Engineering Electron Transfer Pathway of Cytochrome P450s.

Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. P450s can catalyze various regional and stereoselective oxidation reactions, which are widely used in natural product biosynthesis, drug metabolism, and biotechnology. In a typical catalytic cycle, P450s use redox proteins or domains to mediate electron transfer from NAD(P)H to heme iron. Therefore, the main factors determining the catalytic efficiency of P450s include not only the P450s themselves but also their redox-partners and electron transfer pathways. In this review, the electron transfer pathway engineering strategies of the P450s catalytic system are reviewed from four aspects: cofactor regeneration, selection of redox-partners, P450s and redox-partner engineering, and electrochemically or photochemically driven electron transfer.

In Vitro Investigations into the Potential Drug Interactions of Pseudoginsenoside DQ Mediated by Cytochrome P450 and Human Drug Transporters.

Pseudoginsenoside DQ (PDQ), an ocotillol-type ginsenoside, is synthesized with protopanaxadiol through oxidative cyclization. PDQ exhibits good anti-arrhythmia activity. However, the inhibitory effect of PDQ on the cytochrome 450 (CYP450) enzymes and major drug transporters is still unclear. Inhibition of CYP450 and drug transporters may affect the efficacy of the drugs being used together with PDQ. These potential drug-drug interactions (DDIs) are essential for the clinical usage of drugs. In this study, we investigated the inhibitory effect of PDQ on seven CYP450 enzymes and seven drug transporters with in vitro models. PDQ has a significant inhibitory effect on CYP2C19 and P-glycoprotein (P-gp) with a half-inhibitory concentration (IC50) of 0.698 and 0.41 µM, respectively. The inhibition of CYP3A4 and breast cancer-resistant protein (BCRP) is less potent, with IC50 equal to 2.02-6.79 and 1.08 µM, respectively.

Overexpressing CYP81D11 enhances 2,4,6-trinitrotoluene tolerance and removal efficiency in Arabidopsis.

Phytoremediation is a promising technology for removing the high-toxic explosive 2,4,6-trinitrotoluene (TNT) pollutant from the environment. Mining dominant genes is the key research direction of this technology. Most previous studies have focused on the detoxification of TNT rather than plants' TNT tolerance. Here, we conducted a transcriptomic analysis of wild type Arabidopsis plants under TNT stress and found that the Arabidopsis cytochrome P450 gene CYP81D11 was significantly induced in TNT-treated plants. Under TNT stress, the root length was approximately 1.4 times longer in CYP81D11-overexpressing transgenic plants than in wild type plants. The half-removal time for TNT was much shorter in CYP81D11-overexpressing transgenic plants (1.1 days) than in wild type plants (t1/2 = 2.2 day). In addition, metabolic analysis showed no difference in metabolites in transgenic plants compared to wild type plants. These results suggest that the high TNT uptake rates of CYP81D11-overexpressing transgenic plants were most likely due to increased tolerance and biomass rather than TNT degradation. However, CYP81D11-overexpressing plants were not more tolerant to osmotic stresses, such as salt or drought. Taken together, our results indicate that CYP81D11 is a promising target for producing bioengineered plants with high TNT removing capability.

Azole resistance in Aspergillus fumigatus- comprehensive review.

Aspergillus fumigatus is a ubiquitous filamentous fungus commonly found in the environment. It is also an opportunistic human pathogen known to cause a range of respiratory infections, such as invasive aspergillosis, particularly in immunocompromised individuals. Azole antifungal agents are widely used for the treatment and prophylaxis of Aspergillus infections due to their efficacy and tolerability. However, the emergence of azole resistance in A. fumigatus has become a major concern in recent years due to their association with increased treatment failures and mortality rates. The development of azole resistance in A. fumigatus can occur through both acquired and intrinsic mechanisms. Acquired resistance typically arises from mutations in the target enzyme, lanosterol 14-α-demethylase (Cyp51A), reduces the affinity of azole antifungal agents for the enzyme, rendering them less effective, while intrinsic resistance refers to a natural resistance of certain A. fumigatus isolates to azole antifungals due to inherent genetic characteristics. The current review aims to provide a comprehensive overview of azole antifungal resistance in A. fumigatus, discusses underlying resistance mechanisms, including alterations in the target enzyme, Cyp51A, and the involvement of efflux pumps in drug efflux. Impact of azole fungicide uses in the environment and the spread of resistant strains is also explored.

