P450 Enzyme Activity Research Articles

Molecular insights into developmental toxicity induced by PCB77 exposure on zebrafish via integrating transcriptomics with adverse outcome pathway.

Polychlorinated biphenyls (PCBs), a typical type of persistent organic pollutants (POPs), were previously widely employed as insulating and heat exchange fluids in transformers and capacitors. Despite knowledge of its adverse effects, the precise mechanism underlying PCB77 toxicity remains enigmatic. In this study, we utilized zebrafish as a model organism to explore the toxic effects of various concentrations of PCB77 (10, 200, and 1000μg/L) and its molecular toxicity mechanisms. Upon exposure to dosages of PCB77 throughout embryonic and larval stages, the zebrafish exhibited adverse phenotypic manifestations, including deformities, decreased heart rates, increased distances between the bulbus arteriosus (BA) and sinus venosus (SV) and reduced locomotor ability. Transcriptome analysis revealed the common enriched pathways across all PCB77 concentration groups, such as retinol metabolism, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450, which are closely related to the activity of cytochrome P450 (cyp1a) enzymes. Furthermore, Adverse Outcome Pathway (AOP) framework which integrates AOPs and dose-dependent transcriptomics to predict PCB77-induced adverse outcomes (AOs) revealed that aryl hydrocarbon receptor (AhR) associated AOPs triggered by PCB77 exposure may increase early-life stage mortality and decrease cardiac development, indicating that the primary toxic pathways of PCB77 in zebrafish may involve AhR-mediated signaling. Besides, molecular docking modeling demonstrated that PCB77 could bind to the groove within the AhR domain, suggesting that PCB77 induces embryotoxicity in zebrafish through its interaction with AhR. Collectively, these findings not only deliver a thorough examination of PCB77-induced developmental toxicity as well as the underlying mechanisms, but also validate the efficacy of the analytical approach leveraging AOP framework in unraveling toxicity mechanisms of environmental contaminants, which holds promise for risk assessment associated with novel environmental pollutants.

Maize Herbivore-Induced Volatiles Enhance Xenobiotic Detoxification in Larvae of Spodoptera frugiperda and S. litura

The release of herbivore-induced plant volatiles (HIPVs) has been recognized to be an important strategy for plant adaptation to herbivore attack. However, whether these induced volatiles are beneficial to insect herbivores, particularly insect larvae, is largely unknown. We used the two important highly polyphagous lepidopteran pests Spodoptera frugiperda and S. litura to evaluate the benefit on xenobiotic detoxification of larval exposure to HIPVs released by the host plant maize (Zea mays). Larval exposure of the invasive alien species S. frugiperda to maize HIPVs significantly enhanced their tolerance to all three of the well-known defensive compounds 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), chlorogenic acid, and tannic acid in maize and the two commonly used insecticides methomyl and chlorpyrifos. HIPV exposure also improved the larval tolerance of S. litura third instars to chlorogenic and tannic acids. Furthermore, larval exposure to either maize HIPVs or DIMBOA induced the activities of cytochrome P450 enzymes (P450s), glutathione-s-transferase (GST), and carboxylesterase (CarE) in the midguts and fat bodies of the two insects, while the induction was significantly higher by the two components together. In addition, the expression of four genes encoding uridine diphosphate (UDP)-glycosyltransferases (UGT33F28, UGT40L8) and P450s (CYP4d8, CYP4V2) showed similar induction patterns in S. frugiperda. Cis-3-hexen-1-ol, an important component in maize HIPVs, also showed the same functions as maize HIPVs, and its exposure increased larval xenobiotic tolerance and induced the detoxification enzymes and gene expression. Our findings demonstrate that HIPVs released by the pest-infested host plants are conductive to the xenobiotic tolerance of lepidopteran insect larvae. Hijacking the host plant HIPVs is an important strategy of the invasive alien polyphagous lepidopteran pest to counter-defend against the host plant’s chemical defense.

