Cytochrome P450s Research Articles

Mechanism of cytochrome P450s mediated interference with glutathione and amino acid metabolisms from halogenated PAHs exposure

Epidemiological evidence indicates that exposure to halogenated polycyclic aromatic hydrocarbons (HPAHs) is associated with many adverse effects. However, the mechanisms of metabolic disorder of HPAHs remains limited. Herein, effects of pyrene (Pyr), and its halogenated derivatives (1-chloropyrene (1-Cl-Pyr), 1-bromopyrene (1-Br-Pyr)) on endogenous metabolic pathways were investigated, in human hepatoma (HepG2) and HepG2-derived cell lines expressing various human cytochrome P450s (CYPs). Non-targeted metabolomics results suggested that 1-Br-Pyr and Pyr exposure (625 nM) induced disruption in glutathione and riboflavin metabolism which associated with redox imbalance, through abnormal accumulation of oxidized glutathione, mediated by bioactivation of CYP2E1. Conversely, CYP2C9-mediated 1-Cl-Pyr significantly interfered with glutathione metabolism intermediates, including glycine, L-glutamic acid and pyroglutamic acid. Notably, CYP1A1-mediated Pyr-induced perturbation of amino acid metabolism which associated with nutrition and glycolipid metabolism, resulting in significant upregulation of most amino acids, whereas halogenated derivatives mediated by CYP1A2 substantially downregulated amino acids. In conclusion, this study suggested that Pyr and its halogenated derivatives exert potent effects on endogenous metabolism disruption under the action of various exogenous metabolic enzymes (CYPs). Thus, new evidence was provided to toxicological mechanisms of HPAHs, and reveals potential health risks of HPAHs in inducing diseases caused by redox and amino acid imbalances.

Unobtrusive Health Hazard

Oxidative stress is a biological mechanism triggered by an imbalance between the biosynthesis and accumulation of reactive oxygen species (ROS) in a biological system. Normally, ROS are produced as byproducts of oxygen metabolism and fulfill various physiological roles such as cell signaling. However, environmental stressors like UV radiation, ionizing radiation, pollutants, heavy metals, and xenobiotics can also lead to increased ROS production. Common types of ROS include superoxide radicals (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2), which are generated during various metabolic pathways. Key cellular processes such as protein phosphorylation, activation of transcriptional factors, apoptosis, immunity, and differentiation depend on maintaining appropriate levels of ROS within cells. Under conditions of excessive ROS production, which can occur during both normal physiological processes and pathological conditions like inflammation, ischemia, and aging, ROS can damage important cellular components such as proteins, lipids, and nucleic acids. This production is primarily mitochondrial but can also result from enzymatic activities involved in the respiratory chain, prostaglandin synthesis, phagocytosis, and the cytochrome P450 system. Both immune system activation and environmental factors contribute to endogenous and exogenous free radical production. The signs of oxidative stress include fatigue, memory loss, muscle or joint pain, the appearance of wrinkles and grey hair, decreased eyesight, headaches, increased sensitivity to noise, and a heightened susceptibility to infections. To reduce oxidative stress, it is advised to avoid sugar and processed foods while balancing blood sugar levels, prevent infections, incorporate daily stress reduction practices, avoid toxins, and promote the production of antioxidants through diet and herbal supplements that are rich in antioxidants.

A remarkable peroxidase-like behavior of the catalase KatA from the pathogenic bacteria Helicobacter pylori: The oxidation reaction with formate as substrate and the stabilization of an [Fe(IV) = O Trp•] intermediate assessed by multifrequency EPR spectroscopy

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9–285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e− oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H+/2e− oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO2 to regulate the pH in the acidic environment.