Advanced three-dimensional in vitro liver models to study the activity of anticancer drugs.

The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell-cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.

Identification of the cytochrome P450 gene AccCYP6A13 in Apis cerana cerana and its response to environmental stress.

Cytochrome P450 plays a crucial role in regulating insect growth, development, and resisting a variety of stresses. Insect metamorphosis and response to external stress are altered by deleting CYP450 genes. In this study, we identified and analyzed a novel gene of CYP450 family, AccCYP6A13, from Apis cerana cerana, and explored its role in the response of Apis cerana cerana to adverse external stressors. It was found that the expression of AccCYP6A13 was spatiotemporal specificity. The expression level increased with age and reached its highest value in the adult stage. The primarily expressiong location were legs, brain, and epidermis of honeybees. Stress conditions can affect the expression of AccCYP6A13 depending on treatment times. RNA interference experiments have shown that knocking down AccCYP6A13 reduces antioxidant activity and deactivates detoxification enzymes, resulting in oxidative damage accumulation and a decline in detoxification capability in bees, as well as inhibiting numerous antioxidant genes. Additionally, knockdown of the AccCYP6A13 gene in Apis cerana cerana resulted in increased sensitivity to pesticides and increased mortality when treated with neonicotinoid pesticides such as thiamethoxam. AccCYP6A13 overexpression in a prokaryotic system further confirmed its role in resistance to oxidative stress. To summarize, AccCYP6A13 may play an essential role in the normal development and response to environmental stress in Apis cerana cerana. Furthermore, this study contributed to the theoretical understanding of bee resistance biology.

Functional study on candidate regulators mediating the expression of CYP6B7 induced by fenvalerate in a susceptible strain of Helicoverpa armigera.

Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

Various functions of detoxification enzymes against insecticides in Nilaparvata lugens selected by toxicity assays and RNAi methods.

The brown planthopper (BPH), Nilaparvata lugens is a devastating agricultural pest of rice, and they have developed resistance to many pesticides. In this study, we assessed the response of BPH nymphs to nitenpyram, imidacloprid, and etofenprox using contact and dietary bioassays, and investigated the underlying functional diversities of BPH glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 monooxygenase (P450) against these insecticides. Both contact and ingestion toxicity of nitenpyram to BPH were significantly higher than either imidacloprid or etofenprox. Under the LC50 concentration of each insecticide, they triggered a distinct response for GST, CarE, and P450 activities, and each insecticide induced at least one detoxification enzyme activity. These insecticides almost inhibited the expression of all tested GST, CarE, and P450 genes in contact bioassays but induced the transcriptional levels of these genes in dietary bioassays. Silencing of NlGSTD2 expression had the greatest effect on BPH sensitivity to nitenpyram in contact test and imidacloprid in dietary test. The sensitivities of BPH to insecticide increased the most in the contact test was etofenprox after silencing of NlCE, while the dietary test was nitenpyram. Knockdown of NlCYP408A1 resulted in BPH sensitivities to insecticide increasing the most in the contact test was nitenpyram, while the dietary test was imidacloprid. Taken together, these findings reveal that NlGSTD2, NlCE, and NlCYP408A1 play an indispensable role in the detoxification of the contact and ingestion toxicities of different types of insecticides to BPH, which is of great significance for the development of new strategies for the sucking pest control.

S431F mutation on AChE1 and overexpression of P450 genes confer high pirimicarb resistance in Sitobion miscanthi.

