PGC 1α-Mediates Mitochondrial Damage in the Liver by Inhibiting the Mitochondrial Respiratory Chain as a Non-cholinergic Mechanism of Repeated Low-Level Soman Exposure.

This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague-Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I-IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I + II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I-IV, I-III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.

Potentially Cardiotoxic Diterpenoid Alkaloids from the Roots of Aconitum carmichaelii.

Aconitum carmichaelii is a traditional Chinese herbal medicine used for the treatment of pain and inflammation in the joints. However, the strong cardiotoxicity hinders its use. Although diester- and monoester-type diterpenoids, e.g., aconitine, mesaconitine, and hypacaonitine, are commonly considered as the toxic components, the toxicity of A. carmichaelii cannot be completely explained by the compounds reported. To investigate further the cardiotoxic compounds and their potential mechanism, the chemical constituents were first isolated by column chromatography and identified using mass spectrometry and NMR spectroscopy. Two new hetisine-type (1 and 2) and four new aconitine-type alkaloids (3-6) were assigned. The cardiac cytotoxicity assessed on H9c2 cells indicated that the new compound 4 as well as six known alkaloids (7 and 9-13) exhibited significant toxicities. A preliminary structure-toxicity relationship study suggested that substitution at C-8 and C-10 both have a significant influence on cardiotoxicity, and such toxicity decreased in the order OBz-8, OBu-8, and OMe-8. The presence of an OH-10 group abolished the toxicity. Finally, it was found that ion channel disorder and induction of mitochondrial-mediated cell apoptosis are the possible mechanisms of cardiotoxicity among the compounds studied.

Qualitative and quantitative analysis of lipo-alkaloids and fatty acids in Aconitum carmichaelii using LC-MS and GC-MS.

INTRODUCTION: Lipo-alkaloid (LA) is a kind of C19 -norditerpenoid alkaloid in Aconitum species, which usually contains an aconitane skeleton and one or two fatty acid residues. OBJECTIVE: To qualify and quantify the fatty acids and lipo-alkaloids in Aconitum carmichaelii. METHODOLOGY: An ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-QQQ-MS) method was established to quantify LAs, while the free fatty acids were identified by gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF-MS). RESULTS: Six major LAs (1-6) containing linoleic, palmitic, and oleic acid residues as side chains were quantified. Eighteen fatty acids were determined by GC-MS, and 15 were detected as the side chains of LAs. The LAs containing these 15 fatty acid residues accounted for about a third of the total identified LAs. Moreover, the contents of linoleic, palmitic, and oleic acids were highest. In addition, 12 oxygenated fatty acids were also identified by UHPLC-Q-TOF-MS for the first time. CONCLUSION: The positive correlation between free fatty acids and LAs in A. carmichaelii indicated that the types and contents of LAs were influenced by free fatty acids.

The adsorption effects of biochar on carbofuran in water and the mixture toxicity of biochar-carbofuran in rats.

Carbofuran, a widely used carbamate insecticide, is frequently detected in water. In this study, a high-performance adsorbent (WAB4) for carbofuran was obtained from laboratory-synthesized biochars. The maximum adsorption of carbofuran by WAB4 reaches 113.7 mg/g approximately. The adsorption of carbofuran by biochar was a multi-molecular layer and the adsorption process conforms to the pseudo-second-order kinetic model (R2 = 0.9984) and Freundlich isotherm model (R2 = 0.99). Importantly, an in vivo rat model was used to assess the combined toxicological effects of biochar-carbofuran complexes. The toxicity of the complexes (LD50 > 12 mg/kg) is lower than that of carbofuran (LD50 = 7.9 mg/kg) alone. The damage of biochar-carbofuran complex on rat liver and lung is significantly less than that of carbofuran. The Cmax and bioavailability of carbofuran were found to be reduced by 64% and 68%, respectively, when biochar was present, by UPLC-MS/MS analysis of carbofuran in rat plasma. Furthermore, it was confirmed that the biochar-carbofuran complex is relatively stable in the gastrointestinal tract, by performing a carbofuran release assay in artificial gastrointestinal fluids in vitro. Collectively, biochar is a bio-friendly material for the removal of carbofuran from water.

Rapid and efficient removal of multiple aqueous pesticides by one-step construction boric acid modified biochar.

