Metabolic activation and cytotoxicity of 4-methylquinoline mediated by CYP3A4 and sulfotransferases in rats.

4-Methylquinoline (4-MQ) is a quinoline derivative widely present in groundwater and soil and has been reported to be genotoxic. The mechanisms of the toxic action remain unknown. This study aimed to elucidate the metabolic activation of 4-MQ and to determine the possible role of reactive metabolites in 4-MQ-induced liver injury in rats. In the present study, a hydroxylation metabolite (M1), a GSH conjugate (M2) and an NAC conjugate (M3) derived from 4-MQ were detected in vitro and in vivo. The structures of the two conjugates were verified by chemical synthesis, mass spectrometry, and nuclear magnetic resonance. CYP3A4 was found to dominate the hydroxylation of 4-MQ. Sulfotransferases also participated in the metabolic activation of 4-MQ. Pretreatment of primary hepatocytes with ketoconazole (KTC) or 2,6-dichloro-4-nitrophenol (DCNP) not only reduced the production of GSH conjugate M2 but also decreased the susceptibility of hepatocytes to the cytotoxicity of 4-MQ. Urinary NAC conjugate M3 was found in rats given 4-MQ, and M3 may be a potential biomarker for 4-MQ exposure.

Quinoline is more genotoxic than 4-methylquinoline in hiHeps cells and rodent liver.

Environmental pollutants, such as quinoline (QN) and 4-methylquinoline (4-MeQ), may be genotoxic and carcinogenic. Earlier studies, including in vitro genotoxicity tests, indicated that 4-MeQ is more mutagenic than QN. However, we hypothesized that the methyl group of 4-MeQ favors detoxication over bioactivation, and this factor may be overlooked in in vitro tests that do not incorporate supplementation with cofactors for enzymes that catalyze conjugation reactions. We used human induced hepatocyte cells (hiHeps), which express such enzymes, and compared the genotoxicity of 4-MeQ and QN. We also carried out an in vivo micronucleus (MN) test in rat liver, since 4-MeQ is not genotoxic in rodent bone marrow. In the Ames test and the Tk gene mutation assay, with rat S9 activation, 4-MeQ was more mutagenic than QN. However, QN induced significantly higher MN frequencies in hiHeps and rat liver than did 4-MeQ. Furthermore, QN upregulated genotoxicity marker genes much more than did 4-MeQ. We also investigated the roles of two important detoxication enzymes, UDP-glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs). When hiHeps were preincubated with hesperetin (UGT inhibitor) and 2,6-dichloro-4-nitrophenol (SULT inhibitor), MN frequencies were elevated approximately 1.5-fold for 4-MeQ, whereas no significant effects were seen for QN. This study shows that QN is more genotoxic than 4-MeQ, when the roles of SULTs and UGTs in detoxication are considered and our results may improve understanding the structure-activity relationships of quinoline derivatives.

Cardiovascular toxicity associated with the multitargeted tyrosine kinase inhibitor anlotinib.

BACKGROUND: Anlotinib, a multitargeted tyrosine kinase inhibitor, has been shown to have encouraging activity against many tumors, but its cardiovascular toxicity has not been investigated specifically. We reviewed anlotinib-associated cardiovascular adverse events in patients and explored its cardiotoxicity in vitro. METHODS: We retrospectively reviewed all cardiovascular events in 62 patients with unresectable tumors who had taken anlotinib and mainly examined anlotinib's effects on left ventricular ejection fraction (LVEF) and blood pressure. Besides, we investigated its cardiotoxicity in Neonatal Rat Ventricular Myocytes (NRVMs). RESULTS: All-grade hypertension was seen in 60 patients (97%), and 25 individuals (40%) developed grade 3 hypertension. Significant univariate associations for predictors of post-treatment hypertension were age (P<0.001), BMI (P=0.003), ECOG PS(P<0.001), diabetes mellitus (P=0.035), dose of anlotinib (P=0.025). Multivariate analysis suggested that age [odds ratio (OR) 1.079, 95% confidence interval (CI): 1.029-1.130, P= 0.001] and BMI [OR 3.448, 95% CI: 1.410-8.433, P= 0.007] were the only significant independent predictors. No grade 3/4 left ventricular systolic dysfunction was reported. One patient (2%) had acute myocardial infarction, leading to cardiac death. In vitro, western blotting results showed that the levels of ANP, BNP, c-Myc and Cleaved Caspase3 were notably increased and cardiomyocyte apoptosis was strikingly increased in anlotinib group, as detected by TUNEL staining and Annexin V-FITC/PI flow cytometry. CONCLUSIONS: Our study results showed that anlotinib could induce rat cardiomyocytes apoptosis. Nonetheless, anlotinib-associated cardiovascular toxicity was acceptable and manageable for patients with unresectable tumors.

