Cytochrome P450s Research Articles

Omics-centric evidences of fipronil biodegradation by Rhodococcus sp. FIP_B3.

The widespread use of the pesticide fipronil in domestic and agriculture sectors has resulted in its accumulation across the environment. Its use to assure food security has inadvertently affected soil microbiome composition, fertility and, ultimately, human health. Degradation of residual fipronil present in the environment using specific microbial species is a promising strategy for its removal. The present study delves into the omics approach for fipronil biodegradation using the native bacterium Rhodococcus sp. FIP B3. It has been observed that within 40 days, nearly 84% of the insecticide gets degraded. The biodegradation follows a pseudo-first-order kinetics (k = 0.0197/d with a half-life of ∼11 days). Whole genome analysis revealed Cytochrome P450 monooxygenase, peroxidase-related enzyme, haloalkane dehalogenase, 2-nitropropane dioxygenase, and aconitate hydratase are involved in the degradation process. Fipronil-sulfone, 5-amino-1-(2-chloro-4-(trifluoromethyl)phenyl)-4- ((trifluoromethyl)sulfonyl)-1H-pyrazole-3-carbonitrile, (E)-5-chloro-2-oxo-3- (trifluoromethyl)pent-4-enoic acid, 4,4,4-trifluoro-2-oxobutanoic acid, and 3,3,3- trifluoropropanoic acid were identified as the major metabolites that support the bacterial degradation of fipronil. In-silico molecular docking and molecular dynamic simulation-based analyses of degradation pathway intermediates with their respective enzymes have indicated stable interactions with significant binding energies (-5.9 to -9.7 kcal/mol). These results have provided the mechanistic cause of the elevated potential of Rhodococcus sp. FIP_B3 for fipronil degradation and will be advantageous in framing appropriate strategies for the bioremediation of fipronil-contaminated environment.

The Metabolic Characteristics and Bioavailability of Resveratrol Based on Metabolic Enzymes.

The natural polyphenol resveratrol (RV) has garnered fame for its extensive pharmacological properties. Although clinical studies have shown some positive results, many contradictory outcomes remain. An important obstacle to the development of therapeutic applications for RV is its low bioavailability in vivo. This may be partially attributed to biotransformation mediated by phase I and II enzymes, such as cytochrome P450s, UDP-glucuronosyltransferases, and sulfotransferases. To date, more than 20 different types of metabolites have been detected after catalysis by these enzymes. Notably, RV and some of its metabolites serve as substrates for these enzymes. Conversely, RV can directly regulate the expression or activity of these enzymes. Given the increasing number of studies investigating the bioactivity of RV, this review summarizes its physicochemical and pharmacokinetic characteristics and describes the metabolism of RV and the bioactivities of its metabolites, with emphasis on the interaction between RV and its related metabolic enzymes. In addition to hepatic metabolism, the crucial roles of RV metabolism in multiple other tissues and organs cannot be overlooked, and they reveal the relationship between RV metabolism and its biological potential.

PlZAT10 binds to the ABA catabolism gene PlCYP707A2 promoter to mediate seed dormancy release in Paeonia lactiflora.

PlZAT10-PlCYP707A2 module promotes Paeonia lactiflora seeds germination. The herbaceous peony (Paeonia lactiflora) seeds exhibit double dormancy in the epicotyl and hypocotyl, which significantly inhibits the process of cultivation and breeding of new varieties. Nevertheless, the molecular mechanism underlying seed dormancy release in P. lactiflora remains to be fully identified.In this current study, we analyzed differentially expressed genes based on transcriptome data and selected the abscisic acid catabolic gene PlCYP707A2 for further investigation. The conserved domain of the protein indicated that PlCYP707A2 possessed a cytochrome P450 monooxygenase domain. Subcellular localization indicated that PlCYP707A2 was localized on the cytoplasm and cell membrane. Overexpression of PlCYP707A2 in P. lactiflora seeds decreased ABA contents and promoted seeds germination. The silencing of PlCYP707A2 resulted in seed dormancy and an alteration in the content of ABA. Moreover, yeast one-hybrid, electrophoretic mobility shift and dual-luciferase reporter assay revealed that PlZAT10 bound to the promoter of PlCYP707A2. In conclusion, the results demonstrated the mechanism of the PlZAT10-PlCYP707A2 module in regulating the dormancy release of P. lactiflora seeds, enriching relevant theories on seed dormancy and having significant implications for the herbaceous peony industry developing.