Genome-wide identification, molecular evolution and gene expression of P450 gene family in Cyrtotrachelus buqueti

BackgroundInsect Cytochrome P450 monooxygenase (CYPs or P450s) plays an important role in detoxifying insecticides, causing insect populations to develop resistance. However, the molecular functions of P450 gene family in Cyrtotrachelus buqueti genome are still lacking.ResultsIn this study, 71 CbuP450 genes have been identified. The amino acids length of CbuP450 proteins was between 183 aa ~ 1041 aa. They are proteins with transmembrane domains. The main component of their secondary structure is α-helix and random coils. Phylogenetic analysis showed that C. buqueti and Rhynchophorus ferrugineus were the most closely related. This gene family has 29 high-frequency codons, which tend to use A/T bases and A/T ending codons. Gene expression analysis showed that CbuP450_23 in the female adult may play an important role on high temperature resistance, and CbuP450_17 in the larval may play an important role on low temperature tolerance. CbuP450_10, CbuP450_17, CbuP450_23, CbuP450_10, CbuP450_16, CbuP450_20, CbuP450_23 and CbuP450_ 29 may be related to the regulation of bamboo fiber degradation genes in C. buqueti. Protein interaction analysis indicates that most CbuP450 proteins are mainly divided into three aspects: encoding the biosynthesis of ecdysteroids, participating in the decomposition of synthetic insecticides, metabolizing insect hormones, and participating in the detoxification of compounds.ConclusionsWe systematically analyzed the gene and protein characteristics, gene expression, and protein interactions of CbuP450 gene family, revealing the key genes involved in the stress response of CbuP450 gene family in the resistance of C. buqueti to high or low temperature stress, and identified the key CbuP450 proteins involved in important life activity metabolism. These results provided a reference for further research on the function of P450 gene family in C. buqueti.

Cytochrome P450 SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11

Accumulating salinity in soil critically affected growth, development, and yield in plant. However, the mechanisms of plant against salt stress largely remain unknown. Herein, we identified a gene named SmCYP78A7a, which encoded a cytochrome P450 monooxygenase and belonged to the CYP78A sub-family, and its transcript level was significantly up-regulated by salt stress and down-regulated by dehydration stress. SmCYP78A7a located in the endoplasmic reticulum. Silencing of SmCYP78A7a enhanced susceptibility of eggplant to salt stress, and significantly down-regulated the transcript levels of salt stress defense related genes SmGSTU10 and SmWRKY11 as well as increased hydrogen peroxide (H2O2) content and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) enzyme activities. In addition, SmCYP78A7a transient expression enhanced eggplant tolerance to salt stress. By chromatin immunoprecipitation PCR (ChIP-PCR), luciferase reporter assay, and electrophoretic mobility shift assay (EMSA), SmWRKY11 activated SmCYP78A7a expression by directly binding to the W-box 6–8 (W-box 6, W-box 7, and W-box 8) within SmCYP78A7a promoter to confer eggplant tolerance to salt stress. In summary, our finds reveal that SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11, and provide a new insight into regulatory mechanisms of eggplant to salt stress.

The influence of the model pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases in HepaRG cells

Introduction Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the cytochrome P450 system to their corresponding oxon analogues to act as potent inhibitors of acetylcholinesterase. It is known that interactions between cytochrome P450 and pesticides include the inhibition of major xenobiotic metabolizing cytochrome P450 enzymes and changes on the genetic level. Methods In this in vitro study, the influence of the pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases was investigated with a metabolically competent cell line (HepaRG cells). First, the viability of the cells after exposure to parathion and paraoxon was evaluated. The inhibitory effect of both pesticides on cytochrome P450 3A4, which is a pivotal enzyme in the metabolism of xenobiotics, was examined by determining the dose-response curve. Changes on the transcription level of 92 oxygenase associated genes, including those for important cytochrome P450 enzymes, were evaluated. Results The exposure of HepaRG cells to parathion and paraoxon at concentrations up to 100 µM resulted in a viability of 100 per cent. After exposure for 24 hours, pronounced inhibition of cytochrome P450 3A4 enzyme activity was shown, indicating 50 per cent effective concentrations of 1.2 µM (parathion) and 2.1 µM (paraoxon). The results revealed that cytochrome P450 involved in parathion metabolism were significantly upregulated. Discussion Relevant changes of the cytochrome P450 3A4 enzyme activity and significant alteration of genes associated with cytochrome P450 suggest an interference of pesticide exposure with numerous metabolic processes. The major limitations of the work involve the use of a single pesticide and the in vitro model as surrogate to human hepatocytes. Conclusion The data of this study might be of relevance after survival of acute, life-threatening intoxications with organophosphorus compounds, particularly for the co-administration of drugs, which are metabolized by the affected cytochrome P450.