Lung-restricted ALK5 inhibition avoids systemic toxicities associated with TGFß pathway inhibition.

Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.

2-Amino-3-Methylimidazo[4,5-f]quinoline Triggering Liver Damage by Inhibiting Autophagy and Inducing Endoplasmic Reticulum Stress in Zebrafish (Danio rerio).

It is important to note that 2-Amino-3-methylimidazole[4,5-f]quinoline (IQ) is one of the most common heterocyclic amines (HCAs), which is a class of mutagenic/carcinogenic harmful compounds mainly found in high-protein thermal processed foods and contaminated environments. However, the pre-carcinogenic toxicity of IQ to the liver and its mechanism are poorly understood, further research is needed. In light of this, we exposed zebrafish to IQ (0, 8, 80, and 800 ng/mL) for 35 days, followed by comprehensive experimental studies. Histopathological and ultrastructural analysis showed that hepatocytes were damaged. TUNEL results showed that IQ induced apoptosis of liver cells, the expression of apoptosis factor gene was significantly increased, and the expression of Bcl-2 protein was significantly decreased. In addition, upregulated expression of the 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) and endoplasmic reticulum stress (ERS)-related factors transcription levels were elevated obviously, suggesting that IQ induced ERS. Decreased protein expression of autophagy-related 5 (Atg5)-Atg12, Beclin1, and LC3-II, increased protein expression of p62, and autophagy-related factors transcription levels were significantly decreased, suggesting that IQ inhibited autophagy. Overall, our research showed that the potential harm of IQ to the liver before the occurrence of liver cancer was related to ERS and its mediated autophagy and apoptosis pathways.

Metabolism is not a Major Contributor to the Toxicity of Piperaquine, a Long-acting Antimalarial Agent in Artemisinin-based Combination Therapy.

BACKGROUND: Hepatocellular damage has been reported for the antimalarial piperaquine (PQ) in the clinic after cumulative doses. OBJECTIVES: The role of metabolism in PQ toxicity was evaluated, and the mechanism mediating PQ hepatotoxicity was investigated. METHODS: The toxicity of PQ and its major metabolite (PQ N-oxide; M1) in mice was evaluated in terms of serum biochemical parameters. The role of metabolism in PQ toxicity was investigated in mice pretreated with an inhibitor of CYP450 (ABT) and/or FMO enzyme (MMI). The dose-dependent pharmacokinetics of PQ and M1 were studied in mice. Histopathological examination was performed to reveal the mechanism mediating PQ hepatotoxicity. RESULTS: Serum biochemical levels (ALT and BUN) increased significantly (P < 0.05) in mice after three-day oral doses of PQ (> 200 mg/kg/day), indicating hepatotoxicity and nephrotoxicity of PQ at a high dose. Weaker toxicity was observed for M1. Pretreatment with ABT and/or MMI did not increase PQ toxicity. PQ and M1 showed linear pharmacokinetics in mice after a single oral dose, and multiple oral doses led to their cumulative exposures. Histopathological examination showed that a high dose of PQ (> 200 mg/kg/day for three days) could induce hepatocyte apoptosis. The mRNA levels of targets in NF-κB and p53 pathways could be up-regulated by 2-30-fold in mice by PQ or M1. CONCLUSION: PQ metabolism led to detoxification of PQ, but there was a low possibility of altered toxicity induced by metabolism inhibition. The hepatotoxicity of PQ and its N-oxidation metabolite was partly mediated by NF-κB inflammatory pathway and p53 apoptosis pathway.

Lenvatinib exposure induces hepatotoxicity in zebrafish via inhibiting Wnt signaling.