Yeast Synthesis and Herbicidal Activity Evaluation of Aspterric Acid.

Aspterric acid (AA) is a novel natural product herbicide that targets dihydroxyacid dehydratase in plants. In this study, we introduced two distinct AA biosynthesis-related gene clusters into Saccharomyces cerevisiae and screened the core biosynthetic enzyme, sesquiterpene cyclase, from various fungi. The combination of sesquiterpene cyclase from Aspergillus taichungensis IBT 19404 and two cytochrome P450s from Penicillium brasilianum resulted in the optimal AA synthesis efficiency in yeast, with the highest titer of 33.21 mg/L achieved by optimizing fermentation conditions in shake flasks. Moreover, the herbicidal effects of AA on weed germination and growth were evaluated. Notably, AA strongly inhibited the germination of Amaranthus tricolor, Portulaca oleracea, Bidens pilosa, Lolium perenne, and Leptochloa chinensis. Furthermore, AA could also inhibit the shoot and root growth of weeds with a superior inhibitory effect on roots relative to shoots. Our work not only provides a sustainable method for the biosynthesis of AA in yeast but also paves the way for the application of AA as a preemergence herbicide.

Genome-wide analysis of the Cannabis sativa cytochrome P450 monooxygenase superfamily and uncovering candidate genes for improved herbicide tolerance

Genome-wide analysis of the Cannabis sativa cytochrome P450 monooxygenase superfamily and uncovering candidate genes for improved herbicide tolerance

Modification of B-Nor Steroids Mediated by Filamentous Fungus Fusarium culmorum: Focus on 15α-Hydroxylase Activity

The metabolic activities of microorganisms to modify the chemical structures of organic compounds are an effective tool for the production of high-value steroidal drugs or active pharmaceutical ingredients (APIs). The integration of biotransformation into the synthesis of APIs can greatly reduce the number of reaction steps and achieve higher process efficiency, thus enabling their greener production. The current research efforts are focused on either the optimization of existing processes or identification of new potentially useful bioconversions. This study aimed to assess the catalytic abilities of the filamentous fungus Fusarium culmorum AM 282 to transform B-nor analogues (5(6→7)abeo compounds) of steroid hormones: androstenedione (AD), dehydroepiandrosterone (DHEA) and its acetate. Our previous studies have demonstrated that this strain is an active hydroxylating catalyst for many steroidal compounds with diverse structures. The results presented in this work showed that the hydroxylation of B-nor steroids occurred with the regio- and stereoselectivity typical of this strain in relation to the corresponding natural hormones of the standard 6:6 A/B series. After the transformations of B-nor-DHEA and its acetate, 15α-hydroxy-B-nor-DHEA was obtained as the sole product of the reaction, while the transformation of the AD analogue resulted in a mixture of its 15α- and 6α-hydroxy derivatives. A detailed analysis of the transformation course indicated that all the obtained hydroxy derivatives could be the result of the activity of the same enzyme. The presented results may provide a basis for research aimed at understanding the molecular nature of cytochrome P-450 monooxygenase from F. culmorum AM 282 with its ability for 15α-hydroxylation of steroidal compounds. An analysis of the pharmacokinetic and pharmacodynamic properties of the obtained metabolites with cheminformatics tools suggests their potential biological activity.

Molecular cloning and characterization of a brassinosteriod biosynthesis-related gene PtoCYP90D1 from Populus tomentosa

Brassinosteroids (BRs), one of the major classes of phytohormones are essential for various processes of plant growth, development, and adaptations to biotic and abiotic stresses. In Arabidopsis, AtCYP90D1 acts as a bifunctional cytochrome P450 monooxygenase, catalyzing C-23 hydroxylation in the brassinolide biosynthetic pathway. The present study reports the functional characterizations of PtoCYP90D1, one of the AtCYP90D1 homologous genes from Populus tomentosa. The qRT-PCR analysis showed that PtoCYP90D1 was highly expressed in roots and old leaves. Overexpression of PtoCYP90D1 (PtoCYP90D1-OE) in poplar promoted growth and biomass yield, as well as increased xylem area and cell layers. Transgenic plants exhibited a significant increase in plant height and stem diameter as compared to the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoCYP90D1 (PtoCYP90D1-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that cell wall components increased significantly in PtoCYP90D1-OE lines but not in PtoCYP90D1-KO lines, as compared to wild-type plants. Overall, the findings indicate a positive role of PtoCYP90D1 in improving growth rate and elevating biomass production in poplar, which will have positive implications for its versatile industrial or agricultural applications.