Tricyclazole, propiconazole, imidacloprid, and thiamethoxam are commonly used pesticides in paddy fields. It is necessary and practical to remove pesticides from the water environment because the low utilization rate of pesticides will produce residues in the water environment. It is known that there are few studies on the preparation of biochar adsorption pesticides by the walnut shell and few studies on the removal of tricyclazole and propiconazole. Based on this, this paper used the walnut shell as raw material and boric acid as an activator to prepare biochar by the one-step method. The boric acid modified walnut shell biochar (WAB4) with a specific surface area of 640.6 m2 g-1, exhibited the high adsorption capacity of all four pesticides (>70%) at pH 3-9. The adsorption capacities of tricyclazole, propiconazole, imidacloprid, and thiamethoxam were 171.67, 112.27, 156.40, and 137.46 mg g-1, respectively. The adsorption kinetics fitted the pseudo-second-order kinetic model and the adsorption isotherm curves conformed to the Freundlich isotherm model. The adsorption of pesticides by WAB4 was associated with hydrogen bonding, pore filling, hydrophobic effects, and π-π interactions. More significantly, WAB4 has excellent adsorption capacity compared to other adsorbents for real water samples. Finally, walnut shell biochar has no significant acute toxicity to Daphnia magna. This work shows that walnut shell-based biochar has a good effect on the removal of pesticides at a wide range of pH and is economical and safe, providing a new idea for the removal of pesticides in water.

Whole-Transcriptome Analysis of Repeated Low-Level Sarin-Exposed Rat Hippocampus and Identification of Cerna Networks to Investigate the Mechanism of Sarin-Induced Cognitive Impairment.

Sarin is a potent organophosphorus nerve agent that causes cognitive dysfunction, but its underlying molecular mechanisms are poorly understood. In this study, a rat model of repeated low-level sarin exposure was established using the subcutaneous injection of 0.4 × LD50 for 21 consecutive days. Sarin-exposed rats showed persistent learning and memory impairment and reduced hippocampal dendritic spine density. A whole-transcriptome analysis was applied to study the mechanism of sarin-induced cognitive impairment, and a total of 1035 differentially expressed mRNA (DEmRNA), including 44 DEmiRNA, 305 DElncRNA, and 412 DEcircRNA, were found in the hippocampus of sarin-treated rats. According to Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Protein-Protein Interaction (PPI) analysis, these DERNAs were mainly involved in neuronal synaptic plasticity and were related to the pathogenesis of neurodegenerative diseases. The circRNA/lncRNA-miRNA-mRNA ceRNA network was constructed, in which Circ_Fmn1, miR-741-3p, miR-764-3p, miR-871-3p, KIF1A, PTPN11, SYN1, and MT-CO3 formed one circuit, and Circ_Cacna1c, miR-10b-5p, miR-18a-5p, CACNA1C, PRKCD, and RASGRP1 constituted another circuit. The balance between the two circuits was crucial for maintaining synaptic plasticity and may be the regulatory mechanism by which sarin causes cognitive impairment. Our study reveals the ceRNA regulation mechanism of sarin exposure for the first time and provides new insights into the molecular mechanisms of other organophosphorus toxicants.

Toxic effects and bioaccumulation of pinacolyl methylphosphonate acid in zebrafish following soman exposure to a water environment.

Soman has been shown to be highly neurotoxic and can be easily degraded to produce pinacolyl methylphosphonate acid (PMPA). Thus, the perniciousness of PMPA deserved serious attention after soman was exposed to the environment. However, the toxicity of PMPA was not clearly elucidated to date. In this regard, the objective of this study was to determine if PMPA could pose an environmental risk after soman exposure to a water environment. In this study, the toxicity and bioaccumulation assessments of PMPA were carried out on zebrafish. Histological examination was used to assess the toxicity of PMPA in zebrafish and revealed that PMPA has chronic toxicity in view of tissue injury. The contents of PMPA in whole zebrafish and tissues were determined after soman exposure. The result showed that PMPA bioaccumulated in the whole zebrafish and tissue, especially the liver and intestinal tissues. This is the first report showing that the hydrolyzate of a G-series chemical nerve agent could accumulate in organisms. This study offers novel insights into the environmental risk assessments associated with soman exposure to a water environment.

The kisspeptin-GnIH signaling pathway in the role of zebrafish courtship and aggressive behavior induced by azoxystrobin.