Lenvatinib is a multi-kinase inhibitor for widely treating thyroid cancer. However, little studies have been done about it or its toxicity on embryonic development of vertebrate. In this study, we used zebrafish to assess the effect of lenvatinib on early embryonic development. Exposure of zebrafish embryos to 58, 117, 176 nM lenvatinib induced abnormal embryonic development, such as decreased heart rate, pericardial edema, delayed yolk absorption, and bladder atrophy. Lenvatinib exposure reduced liver area and down-regulated liver developmental related genes. The proliferation of hepatocytes and the expression of apoptosis-related genes were significantly reduced.by Lenvatinib. Furthermore, the imbalance of liver metabolism and abnormal liver tissue structure were observed in adult zebrafish after Lenvatinib exposure. Oxidative stress was up-regulated by lenvatinib and astaxanthin partially rescued hepatic developmental defects via downregulating oxidative stress. After lenvatinib exposure, Wnt signaling was down-regulated, and activation of Wnt signaling partially rescued hepatic developmental defects. Therefore, these results suggested that lenvatinib might induce zebrafish hepatotoxicity by down-regulating Wnt signaling related genes and inducing oxidative stress. This study provides a reference for the potential hepatotoxicity of lenvatinib during embryonic development and raises health concern about the potential harm of exposure to lenvatinib for foetuses.

2-Amino-3-methylimidazo[4,5-f]quinoline induced oxidative stress and inflammation via TLR4/MAPK and TLR4/NF-κB signaling pathway in zebrafish (Danio rerio) livers.

2-Amino-3-methylimidazole[4,5-f]quinoline (IQ) is a harmful substance, mainly existing in protein-abundant thermally processed foods and polluted environments. This study investigated the hepatotoxicity of IQ by exposing zebrafish model organisms at 0, 8, 80, and 800 ng/mL concentrations for 35 days and was supposed to reveal the mechanism of IQ-induced oxidative stress and inflammation in the liver. The results showed that, after IQ exposure, alanine aminotransferase (ALT), aspartate aminotransferase (AST), reactive oxygen species (ROS), and malondialdehyde (MDA) levels in zebrafish liver increased significantly; meanwhile, significantly increased tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-12 (IL-12) levels induced severe oxidative stress and inflammation; however, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST) and glutathione peroxidase (GSH-Px) levels significantly decreased. The results indicated that the increased IQ exposure gradually aggravated pathological changes of zebrafish liver tissue (irregular cell morphology, cytoplasmic vacuolation, and inflammatory cell infiltration) and induced significant liver damage at last. Alterations in the expressions of genes and proteins involved in the IQ-induced TLR4/MAPK and TLR4/NF-κB pathways can elucidate the mechanism of its hepatotoxicity. The study provides evidence of IQ-induced hepatotoxicity and helps to draw attention to the health risks of dietary and environmental exposure to IQ.

Quantitative proteomics and bioinformatics analyses reveal the protective effects of cyanidin-3-O-glucoside and its metabolite protocatechuic acid against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced cytotoxicity in HepG2 cells via apoptosis-related pathways.

The aim of this study was to investigate the mechanism of action of cyanidin-3-O-glucoside (C3G) and its metabolite protocatechuic acid (PCA) mediated protection against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced cytotoxicity in HepG2 cells. The effects of C3G and PCA on cell viability, LDH release and apoptosis in IQ-induced HepG2 cells were evaluated using CCK-8, LDH release and flow cytometry assays, respectively. TMT-based proteomics was utilized to characterize the proteins and pathways associated with the improvement after C3G and PCA treatment. Results showed that exposure to IQ significantly increased cytotoxicity and apoptosis in HepG2 cells, which were alleviated by C3G and PCA. C3G was more effective than PCA in protecting HepG2 cells against IQ-induced cytotoxicity and regulating the related signaling pathways. Proteomics and bioinformatics analyses and Western blot validation revealed that apoptosis-related signaling pathways played pivotal roles in protecting against the cytotoxicity of IQ by C3G, and XIAP was identified as the target protein. Molecular docking proved that C3G had strong binding affinity to XIAP and hindered the binding of IQ to the BIR3 domain of XIAP, resulting in the inhibition of apoptosis. Our findings suggested that C3G has potential as a preventive food ingredient to prevent carcinogenic risk of heterocyclic aromatic amines.

Mitochondrial dynamics imbalance and mitochondrial dysfunction contribute to the molecular cardiotoxic effects of lenvatinib.

Lenvatinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of resistant differentiated thyroid cancer, advanced renal cell carcinoma, unresectable hepatocellular carcinoma, and endometrial carcinoma. Although it is successful in cancer treatment, it can cause life-threatening side effects such as cardiotoxicity. The molecular mechanism of cardiotoxicity caused by lenvatinib is not fully known. In this study, the molecular mechanism of lenvatinib's cardiotoxicity was investigated focusing on mitochondrial toxicity in the H9c2 cardiomyoblastic cell line. Lenvatinib inhibited cell viability at 48 and 72 h exposure with three selected concentrations (1.25 µM, 5 µM and 10 µM); and inhibited intracellular ATP after 72 h exposure compared to the control group. Mitochondrial membrane potential was decreased after 48 h and did not show significant changes after 72 h exposure. Evaluated with real-time PCR, mitochondrial dynamics (Mfn1, Mfn2, OPA1, DRP1, Fis1) expression levels after lenvatinib treatment significantly changed. Lenvatinib triggered the tendency from fusion to fission in mitochondria after 48 h exposure, and increased both fusion and fission after 72 h. The mtDNA ratio increased after 48 h and decreased after 72 h. ASK1, JNK and AMPKα2 increased. UCP2 showed downregulation, SOD2 level showed upregulation and Cat levels decreased after drug treatment. Nrf1 and Nrf2 also changed concentration-dependently. Protein carbonyl levels increased significantly after lenvatinib treatments indicating oxidative stress. The protein levels of the electron transport chain complexes, LONP1, UCP2, and P21 showed significant differences after lenvatinib treatment. The outcome of our study is expected to be a contribution to the understanding of the molecular mechanisms of TKI-induced cardiotoxicity.

In vitro and in vivo antiplasmodial activity of novel quinoline derivative compounds by molecular hybridization.

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to produce compound candidates to treat malarial infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic properties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection (IQ5 15 mg/kg = 72.64% and IQ6 15 mg/kg = 71.15% and 25 mg/kg = 93.7%). IQ6 also showed interaction with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield imines is promising and capable of producing molecules with antiplasmodial activity.

Synthesis, characterization and pharmacological evaluation of quinoline derivatives and their complexes with copper(ΙΙ) in in vitro cell models of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system. The main pathophysiological mechanisms involve cholinergic neurotransmission, beta-amyloid (Αß) and Tau proteins, several metal ions and oxidative stress, among others. Current drugs offer only relief of symptoms and not a cure of AD. Accumulating evidence suggests that multifunctional compounds, targeting multiple pathophysiological mechanisms, may have a great potential for the treatment of AD. In this study, we report on the synthesis and physicochemical characterization of four quinoline-based metal chelators and their respective copper(II) complexes. Most compounds were non-toxic at concentrations ≤5 µM. In neuroprotection studies employing undifferentiated and differentiated SH-SY5Y cells, the metal chelator N2,N6-di(quinolin-8-yl)pyridine-2,6-dicarboxamide (H2dqpyca) appeared to exert significant neuroprotection against both, Aß peptide- and H2O2-induced toxicities. The copper(II) complex [CuII(H2bqch)Cl2].3H2O (H2bqch = N,N'-Bis(8-quinolyl)cyclohexane-1,2-diamine) also protected against H2O2-induced toxicity, with a half-maximal effective concentration of 80 nM. Molecular docking simulations, using the crystal structure of the acetylcholinesterase (AChE)-rivastigmine complex as a template, indicated a strong interaction of the metal chelator H2dqpyca, followed by H2bqch, with both the peripheral anionic site and the catalytic active site of AChE. In conclusion, the sufficient neuroprotection provided by the metal chelator H2dqpyca and the copper(II) complex [CuII(H2bqch)Cl2].3H2O along with the evidence for interaction between H2dqpyca and AChE, indicate that these compounds have the potential and should be further investigated in the framework of preclinical studies employing animal models of AD as candidate multifunctional lead compounds for the treatment of the disease.

Long-term exposure to 2-amino-3-methylimidazo[4,5-f]quinoline can trigger a potential risk of Parkinson's disease.

Humans are exposed to heterocyclic amines (HCAs) from a wide range of sources, such as protein-rich thermally processed foods, cigarette smoke, contaminated river water, the atmosphere, soil, and forest fire ash. Although the carcinogenic and mutagenic hazards of HCAs have been widely studied, the potential neurotoxicity of these compounds still needs to be further elucidated. Here, we studied the neurotoxicity of the HCA 2-amino-3-methylimidazole[4,5-f]quinoline (IQ) in vivo by utilizing a zebrafish model. After 35 days of exposure at 8, 80, and 800 ng/mL, zebrafish exploratory behavior and locomotor activity were significantly inhibited, and light/dark preference behaviors were also disturbed. Moreover, the expression of Parkinson's disease (PD)-related genes and proteins, dopamine-related genes, neuroplasticity-related genes, antioxidant enzyme genes and inflammatory cytokine genes in the zebrafish brain was significantly affected. The numbers of NeuN neurons in the midbrain were decreased in exposed zebrafish, while the numbers of apoptotic cells were increased. In summary, our research suggests that IQ is neurotoxic and significantly associated with PD and that long-term exposure to IQ may contribute to PD risk. This risk may be related to IQ-mediated effects on mitochondrial homeostasis and induction of oxidative stress and inflammation.