Microbial degradation mechanisms of the neonicotinoids acetamiprid and flonicamid and the associated toxicity assessments

Extensive use of the neonicotinoid insecticides acetamiprid (ACE) and flonicamid (FLO) in agriculture poses severe environmental and ecological risks. Microbial remediation is considered a feasible approach to address these issues. Many ACE-and FLO-degrading microorganisms have been isolated and characterized, but few reviews have concentrated on the underlying degradation mechanisms. In this review, we describe the microbial degradation pathways of ACE and FLO and assess the toxicity of ACE, FLO and their metabolites. Especially, we focus on the enzymes involved in degradation of ACE and FLO, including cytochrome P450s, nitrile hydratases, amidases, and nitrilases. Those studies reviewed here further our understanding of the enzymatic mechanisms of microbial degradation of ACE and FLO, and aid in the application of microbes to remediate environmental ACE and FLO contamination.

Characterization of insecticide resistance and their mechanisms in field populations of the German cockroach (Blattodea: Ectobiidae) in Taiwan under different treatment regimes.

This study investigated how management strategies influence resistance profiles in German cockroach (Blattella germanica (L.)) populations and their impact on the performance of commercial gel baits containing fipronil, imidacloprid, and indoxacarb. Field populations from premises managed under 3 different strategies: Baiting, random insecticide (RI) used, and insecticide rotation (IR) were tested. Almost all populations under RI and IR were resistant to deltamethrin, but low to moderate resistance was observed under the Baiting approach. Cytochrome P450 monooxygenases (P450) were involved in deltamethrin resistance in these resistant populations. All individuals under Baiting and RI were homozygous for the L993F mutation, but the populations under IR lacked homozygous-resistant individuals. Eighty-three percent of field populations with complete homozygosity for the Rdl mutation displayed low mortality upon exposure to 3× LD95 fipronil. The effect of P450 and the Rdl mutation conferred high fipronil resistance in populations under the Baiting approach, recording moderate performance indices (PI) of 44-67 in fipronil bait. By contrast, those populations under RI and IR, in which involve glutathione S-transferases in fipronil resistance, had high PIs of 78-93. Almost 80% of populations exhibited over 90% mortality at 3× LD95 indoxacarb treatment, accompanied by high PIs of 90-100 in indoxacarb bait. Partial mortality from 1× LD95 imidacloprid occurred across all field populations due to the involvement of P450. PIs of imidacloprid bait ranged 5-57 and 20-94 in populations under RI and IR, respectively. Field populations demonstrate different resistance profiles depending on the treatment regimes, and the resistance mechanisms involved influenced gel bait's effectiveness.

Metabolism of Furanocoumarins by Three Recombinant CYP9A Proteins From the Polyphagous Cotton Bollworm Helicoverpa armigera.

Furanocoumarins are a class of chemical compounds with phototoxic properties. For herbivores, efficient detoxification of such defense compounds is the prerequisite to feed successfully on furanocoumarin-containing plants. The cotton bollworm Helicoverpa armigera is a very important polyphagous pest in agriculture, but how it copes with toxic furanocoumarins in some of its host plants is not well understood. Given that cytochrome P450s are well known for their capacity in xenobiotic metabolism, this study attempted to explore the potential roles of cytochrome P450s in furanocoumarin transformation in this pest. Our data showed that two linear structures (psoralen and xanthotoxin) could be metabolized by three recombinant CYP9A enzymes, but no detectable depletion was observed for the linear one with the 8-dimethylallyloxy substituent on the coumarin moiety (imperatorin) and the angular furanocoumarin (angelicin). Initial epoxidation of the double bond connecting C2' and C3' of the furano ring following by cleavage of the epoxidated furan ring, leading to the formation of more soluble, less reactive and nonphotosensitizing metabolites, was identified as a common mechanism of linear furanocoumarin metabolism using a quadrupole/time-of-flight (Q-TOF) mass spectrometry interfaced with a high performance liquid chromatography (HPLC) system. Our data demonstrated that multiple P450s were involved in the detoxification of linear furanocoumarins in the cotton bollworm. These findings contribute to a better understanding of the biochemical basis of adaptation to plant defense chemicals in this economically important pest.