Mitochondrial ROS, a trigger for mitochondrial dysfunction and inflammasome activation and a therapeutic target in liver diseases

Mitochondria are essential organelles responsible for intracellular energy production and play crucial roles in cellular metabolism, inflammation, and apoptosis. Reactive oxygen species (ROS) are primarily produced in the mitochondria and endoplasmic reticulum of hepatocytes due to the activity of cytochrome P450 enzymes. Under ideal conditions, cells have specific molecular mechanisms that manage oxidative stress levels, thus ensuring a balance between oxidants and antioxidants. The interplay between ROS-induced mitochondrial dysfunction and the activation of the NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome in the context of liver diseases has been extensively studied. However, the exact mechanisms by which mitochondria promote the activation of the NLRP3 inflammasome and contribute to the onset of liver disease remain unclear. This review aims to elucidate the recently discovered mitochondrial regulation of the NLRP3 inflammasome in liver disorders, including alcohol-related liver disease (ALD), metabolic-associated steatotic liver disease (MASLD), and hepatocellular carcinoma (HCC). Finally, it summarizes various natural and pharmaceutical agents that can mitigate liver damage by modulating the activation of the NLRP3 inflammasome through mitochondrial pathways. This work serves as an important resource for identifying new therapeutic approaches and provides further support for advancing the understanding of liver diseases.

Insecticide Resistance of Xenopsylla cheopis in Madagascar: Revision of Diagnostic Doses for Bioassay and Exploration of Biochemical Mechanisms.

The Oriental rat flea, Xenopsylla cheopis, is known worldwide as an efficient plague vector, including in Madagascar, where the disease remains a public health concern. Chemical control is the primary response method against X. cheopis in Madagascar. Previous bioassays focusing on different flea populations from Madagascar showed phenotypic resistance to various insecticides, including deltamethrin and fenitrothion, which, respectively, represent the previous and current chemicals for flea vector control. Despite apparent insecticide resistance, the associated mechanisms of this resistance remain poorly known. The aims of this study were to adjust diagnostic doses of deltamethrin and fenitrothion and to investigate the metabolism-based insecticide resistance of X. cheopis in Madagascar. Five available laboratory-reared flea strains of X. cheopis were selected, and their susceptibility statuses to deltamethrin and fenitrothion were determined using the WHO standard bioassay. Diagnostic doses of each insecticide were determined by the probit method, in accordance with concentration gradients. Biochemical microplate-based assays were performed to detect overproduction of cytochrome P450, alpha-/beta-esterases, and glutathione S-transferase (GST), which are signatures of metabolic resistance. The five tested strains showed different susceptibility statuses against deltamethrin and fenitrothion. The diagnostic doses were estimated to be 0.07% for deltamethrin and 1.56% for fenitrothion. Increased activities of cytochrome P450, beta-esterase, and GST enzymes in the resistant strains were revealed in comparison with those of the susceptible strain. In conclusion, readjusted diagnostic doses will help to better understand the susceptibility status of X. cheopis to deltamethrin and fenitrothion. The overproduction of cytochrome P450, beta-esterase, and GST observed on deltamethrin-resistant flea strains suggests metabolic resistance.

Size matters: the effects of varying zinc oxide nanoparticle sizes on human cytochrome P450 enzyme activity and gene expression

Size matters: the effects of varying zinc oxide nanoparticle sizes on human cytochrome P450 enzyme activity and gene expression

Resistance ratios and enzyme activity of Tetranychus urticae and Tetranychus turkestani (Acari: Tetranychidae) to commonly used acaricides on cotton in Aydin province, Türkiye

This study aims to determine the resistance ratios of Tetranychus urticae Koch red form (RF) and Tetranychus turkestani (Ugarov & Nikolskii) populations collected from cotton fields in Soke, Germencik, Kocarli and Nazilli districts of Aydin province to commonly used acaricides (abamectin, hexythiazox, etoxazole, spiromesifen). The potential role of detoxification enzymes (esterase, glutathione-s-transferase (GST), P450 monooxygenase (P450)) in acaricide resistance was also assessed. Overall populations of T. urticae RF presented varying ratios of resistance to these acaricides with all three populations (G15R21, K24R21, S3R21) showing significant resistance to abamectin (187.1–223.66 fold), hexythiazox (156.31–168.25 fold), etoxazole (409.58–517.20 fold) and spiromesifen (981.77–1246.11 fold). In contrast, all five T. turkestani populations (G4G21, K16G21, N1G21, N11G21, S19G21) were susceptible to abamectin, etoxazole, and spiromesifen, but exhibited some resistance to hexythiazox (164.34–182.39 fold). The resistance ratios of T. turkestani were however lower compared to T. urticae RF. A relationship was also found between detoxification enzymes and resistance. Esterase activity varied among mite populations, with the highest levels recorded in resistant populations of T. urticae (G15R21 and K24R219) with rates ranging between 6.48 and 7 mOD/min/mg protein. GST activity was highest in resistant mite populations (T. urticae S3R21 and T. turkestani K16G21). T. turkestani N11G21 population exhibited the highest P450 enzyme activity, reaching 4.39 RFU/30 min/mg protein. This study was the first to determine the resistance ratios of T. turkestani to hexythiazox in Türkiye, and on a global scale.