RNA-Seq analysis reveals the gene expression changes of Bemisia tabaci in response to destruxin A treatment

ABSTRACT Bemisia tabaci is an important agricultural pest that has been causing significant economic losses to crops across the globe. Destruxins are secondary metabolites of entomopathogenic fungi which can be used as a potential biopesticide against B. tabaci. However, little is known about the molecular mechanism involved in the regulation of the immune response of B. tabaci post-destruxin application. In this study, we explored the molecular responses of B. tabaci exposed to destruxin A (DA) using RNA-Seq. A total of 1702, 616, and 555 differentially expressed genes (DEGs) were identified in B. tabaci after 4, 8, and 12 h of DA treatment. In addition, 39 putative detoxification-related DEGs including 29 cytochrome P450s (P450s), 5 glutathione S-transferases (GSTs), and 5 carboxylesterases (CarEs) were also identified. Quantitative real-time PCR analysis indicated that the expression profiles of 19 random DEGs were consistent with the RNA-Seq results. These findings serve as valuable information for a better understanding of the interaction and molecular mechanisms involved in the defense response of B. tabaci against DA.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases

AbstractSpirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8‐dihydroxynaphthalene to generate the spiroketal linkage and 2,3‐epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C‐5 hydroxy group, widely found in SBNs. PalC catalyzes 1‐keto reduction, the reverse 1‐dehydrogenation, and 2,3‐epoxide reduction. Moreover, an FAD‐dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1‐dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxy group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

The deregulation of arachidonic acid metabolism in ovarian cancer.

Arachidonic acid (AA) is a crucial polyunsaturated fatty acid in the human body, metabolized through the pathways of COX, LOX, and cytochrome P450 oxidase to generate various metabolites. Recent studies have indicated that AA and its metabolites play significant regulatory roles in the onset and progression of ovarian cancer. This article examines the recent research advancements on the correlation between AA metabolites and ovarian cancer, both domestically and internationally, suggesting their potential use as biological markers for early diagnosis, targeted therapy, and prognosis monitoring.

Myostatin deletion alters hormone metabolism shifts in obese skeletal muscle of C57/Bl6 mice

