.22×10(-5) to 2.85×10(-5) cm/s. Then, the pharmacokinetic profile of orally administered AC with or without pre-treatment with verapamil was determined in rats. With pre-treatment of verapamil, the maximum plasma concentration (Cmax) of AC increased sharply, from 39.43 to 1490.7 ng/ml. Accordingly, a 6.7-fold increase in the area under the plasma concentration-time curve (AUC0-12h) of AC was observed when co-administered with verapamil. In silico docking analyses suggested that AC and verapamil possess similar P-gp recognition mechanisms. This work demonstrated that P-gp is involved in limiting the intestinal absorption of AC and attenuating its toxicity to humans. Our data indicate that potential P-gp-mediated drug-drug interactions should be considered carefully in the clinical application of aconite and formulations containing AC., (© 2013.)

The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , β2 and β4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis., (Published 2013. This article is a US Government work and is in the public domain in the USA.)

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)