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.

17α-Hydroxyprogesterone (17α-OHP) is a steroid hormone with significant biological activity that can be obtained by catalyzing progesterone (PROG), the main product of sitosterol, through CYP17A1. However, increasing the catalytic specificity of HCYP17A1 for C17 hydroxylation of progesterone (PROG) poses a formidable challenge due to the close proximity of the C16 and C17 positions. In this study, a rational design was utilized to alter the spatial configuration of the substrate channel, leading to the complete abolition of its 16-hydroxylation activity. Subsequent molecular dynamics simulations revealed that the A105Y mutation heightened the rigidity of the G95-I112 region of CYP17A1, consequently regulating the direction of the entry of PROG into the catalytic pocket. Moreover, the establishment of hydrogen bonding between Y105 and R239, coupled with Pi-stacking of A105Y with F114, effectively immobilizes the substrate PROG in a fixed position, explaining the practically perfect regioselectivity observed in A105Y. Finally, a multifaceted enzymatic cascade system, incorporating A105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase (ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst produced 106 ± 3.2 mg L-1 17α-OHP, a 4.6-fold increase compared with A105Y alone. Thus, this study provides valuable insights for improving the regioselectivity and activity of P450 enzymes.

Systematic metabolic engineering of Yarrowia lipolytica for efficient production of phytohormone abscisic acid

Abscisic acid (ABA) is an important phytohormone with diverse applications. It currently relies on the fermentation of Botrytis cinerea, which suffers from limited availability of genetic engineering tools. Here, Yarrowia lipolytica was engineered to enable de novo biosynthesis of ABA. To overcome the rate-limiting P450 enzymes, systematic engineering strategies were implemented. Firstly, the dissolved oxygen was increased to boost the activity of P450 enzymes. Secondly, the expansion of endoplasmic reticulum was implemented to improve the functional expression of P450 enzymes. Lastly, rate-limiting enzymes were assembled to facilitate substrate trafficking. Moreover, ABA production was further improved by strengthening the mevalonate pathway. Finally, the engineered strain produced 1221.45 mg/L of ABA in a 5-L bioreactor. The study provides effective approaches for alleviating rate-limiting P450 enzymes to enhance ABA production and achieve competitive industrial-level ABA production in Y. lipolytica.

Inhibitory effects characteristics of polysaccharide of Polygonati Rhizome on cytochrome P450 enzymes.

As the major active ingredient of Polygonatum sibriricum Red., polysaccharide of Polygonati Rhizome (PSP) has been reported to possess various pharmacological activities, especially for the treatment of chronic disorders in the elderly. This study evaluated the effect of PSP on the activities of major cytochrome P450 enzymes (CYP450) isoforms, aiming to provide theoretical reference for its co-prescription with other drugs and prevent the risk of adverse drug-drug interaction. The activities of CYP450 isoforms were assessed in human liver microsomes with specific probe substances. Through Lineweaver-Burk fitting models, the effect of PSP on the activity of inhibited CYP450 isoforms was characterized by competitive and non-competitive models. Dose-dependent and time-dependent experiments were also performed to completely understand the inhibition. Among experimental isoforms, PSP significantly inhibited the activities of CYP2C9, 2D6, and 3A4 in a concentration-dependent manner with IC50 values of 14.01, 17.63, and 5.33 μM. The inhibition of CYP2C9 and 2D6 was best fitted with the competitive model with the Ki values of 7.15 and 8.92 μM, respectively, while the inhibition of CYP3A4 was non-competitive with the Ki value of 2.63 μM. Additionally, the inhibition of CYP3A4 by PSP also showed time-dependent characteristics with the inhibition parameters, KI of 1.273 μM-1 and Kinact of 0.036 min-1. PSP exerted moderate inhibition on CYP2C9 and 2D6 and strong inhibition of CYP3A4. The dosage of CYP2C9-, 2D6-, and 3A4-metabolized drugs should be adjusted when co-administrated with PSP and its sourced herbs.