Valifenalate-induced non-adverse thyroid changes via adaptive induction of uridine 5′-diphospho-glucuronosyltransferase (UGT) in the liver of dogs and rats but not humans

Some rat and dog toxicology studies with the fungicide valifenalate showed minimal, non-adverse thyroid changes, mostly above the maximum tolerated dose, and concomitantly with liver effects. This publication describes their mode of action (MOA), combining in vivo and new approach methodologies (NAMs), in a weight of evidence approach.Data demonstrate a MOA of liver enzyme induction via nuclear receptor CAR/PXR activation, increased thyroxine (T4) metabolism and elevated thyroid stimulating hormone (TSH) level, leading to thyroid follicular cell hypertrophy and increased thyroid weight. Non-human relevance of the MOA was demonstrated in in vitro cross species assays in rat, dog and human hepatocytes. Increased gene expression and activity of cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were observed in rat and dog hepatocytes exposed to valifenalate, with increased T4 clearance and/or T4 glucuronidation/T4-UGT activity. Therefore, a causal relationship between increased liver enzyme induction and thyroid effects in dogs and rats is concluded. Rat hepatocytes were most sensitive, while valifenalate did not increase T4-UGT activity above 2-fold of vehicle control or T4 glucuronidation and T4 clearance in human hepatocytes. Consequently, valifenalate exposure in humans is unlikely to lead to decreased T4 levels, and subsequent thyroid and developmental neurotoxicity effects. Alternative human-relevant thyroid MOAs were excluded, i.e. inhibition of deiodinases (DIO), thyroperoxidase (TPO) or the sodium iodide symporter (NIS).Due to known species differences in thyroid homeostasis between humans and laboratory animals and, importantly, based on the presented data, this liver enzyme mediated MOA is considered not relevant for human hazard assessment.

Beta-cypermethrin-induced stress response and ABC transporter-mediated detoxification in Tetrahymena thermophila

β-Cypermethrin (β-CYP), a synthetic pyrethroid pesticide, is widely used for insect management. However, it also affects non-target organisms and pollutes aquatic ecosystems. Tetrahymena thermophila, a unicellular ciliated protist found in fresh water, is in direct contact with aquatic environments and sensitive to environmental changes. The proliferation of T. thermophila was inhibited and the cellular morphology changed under β-CYP stress. The intracellular ROS level significantly increased, and SOD activity gradually rose with increasing β-CYP concentrations. Under 25 mg/L β-CYP stress, 687 genes were up-regulated, primarily enriched in the organic cyclic compound binding and heterocyclic compound binding pathways. These include 8 ATP-binding cassette transporters (ABC) family genes, 2 cytochrome P450 monooxygenase genes, and 2 glutathione peroxidase related genes. Among of them, ABCG14 knockdown affected cellular proliferation under β-CYP stress. In contrast, overexpression of ABCG14 enhanced cellular tolerance to β-CYP. The results demonstrated that Tetrahymena tolerates high β-CYP concentration stress through various detoxification mechanisms, with ABCG14 playing a crucial role in detoxification of β-CYP.

Engineering Regioselectivity of P450 BM3 Enables the Biosynthesis of Murideoxycholic Acid by 6β-Hydroxylation of Lithocholic Acid.

Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.

Akt-FoxO signaling drives co-adaptation to insecticide and host plant stresses in an herbivorous insect

IntroductionOngoing interactions between host and herbivorous insect trigger a co-evolutionary arms race. Genetic diversity within insects facilitates their adaptation to phytochemicals and their derivatives, including plant-derived insecticides. Cytochrome P450s play important roles in metabolizing phytochemicals and insecticides, due to their diversity and almost perfect evolution. ObjectivesThis study aims to uncover a common molecular mechanism in herbivorous insects by investigating the role of kinase-transcription factor regulation of P450s in conferring tolerance to both insecticides and phytochemicals. MethodsRNA interference, transcriptome sequencing, insecticide, and phytochemical bioassays were conducted to validate the functions of Akt, FoxO, and candidate P450s. Dual-luciferase activity assays were employed to identify the regulation of P450s by the Akt-FoxO signaling pathway. Recombinant P450 enzymes were utilized to investigate the metabolism of insecticides and phytochemicals. ResultsWe identified an Akt-FoxO signaling cascade, a representative of kinase-transcription factor pathways. This cascade mediates the expression of eight P450 enzymes involved in the metabolism of insecticides and phytochemicals in Nilaparvata lugens. These P450s are from different families and with different substrate selectivity, enabling them to respectively metabolize insecticides and phytochemicals with structure diversity. Nevertheless, the eight P450 genes were up-regulated by FoxO, which was inhibited in a higher cascade by Akt through phosphorylation. The discovery of the Akt-FoxO signaling pathway regulating a series of P450 genes elucidates the finely tuned regulatory mechanism in insects for adapting to phytochemicals and insecticides. ConclusionThese finding sheds light on the physiological balance maintained by these regulatory processes. The work provides the experimental evidence of co-adaptation to the stresses imposed by host plant and insecticide within the model of the kinase-TF involving various P450s. This model provides a comprehensive view of how pests adapt to multiple environmental stresses.