Sitobion miscanthi is a destructive wheat pest responsible for significant wheat yield losses. Pirimicarb, one of the most important representatives of N, N-dimethylcarbamate insecticides, is widely used to control wheat aphids. In present work, heterozygous S431F mutation of acetylcholinesterase 1 (AChE1) was identified and verified in three pirimicarb-resistant S. miscanthi populations (two field populations (HA and HS, >955.8-fold) and one lab-selected population (PirR, 486.1-fold)), which has not been reported in S. miscanthi yet. The molecular docking results revealed that AChE1 containing the S431F mutation of S. miscanthi (SmAChE1S431F) showed higher free binding energy to three insecticides (pirimicarb, omethoate, and methomyl) than wild-type AChE1 of S. miscanthi (SmAChE1). Enzyme kinetic and inhibition experiments showed that the recombinant SmAChE1S431F was more insensitive to pirimicarb and omethoate than the recombinant SmAChE1. Furthermore, two overexpression P450 genes (CYP6K1 and CYP6A14) associated with pirimicarb resistance of S. miscanthi were verified by RNAi. These results suggested both target alteration and enhanced metabolism contributed to high pirimicarb resistance of S. miscanthi in the field and laboratory. These findings lay a foundation for further elucidating the mechanism of pirimicarb resistance in S. miscanthi, and have important implications for the resistance management of S. miscanthi control.

microRNA-3037 targeting CYP6CY2 confers imidacloprid resistance to Sitobion miscanthi (Takahashi).

The wheat aphid Sitobion miscanthi is a dominant and destructive pest in agricultural production. Insecticides are the main substances used for effective control of wheat aphids. However, their extensive application has caused severe resistance of wheat aphids to some insecticides; therefore, exploring resistance mechanisms is essential for wheat aphid management. In the present study, CYP6CY2, a new P450 gene, was isolated and overexpressed in the imidacloprid-resistant strain (SM-R) compared to the imidacloprid-susceptible strain (SM-S). The increased sensitivity of S. miscanthi to imidacloprid after knockdown of CYP6CY2 indicates that it could be associated with imidacloprid resistance. Subsequently, the posttranscriptional regulation of CYP6CY2 in the 3' UTR by miR-3037 was confirmed, and CYP6CY2 participated in imidacloprid resistance. This finding is critical for determining the role of P450 in relation to the resistance of S. miscanthi to imidacloprid. It is of great significance to understand this regulatory mechanism of P450 expression in the resistance of S. miscanthi to neonicotinoids.

Investigation of lambda-cyhalothrin resistance in Spodoptera frugiperda: Heritability, cross-resistance, and mechanisms.

Lambda-cyhalothrin, a representative pyrethroid insecticide widely used for Spodoptera frugiperda control in China, poses challenges due to the development of resistance. This study investigates the realized heritability, inheritance pattern, cross-resistance, and resistance mechanisms to lambda-cyhalothrin. After 21 generations of selection, the lambda-cyhalothrin-resistant strain (G21) developed a 171.11-fold resistance compared to a relatively susceptible strain (RS-G9), with a realized heritability (h2) of 0.11. Cross-resistance assays revealed that lambda-cyhalothrin-resistant strains showed no significant cross-resistance to the majority of tested insecticides. Genetic analysis indicated that lambda-cyhalothrin resistance in S. frugiperda was autosomal, incompletely dominant, and polygenic inheritance. The P450 enzyme inhibitor PBO significantly enhanced lambda-cyhalothrin toxicity in the resistant strains. Compared with the RS-G9 strain, the P450 enzyme activity was significantly increased and multiple P450 genes were significantly up-regulated in the lambda-cyhalothrin-resistant strains. RNAi targeting the most overexpressed P450 genes (CYP337B5 and CYP321B1) significantly increased the susceptibility of resistant S. frugiperda larvae to lambda-cyhalothrin. This study provides comprehensive insights into lambda-cyhalothrin resistance in S. frugiperda, and the results are helpful for developing effective resistance management strategies of this pest.

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines.