Effects of zero-valent iron nanoparticles and quinclorac coexposure on the growth and antioxidant system of rice (Oryza sativa L.).

Quinclorac (3,7-dichloroquinoline-8-carboxylic acid, QNC) is a highly selective auxin herbicide that is typically applied to paddy rice fields. Its residue is a serious problem in crop rotations. In this study, Oryza sativa L. seedlings was used as a model plant to explore its biochemical response to abiotic stress caused by QNC and nZVI coexposure, as well as the interactions between QNC and nZVI treatments. Exposure to 5 and 10 mg/L QNC reduced the fresh biomass by 26.6% and 33.9%, respectively, compared to the control. The presence of 50 and 250 mg/L nZVI alleviated the QNC toxicity, but the nZVI toxicity was aggravated by the coexist of QNC. Root length was enhanced upon exposure to low or medium doses of both QNC and nZVI, whereas root length was inhibited under high-dose coexposure. Both nZVI and QNC, either alone or in combination, significantly inhibited the biosynthesis of chlorophyll, and the inhibition rate increased with elevated nZVI and QNC concentration. It was indicated that nZVI or QNC can affect the plant photosynthesis, and there was a significant interaction between the two treatments. Effects of QNC on the antioxidant response of Oryza sativa L. differed in the shoots and roots; generally, the introduction of 50 and 250 mg/L nZVI alleviated the oxidative stress (POD in shoots, SOD and MDA in roots) induced by QNC. However, 750 mg/kg nZVI seriously damaged Oryza sativa L. seedlings, which likely resulted from active iron deficiency. QNC could be removed from the culture solution by nZVI; as a result, nZVI suppressed QNC uptake by 20%-30%.

Synthesis, biological evaluation and toxicity of novel tetrandrine analogues.

In this work, we present the design and synthesis of novel fully synthetic analogues of the bisbenzylisoquinoline tetrandrine, a molecule with numerous pharmacological properties and the potential to treat life-threatening diseases, such as viral infections and cancer. Its toxicity to liver and lungs and the underlying mechanisms, however, are controversially discussed. Along this line, novel tetrandrine analogues were synthesized and biologically evaluated for their hepatotoxicity, as well as their antiproliferative and chemoresistance reversing activity on cancer cells. Previous studies suggesting CYP-mediated toxification of tetrandrine prompted us to amend/replace the suspected metabolically instable 12-methoxy group. Of note, employing several in vitro models showed that the proposed CYP3A4-driven metabolism of tetrandrine and analogues is not the major cause of hepatotoxicity. Biological characterization revealed that some of the novel tetrandrine analogues sensitized drug-resistant leukemia cells by inhibition of the P-glycoprotein. Interestingly, direct anticancer effects improved in comparison to tetrandrine, as several compounds displayed a markedly enhanced ability to reduce proliferation of drug-resistant leukemia cells and to induce cell death of liver cancer cells. Those enhanced anticancer properties were linked to influences on activation of the kinase Akt and mitochondrial events. In sum, our study clarifies the role of CYP3A4-mediated toxicity of the bisbenzylisoquinoline alkaloid tetrandrine and provides the basis for the exploitation of novel synthetic analogues for their antitumoral potential.

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 µM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (Si) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 µM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 µM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

Assessment of the cytotoxic and genotoxic potential of pillar[5]arene derivatives by Allium cepa roots and Drosophila melanogaster haemocytes.