Nephro- and Cardiotoxic Effects of Etoricoxib: Insights into Arachidonic Acid Metabolism and Beta-Adrenergic Receptor Expression in Experimental Mice

Background: Etoricoxib is a widely used anti-inflammatory drug, but its safety profile concerning cardiovascular and renal health remains inadequately explored. This study aimed to assess the nephro- and cardiotoxic effects of etoricoxib in a murine model, with a focus on its impact on arachidonic acid-metabolizing enzymes and beta-adrenergic receptors associated with drug-induced toxicity. Methods: Thirty-five BALB/C mice were randomly assigned to five groups: control, low-dose etoricoxib, high-dose etoricoxib, low-dose celecoxib, and high-dose celecoxib (a well-known nephro- and cardiotoxic NSAID). The treatments were administered for 28 days, after which hearts and kidneys were excised for physical and histopathological analysis, and the expression of arachidonic acid-metabolizing enzymes (cytochrome P450s, lipoxygenases, cyclooxygenases) and beta-1 adrenergic receptor (adrb1) and angiotensin-converting enzyme (ace2) genes were quantified using quantitative reverse transcription PCR (qRT-PCR). Results: Etoricoxib administration resulted in dose-dependent nephro- and cardiotoxic effects. Renal histology revealed glomerular atrophy or hypertrophy and significant damage to the proximal and distal convoluted tubules, including epithelial flattening, cytoplasmic vacuolation, and luminal widening. Cardiac analysis showed disorganized muscle fibers and hyaline degeneration. These changes were associated with altered gene expression: the downregulation of cox2, cyp1a1, and cyp2c29 in the kidneys and the upregulation of cyp4a12, cox2, and adrb1, along with the downregulation of cyp2c29 and ace2 in the heart. Conclusions: Etoricoxib induces nephro- and cardiotoxicity, marked by alterations in arachidonic acid metabolism and beta-adrenergic signaling pathways. The drug affects the expression of arachidonic acid-metabolizing enzymes and adrb1 in the heart while downregulating cox2 and other related enzymes in the kidneys. These findings underscore the need for caution when prescribing etoricoxib, particularly in patients with pre-existing renal or cardiac conditions.

Resistance of Anopheles gambiae s.s. against commonly used insecticides and implication of cytochrome P450 monooxygenase in resistance to pyrethroids in Lambaréné (Gabon)

BackgroundInsecticides are a crucial component of vector control. However, resistance constitute a threat on their efficacy and the gains obtained over the years through malaria vector control. In Gabon, little data on phenotypic insecticide resistance in Anopheles vectors are published, compromising the rational implementation of resistance management strategies. We assessed the susceptibility to pyrethroids, carbamates and organophosphates of Anopheles gambiae sensu lato (s.l.) and discuss the mechanisms involved in the pyrethroid resistance-phenotype.MethodsA. gambiae s.l. larvae were collected from breeding sites in Lambaréné. Emerging adults were used in WHO tube assays at an insecticide concentration that defines resistance (diagnostic concentration). Subsequently, deltamethrin and permethrin were used at 5x and 10x diagnostic concentrations and after preexposure with the cytochrome p450 (and glutathione S-transferase) inhibitor piperonyl butoxide (PBO). A subset of mosquitoes was typed by molecular methods and screened using Taqman assays for mutations conferring target site resistance at the Voltage-gated sodium channel 1014 (Vgsc-1014) locus and the acetylcholinesterase (Ace-1) gene.ResultsAll mosquitoes were A. gambiae sensu stricto (s.s.) and resistant to permethrin, deltamethrin and alphacypermethrin (mortality less than 98%). However, mosquitoes were susceptible to malathion but resistant to bendiocarb. The level of resistance was high for permethrin and at least moderate for deltamethrin. Pre-exposure to PBO significantly increased the mortality of resistant mosquitoes (P < 0.0001). They became fully susceptible to deltamethrin and permethrin-induced mortality increased 4-fold. The G119S Ace-1 resistance allele, which confers resistance to both organophosphates and carbamates, was not present. All sampled mosquitoes were either homozygous for the Vgsc-L1014F or heterozygous for Vgsc-L1014F/L1014S, a marker for resistance to pyrethroids and organochlorides.ConclusionThese findings demonstrate a role of cytochrome P450 monooxygenases in the pyrethroid-resistance of A. gambiae s.s. from Lambaréné. Combining PBO with pyrethroids, as done in second generation bednets, may be used to revert resistance. In addition, malathion could also be used in combination with pyrethroids-based methods for resistance management.