Objective: Myostatin deletion improves cardiometabolic disease outcomes observed in obesity without any changes in activity in mice. Hormone balance is dysregulated in obesity, resulting in low testosterone and high estradiol levels, which have been shown to correlate with higher risk for developing associated cardiometabolic disease outcomes. However, the basis of these alterations and how they impact factors that improve obese cardiometabolic outcomes are unknown. Methods: Control and obese mice ( db/db) with or without myostatin deletion to induce hypermuscularity were used. Plasme from mice were analyzed for hormone levels via radioimmunoassay (RIA). Aromatase inhibition was performed via dosing 10mg/kg/day via water. Quantitative PCR was used cytochrome P450 enzymes involved in estrogen metabolism in the liver, skeletal muscle, and visceral white adipose tissue. C2C12 overexpression of CYP1B1 and treatment with hydroxyestrogens in vitro was performed and respective enzyme mRNA expression assessed. Results: Deletion of myostatin increased muscle mass by >30% and resulted in and improved glucose tolerance in the context of obesity without any changes in activity. Obesity decreased plasma testosterone 30% and increased estrogen 20% with no change in gonadal weight, which was improved in the absence of myostatin. CYP1B1, an enzyme that converts estradiol to toxic hydroxy estrogens, showed 2.5-fold expression in obese skeletal muscle, which is reduced in the absence of myostatin, with no change in other cytochrome P450s involved in estrogen metabolism. Inhibition of aromatase reduces estrogen and improves testosterone plasma concentration, indicating the excess estrogen in obesity is derived primarily from testosterone conversion. Aromatase expression was increased in obesity but unaffected by changes in muscle mass, this indicates an alteration to downstream estrogen metabolism. In C2C12 cells, overexpression of myostatin recapitulated changes in CYP1B1 expression seen in vivo, suggesting a direct link between the myokine and sex hormone metabolism. In addition, treatment of C2C12 cells with CYP1B1 derived hydroxyestrogens caused a tandem increase in SCD1 expression, indicating a causal link between CYP1B1 activity in obesity to increased triglyceride synthesis. Conclusion: In summary, obesity causes a shift in sex hormone levels towards estrogen production, which is improved by myostatin deletion. A potential mechanism responsible could be altered estrogen metabolism, leading to decreased estrogen levels without restoration of androgen levels. These data suggest CYP1B1 drives the conversion of estrogen to hydroxestrogens, known to harbor carcinogenic and pro-lipidemic influences, in skeletal muscle which may also contribute to cardiometabolic disease. Whether targeting CYP1B1 improves cardiometabolic function in obesity remains to be determined. D.W. Stepp and D.J. Fulton are supported by NIH 1R01HL147159. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Anti-inflammatory and Neuroprotective α-Pyrones from a Marine-Derived Strain of the Fungus Arthrinium arundinis and Their Heterologous Expression.

Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aβ treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.

Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Pyrethroid resistance and gene expression profile of a new resistant An. gambiae colony from Uganda reveals multiple resistance mechanisms and overexpression of Glutathione-S-Transferases linked to survival of PBO-pyrethroid combination.

The effectiveness of long-lasting insecticidal nets (LLINs) are being threatened by growing resistance to pyrethroids. To restore their efficacy, a synergist, piperonyl butoxide (PBO) which inhibits cytochrome P450s has been incorporated into pyrethroid treated nets. A trial of PBO-LLINs was conducted in Uganda from 2017 and we attempted to characterize mechanisms of resistance that could impact intervention efficacy. We established an Anopheles gambiae s.s colony in 2018 using female mosquitoes collected from Busia district in eastern Uganda. We first assessed the phenotypic resistance profile of this colony using WHO tube and net assays using a deltamethrin dose-response approach. The Busia colony was screened for known resistance markers and RT-qPCR targeting 15 genes previously associated with insecticide resistance was performed. The Busia colony had very high resistance to deltamethrin, permethrin and DDT. In addition, the colony had moderate resistance to alpha-cypermethrin and lambda-cyhalothrin but were fully susceptible to bendiocarb and fenitrothion. Exposure to PBO in combination with permethrin and deltamethrin resulted in higher mortality rates in both net and tube assays, with a higher mortality observed in net assays than tube assays. The kdr marker, Vgsc-995S was at very high frequency (91.7-98.9%) whilst the metabolic markers Coeae1d and Cyp4j5-L43F were at very low (1.3% - 11.5%) and moderate (39.5% - 44.7%) frequencies respectively. Our analysis showed that gene expression pattern in mosquitoes exposed to deltamethrin, permethrin or DDT only were similar in comparison to the susceptible strain and there was significant overexpression of cytochrome P450s, glutathione-s-transferases (GSTs) and carboxyl esterases (COEs). However, mosquitoes exposed to both PBO and pyrethroid strikingly and significantly only overexpressed closely related GSTs compared to unexposed mosquitoes while major cytochrome P450s were underexpressed. The high levels of pyrethroid resistance observed in Busia appears associated with a wide range of metabolic gene families.

