Unveiling the ecotoxicological effects of azoxystrobin-based fungicides at realistic concentrations on the land snail, Theba pisana.

The ecotoxicological consequences of azoxystrobin on land snails have not yet been addressed. Therefore, the present study aims to provide novel data on the threat of a commercial grade azoxystrobin (AMISTAR) at two environmentally relevant concentrations (0.3 µg/ml) and tenfold (3 µg/ml) on the model species, Theba pisana by physiological, biochemical, and histopathological markers for 28 days. Our results showed a reduction in animal food consumption and growth due to exposure to both azoxystrobin concentrations. It also induced oxidative stress and led to a significant decrease in lipid peroxidation (LPO) levels after 7 days of exposure, while the opposite effect occurred after 28 days. Except for the 7-day exposure, all treated snails had significantly reduced glutathione (GSH) content and increased catalase (CAT) activity at all-time intervals. Glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and protein content (PC) were elevated in treated snails at all-time intervals. Moreover, alterations in acetylcholinesterase (AChE) activity between a decrease and an increase were noticed. Additionally, azoxystrobin exerted changes in T. pisana hepatopancreas architecture. Our study suggests that azoxystrobin may have negative ecological consequences for T. pisana and highlights its potential risks to the natural environment.

Resveratrol ameliorates cyprodinil-induced zebrafish cardiac developmental defects as an aryl hydrocarbon receptor antagonist.

Cyprodinil, a globally utilized broad-spectrum pyrimidine amine fungicide, has been observed to elicit cardiac abnormality. Resveratrol (RSV), a naturally occurring polyphenolic compound, showcases remarkable defensive properties in nurturing cardiac development. To investigate whether RSV could protect against cyprodinil-induced cardiac defects, we exposed zebrafish embryos to cyprodinil (500 µg/L) in the presence or absence of RSV (1 µM). Our results showed that RSV significantly mitigated the decrease of survival rate and embryo movement and the hatching delay induced by cyprodinil. In addition, RSV also improved cyprodinil-induced zebrafish cardiac developmental toxicity, including pericardial edema and cardiac function impairment. In mechanism, RSV attenuated the cyprodinil-induced changes in mRNA expression involved in cardiac development, such as myh6, myl7, tbx5, and gata4, and calcium ion channels, such as ncx1h, slc8a4a, and atp2a2b. We further showed that RSV might inhibit the activity of aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. In summary, our findings establish that the protective effects of RSV against the cardiac developmental toxicity are induced by cyprodinil due to its remarkable ability to inhibit AhR activity. Our findings not only shed light on a new avenue for regulating and ensuring the safe utilization of cyprodinil but also presents a novel concept to promote its responsible use.

Interaction behavior, mechanisms and hazardous changes of microplastics on single and binary component pesticide in the environment and food: Diethofencarb and pyrimethanil.

In order to investigate the adsorption behavior and mechanism of microplastics (MPs) on multiple coexisting pesticides in practical systems, as well as their hazardous changes upon binding, diethofencarb and pyrimethanil were selected to be studied with four MPs. The adsorption rate of both pesticides would be faster in the binary-component case, conforming to pseudo-second-order kinetics, with adsorption sites and chemical adsorption dominating. And the more hydrophobic the pesticide, the faster the adsorption rate and the higher the adsorption capacity. Diethofencarb belonged to monolayer adsorption, whereas pyrimethanil belonged to monomolecular combined with multilayer adsorption, depending on the size of pesticides. And the adsorption process was both competitive and synergistic when pesticides coexist. In addition, the adsorption process was a spontaneous heat absorption process. Electrostatic forces have little effect on adsorption, while the adsorption capacity can be altered by the adsorption sites and hydrophobicity of MPs. The salting-out effect also facilitated the adsorption process. As for changes in hazard, the bioluminescence of A. fischeri wasn't significantly inhibited, lacking of acute environmental toxicity. However, in vitro digestion experiments demonstrated a significant increase in bioavailability of diethofencarb and pyrimethanil in combination with MPs. These findings suggest the stronger adsorption behaviors and higher loading capacities between pesticides and MPs could lead more serious hazards to the human body, which deserves further attention.

Toxicogenetic, biochemical, and physiological effects of azoxystrobin and carbendazim fungicides over Lactuca sativa L. and Phaseolus vulgaris L.