New CYP154C4 from Streptomyces cavourensis YBQ59 performs regio- and stereo- selective 3β-hydroxlation of nootkatone

Nootkatone, a sesquiterpenoid widely used in the food and cosmetics industries, exhibits diverse biological activities and pharmaceutical prospects. Modification of nootkatone to create new derivatives with desirable activities has attracted significant attention. For this purpose, cytochrome P450 monooxygenases (P450 or CYP) are attractive candidates due to their ability to perform regio- and stereoselective hydroxylation at allylic C–H bonds. In this study, CYP154C4 from Streptomyces cavourensis YBQ59 was cloned and expressed in Escherichia coli. By screening 64 candidate substrates, this P450 was found to catalyze the regio- and stereoselective hydroxylation of nootkatone, yielding a single product, 3β-hydroxynootkatone. Using a whole-cell E. coli system expressing CYP154C4, supported by the heterologous redox partners YkuN from Bacillus subtilis and FdR from E. coli, 3β-hydroxynootkatone was produced on a preparative scale. The structure of this compound was determined by 1H NMR, 13C NMR, NOESY, HMBC, and HSQC. The kinetics of product formation were analyzed using HPLC, and the Km and Kcat values were calculated. Furthermore, structural insights into the selective hydroxylation of nootkatone were elucidated by molecular docking. 3β-Hydroxynootkatone, recently synthesized semi-synthetically from nootkatone, has been reported to exhibit a higher insecticidal activity than its parent compound. Additionally, the functionalization of nootkatone with N-acyl-2-aminothiazole at the C3 and C2 positions, yielding an α-glucosidase inhibitor, has also been previously described. Therefore, 3β-hydroxynootkatone has great potential for further research and for synthesizing new derivatives with valuable biological activities for agricultural and medicinal applications.

Changes in Plasma Clearance of CYP450 Probe Drugs May Not be Specific for Altered In Vivo Enzyme Activity Under (Patho)Physiological Conditions: How to Interpret Findings of Probe Cocktail Studies.

CYP450 (CYP) phenotyping involves quantifying an individual's plasma clearance of CYP-specific probe drugs, as a proxy for in vivo CYP enzyme activity. It is increasingly applied to study alterations in CYP enzyme activity under various (patho)physiological conditions, such as inflammation, obesity, or pregnancy. The phenotyping approach assumes that changes in plasma clearance of probe drugs are driven by changes in CYP enzyme activity. However, plasma clearance is also influenced by protein binding, blood-to-plasma ratio, and hepatic blood flow, all of which may change under (patho)physiological conditions. Using a physiologically based pharmacokinetic (PBPK) workflow, we aimed to evaluate whether the plasma clearance of commonly used CYP probe drugs is indeed directly proportional to alterations in CYP enzyme activity (sensitivity), and to what extent alterations in protein binding, blood-to-plasma ratio, and hepatic blood flow observed under (patho)physiological conditions impact plasma clearance (specificity). Plasma clearance of CYP probe drugs is sensitive to alterations in CYP enzyme activity, since alterations in intrinsic clearance between - 90% and + 150% resulted in near-proportional changes in plasma clearance, except for midazolam in the case of > 50% CYP3A4 induction. However, plasma clearance also changed near-proportionally with alterations in the unbound drug fraction, diminishing probe specificity. This was particularly relevant for high protein-bound probe drugs, as alterations in plasma protein binding resulted in larger relative changes in the unbound drug fraction. Alterations in the blood-to-plasma ratio and hepatic blood flow of ± 50% resulted in plasma clearance changes of less than ± 16%, meaning they limitedly impacted plasma clearance of CYP probe drugs, except for midazolam. In order to correct for the impact of non-metabolic determinants on probe drug plasma clearance, an R script was developed to calculate how much the CYP enzyme activity is actually altered under (patho)physiological conditions, when alterations in the unbound drug fraction, blood-to-plasma ratio, and/or hepatic blood flow also impact probe drug plasma clearance. As plasma protein binding can change under (patho)physiological conditions, alterations in unbound drug fraction should be accounted for when using CYP probe drug plasma clearance as a proxy for CYP enzyme activity in patient populations. The tool developed in this study can support researchers in determining alterations in CYP enzyme activity in patients with (patho)physiological conditions.