Azoxystrobin, a strobilurin widely used to control rice diseases, has raised concerns about possible adverse effects on aquatic ecosystems. At present, very little is known about the effects of azoxystrobin on courtship and aggressive behavior and the potential underlying mechanisms. In the present study, after exposing adult male and female zebrafish to worst-case scenario concentrations of azoxystrobin (0, 2 µg/L, 20 µg/L, and 200 µg/L) for 42 d, we observed a decrease in courtship behavior and an increase in aggressive behavior in both male and female zebrafish. In addition, to elucidate the molecular mechanism of the behavioral effects of azoxystrobin, we quantified the changes in the concentrations of kisspeptin, 5-HT, GnIH, and their corresponding receptor mRNA expression in the brain. The results showed that 200 µg/L azoxystrobin decreased the concentrations of kisspeptin and increased the concentration of GnIH in both male and female zebrafish brain. In addition, azoxystrobin also significantly reduced 5-HT concentration in female zebrafish brain. Further investigation revealed that altered courtship and aggressive behavior were associated with the expression levels of genes (kiss1, kiss2, gnrh3, gnrhr3, 5ht1a, and 5ht2a) involved in kisspeptin-GnIH signaling pathway. In conclusion, our study suggested that azoxystrobin may impair courtship and aggressive behavior in zebrafish by interfering with the kisspeptin-GnIH signaling pathway, which may have more profound effects on natural zebrafish populations.

A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput screening of hTRPV1 agonists.

BACKGROUND: Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists. METHODS: The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared. RESULTS: A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) < nonivamide (67 ± 3.05 nM) < piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists. CONCLUSION: The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.

Characterization of alkaloids in radix Sophora tonkinensis by UPLC-Q-TOF-MS/MS and its application in the comparison of two different habitats.

Sophora tonkinensis is widely used as traditional Chinese medicine for treating the swelling of the gums and tongue and mouth sores due to flame stomach fire. It is mainly origin from Guangxi, Sichuan provinces of China. Alkaloids are considered as the major bioactive components. A method was established for identifying alkaloids in S. tonkinensis root by UPLC-Q-TOF-MS/MS and was applied in characterizing alkaloids in S. tonkinensis root of two different habitats. Consequently, twenty-four alkaloids including six new compounds were identified in S. tonkinensis root. Additionally, the difference of alkaloids in S. tonkinensis from Guozhou, Sichuan province was investigated. In the present study, we firstly characterize total alkaloids in S. tonkinensis root by UPLC-Q-TOF-MS/MS and firstly established the characteristic fragmentation pathway of alkaloids with hydroxy in S. tonkinensis root.

Subacute sarin exposure disrupted the homeostasis of purine and pyrimidine metabolism in guinea pig striatum studied by integrated metabolomic, lipidomic and proteomic analysis.

Sarin was used as a chemical weapon due to its high neurotoxicity and mortality. Subacute sarin induced cognitive and behavioral disorder. However, the underlying mechanism is still unclear. Here we offered a multi-omic approach for the analysis of altered metabolites, lipids, and proteins to explore the neurotoxicity of subacute sarin. Guinea pigs were administered between the shoulder blades 16.8 µg/kg of sarin in a volume of 1.0 ml/kg body weight by subcutaneous injection once daily for 14 days. At the end of the final injection, guinea pigs were sacrificed, and striatum were dissected for analysis. A total of 138 different metabolites were identified in the metabolome analysis. Lipids and lipid-like molecules is the largest group (38.41%). For lipidomic analysis, a total of 216 lipids were identified. In proteomic study, over 4300 proteins were identified and quantified. By integrating these enriched components, we demonstrated that the joint pathways disturbed by subacute sarin mainly involving lipid, purine and pyrimidine metabolism in guinea pig striatum. Overall, this study highlights the powerfulness of omics platforms to deepen the understanding of nerve agents caused neurotoxicity.

Pharmacokinetics and tissue distribution of eighteen major alkaloids of Aconitum carmichaelii in rats by UHPLC-QQQ-MS.

Aconitum carmichaelii Debeaux is a widely used herbal medicine, which has anti-inflammatory and analgesic activities. However, due to its high toxicity, poisoning incidents often occur all over the world. To systematically understand the pharmacokinetics (PK) and tissue distribution of A. carmichaelii, 18 representative alkaloids, including 8 amine- (ADA), 4 monoester- (MDA) and 6 diester-type (DDA) diterpenoid alkaloids, were simultaneously quantified by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QQQ-MS) with dynamic multiple reaction monitoring (MRM) mode. PK results suggested that benzoylmesaconine, mesaconitine, 10-OH-aconitine and aconitine had lower bioavailability, which might relate to the substitution at C-3. In tissue distribution, alkaloids present higher concentrations in the liver, kidney, and only songorine, neoline and benzoyldeoxyaconine were detected in the brain. Moreover, the concentrations of extremely toxic DDAs in high-dose group were much higher than that of low-dose group, indicating that these DDAs might be the main reason for the toxicity of Aconitum. The results also suggested that benzoyldeoxyaconine and deoxyaconitine should be determined for the quality control of A. carmichaelii due to their high concentrations in both herbal extract and tissues. The systematic investigation into these 18 representative alkaloids could basically illuminate the PK and distribution of A. carmichaelii in rats, and provide some information for clinical studies.