Investigation of resistance ratios and resistance mechanisms of Cydia pomonella (L.) (lepidoptera: Tortricidae) populations collected from apple orchards in Isparta (Türkiye) against some insecticides

The codling moth, Cydia pomonella is an important pest that causes significant economic losses in apples and walnuts in the world. The aim of this study was to investigate, for the first time in Türkiye, the resistance status and resistance mechanisms of codling moth, Cydia pomonella against indoxacarb, deltamethrin and emamectin benzoate. All apple orchard populations developed resistance ratios ranging from 3.38 to 22.37-fold to deltamethrin, 5.67–29.87-fold to indoxacarb and 1.46–3.05-fold to emamectin benzoate. The interaction of some synergists triphenyl phosphate (TPP), piperonyl butoxide (PBO) and diethyl maleate (DEM) with indoxacarb and deltamethrin was analyzed in the susceptible, MAREM and Tepeli populations with moderate resistance to indoxacarb and deltamethrin. While indoxacarb + TPP showed a significant synergistic effect only in MAREM population, a significant synergistic effect was observed with indoxacarb + TPP and indoxacarb + PBO in Tepeli population. The activities of detoxifying enzymes [esterase, glutathion –S– transferase (GST) and cytochrome P450 monooxygenase (P450)] studied by biochemical methods showed some variation depending on the population. The results of biochemical analyses showed that esterase and GST enzyme activities of all populations was between 0.51 and 0.94, 0.77 and1.29 mOD min−1mg−1 proteins, respectively. The P450 enzyme activities ranged from 0.53 to 0.78, RFU min−1mg−1 proteins. In addition, the L1014F knockdown mutation (CTT to TTT) corresponding to leucine to phenylalanine amino acid substitution of the voltage-gated sodium channel in Cydia pomonella was determined in MAREM and Tepeli populations. It was determined that while MAREM and Tepeli populations developed moderate resistance, the other populations developed a low level resistance to deltamethrin and indoxacarb. On the other hand, all populations developed a low level resistance to emamectin benzoate. The P450 and esterase enzyme activities were significantly higher in MAREM and Tepeli populations which were resistant to deltamethrin and indoxacarb than the susceptible population. In addition, a Kdr point mutation L1014F was detected in the deltamethrin resistant MAREM and Tepeli populations.

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.

Evaluation of Drug-Drug Interaction Potential of Talquetamab, a T-Cell-Redirecting GPRC5D×CD3 Bispecific Antibody, as a Result of Cytokine Release Syndrome in Patients with Relapsed/Refractory Multiple Myeloma in MonumenTAL-1, Using a Physiologically Based Pharmacokinetic Model.