Fungicides are pesticides that are frequently used in agriculture because of their action against fungal diseases. However, the widespread application of pesticides around the world raises environmental and public health concerns, since these compounds are toxic and can pose risks to ecosystems and human health. The aim of this study was to evaluate the phytotoxic, cytogenotoxic, and biochemical effects of azoxystrobin and carbendazim on Lactuca sativa L. and their physiological effects on Phaseolus vulgaris L. by analyzing the cell cycle and chromosomal and nuclear alterations in L. sativa; the biochemical effects of azoxystrobin and carbendazim on Phaseolus vulgaris L. and their physiological effects on Phaseolus vulgaris L. by analyzing the cell cycle and chromosomal and nuclear alterations in L. sativa; the biochemical effects by analyzing the activity of antioxidant enzymes in L. sativa; and the physiological effects by analyzing chlorophyll content and chlorophyll a fluorescence in P. vulgaris. It was observed that both fungicides were phytotoxic and cytotoxic, reducing root growth and the mitotic index, cytogenotoxic, increasing the occurrence of chromosomal alterations, as well as inducing oxidative stress and an increase in chlorophyll fluorescence emission and altered energy absorption in the plants used as a test system. In view of this, studies such as the one presented here indicate that the use of pesticides, even in small quantities, can lead to damage to the metabolism of plant organisms.

Phosphoproteomics reveals a novel mechanism underlying the proarrhythmic effects of nilotinib, vandetanib, and mobocertinib.

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 µM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.

Discovery of a Novel Thienopyrimidine Compound as a Urate Transporter 1 and Glucose Transporter 9 Dual Inhibitor with Improved Efficacy and Favorable Druggability.

Gout and hyperuricemia are metabolic diseases characterized with high serum uric acid (SUA) levels that significantly impact human health. Lesinurad, a uricosuric agent, is limited to concurrent use with xanthine oxidase inhibitors (XOIs) in clinical practice due to its restricted efficacy and potential nephrotoxicity. Herein, extensive structural modifications of lesinurad were conducted through scaffold hopping and substituent modification strategies, affording 54 novel derivatives containing pyrimidine-fused cyclic structures. Notably, the thienopyrimidine compound 29 demonstrated a remarkable 2-fold increase in SUA-lowering in vivo activity compared to lesinurad, while exhibiting potent inhibitory activity against the urate transporter 1 (URAT1, IC50 = 2.01 µM) and glucose transporter 9 (GLUT9, IC50 = 18.21 µM). Furthermore, it possessed a lower effective dosage of 0.5 mg/kg, favorable safety profile without any apparent acute toxicity at doses of 1000 mg/kg, and improved pharmacokinetic properties. Overall, we have discovered an efficacious URAT1/GLUT9 dual inhibitor for inhibiting urate reabsorption with favorable pharmacokinetic profiles.

Hedgehog pathway negatively regulated depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.

Synthesis, toxicological and in silico evaluation of novel spiro pyrimidines against Culex pipiens L. referring to chitinase enzyme.

The exponential development of resistance to conventional chemical insecticides adds another important motive for the creation of novel insecticidal active agents. One of the keys to meeting this challenge is the exploration of novel classes of insecticidal molecules with different modes of action. Herein, a novel series of spiro pyrimidine derivatives was prepared using some green synthetic methodologies such as microwave irradiation, and sonication under ultrasound waves. Spiro pyrimidine aminonitrile 1 is a key starting material for the synthesis of targets 2-9 by reaction with different carbon electrophiles and nitrogen nucleophiles. The structures of all the newly synthesized compounds were approved using spectral data. The toxicological efficiency and biological impacts of the synthesized spiro pyrimidine derivatives were assessed against Culex pipiens L. larvae. The toxicity of synthesized compounds showed remarkable variations against the C. pipiens larvae. Where, 3, 4 and 2 were the most efficient compounds with LC50 values of 12.43, 16.29 and 21.73 µg/mL, respectively. While 1 was the least potent compound with an LC50 value of 95.18 µg/mL. As well, other compounds were arranged according to LC50 values as follows 5 > 7 > 6 > 9 > 8. In addition, 3 and 4 exhibited significant prolongation of the developmental duration and greatly inhibited adult emergence. Moreover, many morphological deformities were observed in all developmental stages. Furthermore, cytotoxicity of the most effective compounds was assessed against the normal human cells (WI-38) as non-target organisms, where compounds 2, 4 and 3 showed weak to non-toxic effects. The study of binding affinity and correlation between chemical structure and reactivity was carried out using molecular docking study and DFT calculations to investigate their mode of action. This study shed light on promising compounds with larvicidal activity and biological impacts on the C. pipiens life cycle.

Nilotinib exhibits less toxicity than imatinib and influences the immune state by modulating iNOS, p-p38 and p-JNK in LPS/IFN gamma-activated macrophages.