In this study pillar[5]arene (P5) and a quinoline-functionalized pillar[5]arene (P5-6Q) which is used for detecting radioactive element, gas adsorption and toxic ions were synthesized. These materials were characterized by Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FTIR), elemental analysis, melting point, Mass Spectroscopy, Scanning Electron Microscopy (SEM) and Zeta Potential. The cytotoxic and genotoxic potential of P5 and P5-6Q at distinct concentrations of 12.5, 25, 50, and 100 µg/mL were also investigated by Allium ana-telophase and comet assays on Allium cepa roots and Drosophila melanogaster haemocytes. P5 and P5-6Q showed dose dependent cytotoxic effect by decreasing mitotic index (MI) and genotoxic effect by increasing chromosomal aberrations (CAs such as disturbed anaphase-telophase, polyploidy, stickiness, chromosome laggards and bridges) and DNA damage at the exposed concentrations. These changes in P5-6Q were lower than P5. Further research is necessary to clarify the cytotoxic and genotoxic action mechanisms of P5 and P5-6Q at molecular levels.

Ethylene Biosynthesis Inhibition Combined with Cyanide Degradation Confer Resistance to Quinclorac in Echinochloa crus-galli var. mitis.

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of ß-cyanoalanine synthase (ß-CAS). Resistant biotypes exhibited higher ß-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced ß-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.

Characterization of Quinoline Yellow Dyes As Transient Aryl Hydrocarbon Receptor Agonists.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are two ligand activated transcription factors that are targeted by a wide range of anthropogenic compounds. Crosstalk between both receptors is well established but little understood. We previously developed a dual color luciferase assay (i.e., XEER) which allows time dissolved monitoring of the activation of both receptors in situ. The system was now used in conjunction with HPLC-qTOF to identify several quinophthalone dyes as transient receptor agonists of the AHR. Altogether the approach identified three widely used dyes, that is the plastic colorant latyl yellow 3G (LY), the structurally related textile dye disperse yellow 64 (DY), and the cosmetic dye quinoline yellow (QY). The latter was the most potent agonist followed by LY and DY as confirmed by the XEER assay and CYP1A1 gene induction in MCF7 cells. In addition QY, LY, and DY also inhibited ER signaling in an AHR-dependent manner. This establishes some evidence for quinoline yellow dyes as potential disruptors of AHR/ER signaling, raising potential toxicological concern. Although none of the dyes featured any signs of genotoxicity in vitro, our data point to the need for a systematic approach when screening for substances of potential toxicological and endocrine relevance.

Integration of transcriptomics and metabolomics profiling reveals the metabolic pathways affected in dictamnine-induced hepatotoxicity in mice.

Dictamnine (DTN), a major furoquinoline alkoloid from Dictamni Cortex, was reported to induce hepatotoxicity. However, the underlying mechanism is unclear. In the present study, integrated transcriptomic and metabolomics analysis of mouse liver was performed in combination with serum biochemical and histopathological evaluation to investigate the potential mechanism. The results suggested that 640 mg/kg DTN significantly increased serum alanine transaminase and aspartate transaminase levels and induced serious cellular degeneration, with no changes occurring with 4 mg/kg DTN. Integrated analysis suggested that the metabolism of xenobiotics by cytochrome P450, drug metabolism-other enzymes, bile secretion and glutathione metabolism were the major metabolic pathways involved in DTN-induced hepatotoxicity. Notably, 640 mg/kg DTN exposure increased hepatic GSH, GSH peroxidase, superoxide dismutase and malondialdehyde, and decreased ROS, together with altered expression of Idh2 and Nedd9. Representative genes, including Mup12, Lipc, NTCP, MRP3, MRP4, CYP2E1, CYP2D9 and UGT1A9, in altered pathways were verified through PCR and Western blot. Collectively, the combined strategy of transcriptomics and metabolomics profiling could facilitate a better understanding for the discovery of metabolic pathways and that oxidative damage, ABC transporters and lipid metabolism might be the mechanisms linked to DTN-induced hepatotoxicity in mice. SIGNIFICANCE: Dictamnine (DTN) was reported to induce hepatotoxicity. Nevertheless, the underlying mechanism is unknown. This study is the first to utilize integrated transcriptomics and metabolomics in combination with general toxicity evaluation to characterize the potential molecular mechanism in DTN-induced hepatotoxicity in mice. We found that acute exposure to higher dose of DTN induced hepatocellular liver injury with more changes in biochemical parameters, genes and metabolites. Gene expression and metabolite profiles were more sensitive than general toxicity studies for detecting earlier hepatotoxicity. Integrated analysis suggested that oxidative damage, ABC transporters and lipid metabolism were closely correlated with DTN-induced hepatotoxicity. Overall, our results provide insights into the mechanism responsible for DTN-induced hepatotoxicity.