Cytokine release syndrome, commonly associated with T-cell immunotherapies, was observed with talquetamab, a T-cell-redirecting bispecific antibody, in the phase I/II MonumenTAL-1 study, leading to elevated interleukin (IL)-6, which can suppress cytochrome P450 (CYP) enzyme activity. We aimed to evaluate the potential impact of elevated IL-6 on the exposure of co-administered CYP450 substrates for two scenarios: (1) the observed median IL-6 profile and (2) a profile with the highest IL-6 maximum concentration following talquetamab treatment. A physiologically based pharmacokinetic model was developed based on the literature and simulations performed using observed IL-6 profiles from patients in MonumenTAL-1 who received the subcutaneous recommended phase 2 doses (RP2Ds) of talquetamab: 0.4 mg/kg weekly (QW) and 0.8 mg/kg every other week (Q2W). Median IL-6 maximum concentration was 18.4 and 7.1 pg/mL, and maximum IL-6 maximum concentration was 213 and 3503 pg/mL for talquetamab QW and Q2W RP2Ds, respectively. For the median IL-6 profile, no interaction between IL-6 and studied CYP substrates was predicted at either RP2D. The maximum IL-6 profile predicted weak-to-moderate impact on exposure of CYP2C19, CYP3A4, and CYP3A5 substrates and minimal impact on exposure of CYP1A2 substrates at both RP2Ds. Impact on exposure of CYP2C9 substrates was predicted as minimal at QW and minimal-to-weak at Q2W RP2Ds. Time to return to 20% difference from baseline enzymatic activity was predicted as 7 and 9 days after start of cycle 1 for QW and Q2W RP2Ds, respectively. These modeling results suggest that IL-6 release due to talquetamab-induced cytokine release syndrome has limited impact on potential drug-drug interactions, with the highest likelihood of impact occurring from initiation of talquetamab step-up dosing up to 7 (QW) or 9 (Q2W) days after first treatment dose in cycle 1 and during and after cytokine release syndrome. Multiple myeloma can be treated with immunotherapies such as the bispecific antibody, talquetamab, which binds the novel antigen G protein-coupled receptor family C group 5 member D on multiple myeloma cells and CD3 on T cells and induces T-cell-mediated lysis of multiple myeloma cells. Following talquetamab treatment, many patients experience cytokine release syndrome, an inflammatory immune response where levels of proinflammatory cytokines, including interleukin (IL)-6, are increased. Interleukin-6 can suppress the activity of important enzymes in the body (cytochrome [CYP] P450s) that are involved in drug clearance. This study used a physiologically based pharmacokinetic computer model to investigate the potential impact of increased IL-6 levels on CYP450 enzymes to determine subsequent impact on drugs that are metabolized by CYP450 enzymes. The results showed no predicted interaction between median levels of IL-6 observed in patients and CYP substrates (such as caffeine and omeprazole) with talquetamab. In a simulation that assessed higher (maximum) IL-6 levels observed in patients, the predicted impact of IL-6 was minimal to weak for most of the CYP substrates assessed. The effect on CYP450 enzymatic activity was highest from initiation of talquetamab step-up dosing up to 7-9 days after the first treatment dose of talquetamab. These results suggest that, in this treatment time period, elevated IL-6 levels due to talquetamab-induced cytokine release syndrome have limited impact on drugs that are CYP substrates that may be used in conjunction with talquetamab, but that the concentration and toxicity of these drugs should be monitored and the dose of CYP substrate adjusted as required.

Whole Body PhysiologicallyBased Pharmacokinetic Model to Explain A Patient With Drug-Drug Interaction Between Voriconazole and Flucloxacillin.

Voriconazole administered concomitantly with flucloxacillin may result in subtherapeutic plasma concentrations as shown in a patient with Staphylococcus aureus sepsis and a probable pulmonary aspergillosis. After switching our patient to posaconazole, therapeutic concentrations were reached. The aim of this study was to first test our hypothesis that flucloxacillin competes with voriconazole not posaconazole for binding to albumin ex vivo, leading to lower total concentrations in plasma. A physiologicallybased pharmacokinetic (PBPK) model was then applied to predict the mechanism of action of the drug-drug interaction (DDI). The model included non-linear hepatic metabolism and the effect of a severe infectious disease on cytochrome P450 (CYP) enzymes activity. The unbound voriconazole concentration remained unchanged in plasma after adding flucloxacillin, thereby rejecting our hypothesis of albumin-binding site competition. The PBPK model was able to adequately predict the plasma concentration of both voriconazole and posaconazole over time in healthy volunteers. Upregulation of CYP3A4, CYP2C9, and CYP2C19 through the pregnane X receptor (PXR) gene by flucloxacillin resulted in decreased voriconazole plasma concentrations, reflecting the DDI observations in our patient. Posaconazole metabolism was not affected, or was only limitedly affected, by the changes through the PXR gene, which agrees with the observed plasma concentrations within the target range in our patient. Ex vivo experiments reported that the unbound voriconazole plasma concentration remained unchanged after adding flucloxacillin. The PBPK model describes the potential mechanism driving the drug-drug and drug-disease interaction of voriconazole and flucloxacillin, highlighting the large substantial influence of flucloxacillin on the PXR gene and the influence of infection on voriconazole plasma concentrations, and suggests a more limited effect on other triazoles.

Maternal obesogenic diet during pregnancy and its impact on fetal hepatic function in baboons.