Determination of insecticide resistance in cotton whitefly in north India

The whitefly, Bemisia tabaci (Gennadius) considered as most destructive pest, poses a significant threat to various crop species globally has developed resistance due to the indiscriminate use of synthetic chemicals. A study was carried out in 2018 at ICAR-Indian Agricultural Research Institute, New Delhi to determine insecticide resistance, in five Asia II 1 populations of B. tabaci from different cotton (Gossypium hirsutum L.) growing regions of north India. The susceptibility of the populations to different insecticide classes, viz. synthetic pyrethroid (cypermethrin); neonicotinoids (imidacloprid and thiamethoxam); thiourea (diafenthiuron) and diamide (cyantraniliprole) were assessed. Results revealed substantial heterogeneity in the responses of these populations to the insecticides. Sriganganagar and Bathinda populations exhibited moderate resistance to cypermethrin, imidacloprid, and thiamethoxam. Low level of resistance was observed in Bathinda and Sriganganagar populations against diafenthiuron. All populations were highly susceptible to cyantraniliprole. No cross-resistance was observed between cyantraniliprole and other insecticides, suggesting its potential as an alternative for managing insecticide resistance. High levels of detoxification enzymes (esterase, cytochrome P450 monooxygenase, and glutathione-S-transferase) in Sriganganagar and Bathinda populations indicated a positive correlation between insecticide resistance and detoxifying enzymes. These findings offer valuable insight for implementing insecticide rotation strategies to combat B. tabaci resistance in India.

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.

Differential Tissue Abundance of Membrane-Bound Drug Metabolizing Enzymes and Transporter Proteins by Global Proteomics.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are useful for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics that relies on synthetic stable isotope-labeled surrogate peptides as calibrators is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA does not consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage, which was applied to quantify 54 DMETs for characterization of 1) differential tissue DMET abundance in the human liver, kidney, and intestine, and 2) interindividual variability of DMET proteins in individual intestinal samples (n = 13). Uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7), microsomal glutathione S-transferases (MGST1, MGST2, and MGST3) carboxylesterase 2 (CES2), and multidrug resistance-associated protein 2 (MRP2) were expressed in all three tissues, whereas, as expected, four cytochrome P450s (CYP3A4, CYP3A5, CYP2C9, and CYP4F2), UGT1A1, UGT2B17, CES1, flavin-containing monooxygenase 5, MRP3, and P-glycoprotein were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic prediction of systemic and tissue concentration of drugs. SIGNIFICANCE STATEMENT: This study quantified the abundance and compositions of drug-metabolizing enzymes and transporters in pooled human liver, intestine, and kidney microsomes as well as individual intestinal microsomes using an optimized global proteomics approach. The data revealed large intertissue differences in the abundance of these proteins and high intestinal interindividual variability in the levels of cytochrome P450s (e.g., CYP3A4 and CYP3A5), uridine diphosphate-glucuronosyltransferase 2B17, carboxylesterase 2, and microsomal glutathione S-transferase 2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance.

AP-1 and SP1 trans-activate the expression of hepatic CYP1A1 and CYP2A6 in the bioactivation of AFB1 in chicken.

Dietary exposure to aflatoxin B1 (AFB1) is harmful to the health and performance of domestic animals. The hepatic cytochrome P450s (CYPs), CYP1A1 and CYP2A6, are the primary enzymes responsible for the bioactivation of AFB1 to the highly toxic exo-AFB1-8,9-epoxide (AFBO) in chicks. However, the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1 metabolism remains unknown. Dual-luciferase reporter assay, bioinformatics and site-directed mutation results indicated that specificity protein 1 (SP1) and activator protein-1 (AP-1) motifs were located in the core region -1,063/-948, -606/-541 of the CYP1A1 promoter as well as -636/-595, -503/-462, -147/-1 of the CYP2A6 promoter. Furthermore, overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6. Moreover, bioactivation of AFB1 to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression, which was trans-activated owing to the upregulalion of AP-1, rather than SP1, stimulated by AFB1-induced reactive oxygen species. Additionally, nano-selenium could reduce ROS, downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6, thus alleviating the toxicity of AFB1. In conclusion, AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1 to AFBO in chicken liver, which could provide novel targets for the remediation of aflatoxicosis in chicks.