ApCarE4 and ApPOD3 participate in the adaptation of pea aphids to different alfalfa varieties

The adaptability of insects to hosts has long been a focal point in the study of insect-plant interactions. The pea aphid (Acythosiphon pisum), a significant pest of numerous leguminous crops, not only inflicts direct economic losses but also disseminates various plant viruses. To understand how pea aphids adapt to diverse alfalfa varieties. We analyzed the differentially expressed genes (DEGs) of pea aphids in distinct alfalfa varieties using transcriptome sequencing, and subsequently conducted functional validation of these genes. Comparative analysis between pea aphids feeding on susceptible and resistant strains revealed that DEGs in aphids feeding on resistant strains were primarily associated with transcriptional enrichment in the sugar, amino acid, protein, and lipid metabolism pathways. Fourteen DEGs related to adaptation of the pea aphid to alfalfa were chosen, including five carboxylesterases (CarE), four cytochrome P450s, three glutathione S-transferases, and two peroxidases (POD). RT-qPCR results indicated significant up-regulation of two carboxylesterase genes and two peroxidase genes after 24 h of feeding resistant alfalfa (Gannong 5, GN5) compared to the susceptible varieties (Hunter River, LRH), particularly highlighting the high expression levels of ApCarE4 and ApPOD3. Simultaneously, RNAi-induced knockdown of ApCarE4 and ApPOD3 led to a higher mortality of pea aphids in the alfalfa Hunter River. These results indicate that ApPOD3 and ApCarE4 are involved in the detoxification of metabolic functions in the adaptation of pea aphids to host switching. These findings contribute to the understanding of pea aphid adaptation to host plants and lay a foundation for further exploration of the physiological roles of carboxylesterase and peroxidase genes in pea aphids.

Integrated Analysis of Metabolome and Transcriptome Reveals the Effect of Burdock Fructooligosaccharide on the Quality of Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).

Burdock fructooligosaccharide (BFO) is fructose with a low polymerization degree, which could improve the immunity to pathogens, quality, and stress resistance of vegetables. Still, there are no studies on applying BFO in Chinese cabbage. In this study, the effects of exogenous BFO sprayed with different concentrations (0, 5, 10, 20, 30 g·L-1) on the growth and soluble sugar content of Chinese cabbage seedlings were determined. The result showed that 10 g·L-1 was the appropriate spraying concentration. Based on metabolome analysis, a total of 220 differentially accumulated metabolites (DAMs) were found, among which flavonoid metabolites, glucosinolate metabolites, and soluble sugar-related metabolites were the key metabolites involved in improving the quality of Chinese cabbage caused by BFO. Further combination analysis with transcriptome, trans-cinnamate 4-monooxygenase (CYP73A5), and chalcone synthase 1 (CHS1) were more closely associated with the DAMs of flavonoid biosynthesis. Sulfotransferases 18 (SOT18), Branched-chain amino acid amino transferases 6 (BCAT6), and cytochrome P450 monooxygenase (CYP83A1) were the key genes in glucosinolate biosynthesis. Hexokinase (HxK1), beta-glucosidase 8 (BGL08), invertase 3 (INV3), beta-glucosidase 3B (BGL3B), and sucrose phosphate synthase 1 (SPS1) were significantly upregulated, potentially playing crucial roles in the soluble sugar metabolism. In conclusion, these results provided an understanding of the effects of BFO on the expression of genes and the accumulation of metabolites related to quality formation in Chinese cabbage.