In this study, we aimed to analyze the effects of first and second-generation Bcr-Abl tyrosine kinase inhibitors, imatinib and nilotinib on LPS/IFN gamma activated RAW 264.7 macrophages. Our data revealed that imatinib was less effective on nitrite levels and more toxic on macrophages compared to nilotinib. Therefore, we further analysed the effect of nilotinib on various inflammatory markers including iNOS, COX-2, NFkB, IL-6, p-ERK, p-p38 and p-JNK in LPS/IFN gamma activated RAW264.7 macrophages. Spectrophotometric viability test and Griess assay,western blot, RT-PCR and luciferase reporter assays were used to analyze the biological activity of nilotinib. Our findings revealed that nilotinib decreases nitrite levels, iNOS mRNA, iNOS and p-p38 protein expressions significantly whereas induces IL-6 mRNA and p-JNK protein expressions at particular doses. We did not find significant effect of nilotinib on COX-2, p-ERK and nuclear p65 proteins and NFkB transcriptional activity. In addition, the binding mode of nilotinib to iNOS protein was predicted by molecular docking. According to the docking analyses, nilotinib exhibited hydrophobic interactions between MET349, ALA191, VAL346, PHE363, TYR367, MET368, CYS194, TRP366 residues at the binding pocket and the molecule as well as van der Waals interactions at specific residues. In conclusion, our results reveal that, in addition to its anticancer activity, nilotinib can exhibit immune modulatory effects on macrophages through its effects on iNOS, IL-6, p-p38 and p-JNK.

Salmonella mutant strains resistant to herbicides - Acetohydroxyacid synthase inhibitors and their use at the Ames test.

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs. A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05-0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes - inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine. Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.

Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells.

Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 µM), DAS (≥0.006 µM), and REG (≥0.8 µM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs.

Lack of direct bone effect of tofacitinib, a JAK inhibitor, in juvenile rats and evaluation of the association between offspring growth and femur length.

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.

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.

Australindolones, New Aminopyrimidine Substituted Indolone Alkaloids from an Antarctic Tunicate Synoicum sp.

Five new alkaloids have been isolated from the lipophilic extract of the Antarctic tunicate Synoicum sp. Deep-sea specimens of Synoicum sp. were collected during a 2011 cruise of the R/V Nathanial B. Palmer to the southern Scotia Arc, Antarctica. Crude extracts from the invertebrates obtained during the cruise were screened in a zebrafish-based phenotypic assay. The Synoicum sp. extract induced embryonic dysmorphology characterized by axis truncation, leading to the isolation of aminopyrimidine substituted indolone (1-4) and indole (5-12) alkaloids. While the primary bioactivity tracked with previously reported meridianins A-G (5-11), further investigation resulted in the isolation and characterization of australindolones A-D (1-4) and the previously unreported meridianin H (12).

Identification of 2-Aminopyrimidine Derivatives as FLT3 Kinase Inhibitors with High Selectivity over c-KIT.

Herein, we report two promising compounds 30 and 36 possessing nanomolar FLT3 inhibitory activities (IC50 = 1.5-7.2 nM), high selectivity over c-KIT (>1000-fold), and excellent anti-AML activity (MV4-11 IC50 = 0.8-3.2 nM). Furthermore, these two compounds efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-ITD, FLT3-D835V/F, FLT3-F691L, FLT3-ITD-F691L, and FLT3-ITD-D835Y. Oral administration of 30 and 36 at 6 mg/kg/d could significantly suppress tumor growth in the MV4-11 cell-inoculated xenograft model, exhibiting tumor growth inhibitory rates of 83.5% and 95.1%, respectively. Importantly, 36 could prolong the mouse survival time in the FLT3-ITD-TKD dual mutation syngeneic mouse model (BaF3-FLT3-ITD-D835Y) at a dose of 6 mg/kg p.o. bid/4W. No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 µM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.

Evaluation of acetamiprid and azoxystrobin residues and their hormonal disrupting effects on male rats using liquid chromatography-tandem mass spectrometry.

Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).

Co-formulant in a commercial fungicide product causes lethal and sub-lethal effects in bumble bees.

Pollinators, particularly wild bees, are suffering declines across the globe, and pesticides are thought to be drivers of these declines. Research into, and regulation of pesticides has focused on the active ingredients, and their impact on bee health. In contrast, the additional components in pesticide formulations have been overlooked as potential threats. By testing an acute oral dose of the fungicide product Amistar, and equivalent doses of each individual co-formulant, we were able to measure the toxicity of the formulation and identify the ingredient responsible. We found that a co-formulant, alcohol ethoxylates, caused a range of damage to bumble bee health. Exposure to alcohol ethoxylates caused 30% mortality and a range of sublethal effects. Alcohol ethoxylates treated bees consumed half as much sucrose as negative control bees over the course of the experiment and lost weight. Alcohol ethoxylates treated bees had significant melanisation of their midguts, evidence of gut damage. We suggest that this gut damage explains the reduction in appetite, weight loss and mortality, with bees dying from energy depletion. Our results demonstrate that sublethal impacts of pesticide formulations need to be considered during regulatory consideration, and that co-formulants can be more toxic than active ingredients.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons.

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 µM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.