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Phytomediated stress modulates antioxidant status, induces overexpression of CYP6M2, Hsp70, α-esterase, and suppresses the ABC transporter in Anopheles gambiae (sensu stricto) exposed to Ocimum tenuiflorum extracts.

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

Evaluation of surfactant-aided polycyclic aromatic hydrocarbon biodegradation by molecular docking and molecular dynamic simulation in the marine environment.

Marine oil spills directly cause polycyclic aromatic hydrocarbons (PAHs) pollution and affect marine organisms due to their toxic property. Chemical and bio-based dispersants composed of surfactants and solvents are considered effective oil spill-treating agents. Dispersants enhance oil biodegradation in the marine environment by rapidly increasing their solubility in the water column. However, the effect of dispersants, especially surfactants, on PAHs degradation by enzymes produced by microorganisms has not been studied at the molecular level. The role of the cytochrome P450 (CYP) enzyme in converting contaminants into reactive metabolites during the biodegradation process has been evidenced, but the activity in the presence of surfactants is still ambiguous. Thus, this study focused on the evaluation of the impact of chemical and bio-surfactants (i.e., Tween 80 (TWE) and Surfactin (SUC)) on the biodegradation of naphthalene (NAP), chrysene (CHR), and pyrene (PYR), the representative components of PAHs, with CYP enzyme from microalgae Parachlorella kessleri using molecular docking and molecular dynamics (MD) simulation. The molecular docking analysis revealed that PAHs bound to residues at the CYP active site through hydrophobic interactions for biodegradation. The MD simulation showed that the surfactant addition changed the enzyme conformation in the CYP-PAH complexes to provide more interactions between the enzyme and PAHs. This led to an increase in the enzyme's capability to degrade PAHs. Binding free energy (ΔG||Bind) calculations confirmed that surfactant treatment could enhance PAHs degradation by the enzyme. The SUC gave a better result on NAP and PYR biodegradation based on ΔG||Bind, while TWE facilitated the biodegradation of CHR. The research outputs could greatly facilitate evaluating the behaviors of oil spill-treating agents and oil spill response operations in the marine environment.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

St. John's wort extract with a high hyperforin content does not induce P-glycoprotein activity at the human blood-brain barrier.

St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P-gp activity at the human blood-brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)-targeted P-gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P-gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3-6%) for 12-19 days (1800 mg/day), the activity of P-gp at the BBB was assessed by means of PET imaging with the P-gp substrate [11C]metoclopramide and the activity of peripheral P-gp and CYPs was assessed by administering a low-dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P-gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P-gp-mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P-gp induction at the human BBB despite its ability to induce peripheral P-gp and CYPs. Simultaneous intake of SJW with CNS-targeted P-gp substrate drugs is not expected to lead to P-gp-mediated drug interactions at the BBB.

Rational Design of Triazinone Derivatives with Low Bee Toxicity Based on the Binding Mechanism of Neonicotinoids to Apis mellifera.

Bees, one of the most vital pollinators in the ecosystem and agriculture, are currently threatened by neonicotinoids. To explore the molecular mechanisms of neonicotinoid toxicity to bees, the different binding modes of imidacloprid, thiacloprid, and flupyradifurone with nicotinic acetylcholine receptor (nAChR) α1ß1 and cytochrome P450 9Q3 (CYP9Q3) were studied using homology modeling and molecular dynamics simulations. These mechanisms provided a basis for the design of compounds with a potential low bee toxicity. Consequently, we designed and synthesized a series of triazinone derivatives and assessed their bioassays. Among them, compound 5a not only displayed substantially insecticidal activities against Aphis glycines (LC50 = 4.40 mg/L) and Myzus persicae (LC50 = 6.44 mg/L) but also had low toxicity to Apis mellifera. Two-electrode voltage clamp recordings further confirmed that compound 5a interacted with the M. persicae nAChR α1 subunit but not with the A. mellifera nAChR α1 subunit. This work provides a paradigm for applying molecular toxic mechanisms to the design of compounds with low bee toxicity, thereby aiding the future rational design of eco-friendly nicotinic insecticides.