Maternal obesity (MO) increases the risk of later-life liver disease in offspring, especially in males. This may be due to impaired cytochrome P450 (CYP) enzyme activity driven by an altered maternal-fetal hormonal milieu. MO increases fetal cortisol concentrations that may increase CYP activity; however, glucocorticoid receptor (GR)-mediated signaling can be modulated by alternative GR isoform expression. We hypothesized that MO induces sex-specific changes in GR isoform expression and localization that contribute to reduced hepatic CYP activity. Nonpregnant, nulliparous female baboons were assigned to either an ad libitum control diet or a high-fat, high-energy diet (HF-HED) at 9 months pre pregnancy. At 165 days' gestation (term = 180 days), fetal liver samples were collected (n = 6/sex/group). CYP activity was quantified using functional assays, and GR was measured using quantitative RT-PCR and Western blot. CYP3A activity was reduced in the HF-HED group, whereas CYP2B6 activity was reduced in HF-HED males only. Total GR expression was increased in the HF-HED group. Relative nuclear expression of the antagonistic GR isoform GRβ was increased in HF-HED males only. Reduced CYP activity in HF-HED males may be driven in part by dampened hepatic-specific glucocorticoid signaling via altered GR isoform expression. These findings highlight targetable mechanisms that may reduce later-life sex-specific disease risk.

Methyltransferase Like-3-Mediated N6-Methyladenosine Modification of Long Noncoding RNA Hepatocyte Nuclear Factor 1a Antisense RNA 1/Hepatocyte Nuclear Factor 4a Antisense RNA 1 Regulates Cytochrome P450 Enzyme Expression.

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long noncoding RNAs (lncRNAs) hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the downregulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the downregulation of some CYPs proteins, and upregulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. SIGNIFICANCE STATEMENT: Although the impact of long noncoding RNAs (lncRNAs) including hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and hepatocyte nuclear factor 4a antisense RNA 1 (HNF4A-AS1) on the downstream transcription factor (TF) and cytochrome P450 enzyme (CYP) expression is well studied, the upstream regulation of these two lncRNAs by methyltransferase-like 3 (METTL3) remains unexplored. This study reveals that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1 in HepG2 and Huh7 cells.

Homogeneously complexed alginate-chitosan hydrogel microspheres for the viability enhancement of entrapped hepatocytes

The future deployment of biomedicine fields will require a new generation of biodegradable, biocompatible, and non-toxic hydrogels. Alginate and chitosan, naturally occurring polymers, have gained significant interest for hydrogel applications. However, integrating chitosan within alginate-based hydrogels to form microspheres with homogeneous distribution and a tailored surface charge remains challenging. Herein, we report the design and fabrication of homogeneously complexed alginate-chitosan hydrogel microspheres, demonstrating their ability to enhance the viability and liver-specific functionalities of entrapped hepatocytes. By exploring and optimizing the pH and ratio of alginate and chitosan solutions, we achieved well-controlled physicochemical properties, including the degree of sphericity, hydrophilicity, charge property, and surface roughness. Unlike traditional alginate-based hydrogel microspheres, hepatocytes entrapped in homogeneous alginate-chitosan microspheres displayed enhanced viability and liver-specific functions, including albumin secretion, urea synthesis, and cytochrome P-450 enzymatic activity. This work illustrates a potential pathway for manufacturing functionalized microspheres with tunable mechanical properties and functionalities based on biocompatible alginate and chitosan for hepatocyte applications.

Identification and Functional Characterization of Carboxylesterase Genes Involved in Spirodiclofen Resistance in Panonychus citri (McGregor).

Overexpression of carboxyl/cholinesterase (CCE) genes has been reported to be associated with many cases of pesticide resistance in arthropods. However, it has been rarely documented that CCE genes participate in spirodiclofen resistance in Panonychus citri. In previous research, we found that spirodiclofen resistance is related to increased P450 and CCE enzyme activities in P. citri. In this study, we identified two CCE genes, PcCCE3 and PcCCE5, which were significantly upregulated in spirodiclofen-resistant strain and after exposure to spirodiclofen. RNA interference of PcCCE3 and PcCCE5 increased the spirodiclofen susceptibility in P. citri. In vitro metabolism indicated that PcCCE3 and PcCCE5 could interact with spirodiclofen, but metabolites were detected only in the PcCCE3 treatment. Our results indicated that PcCCE3 participates in spirodiclofen resistance through direct metabolism, and PcCCE5 may be involved in the spirodiclofen resistance by passive binding and sequestration, which provides new insights into spirodiclofen resistance in P. citri.

Involvement of P450s in the metabolic resistance of Digitaria sanguinalis (L.) Scop. To ALS-inhibiting herbicides

Weed resistance to a range of herbicides has rapidly evolved, often with different mechanisms of action. The resulting uninhibited growth of weeds poses demonstrable threats to crop production and sustainable agriculture. Digitaria sanguinalis (L.) Scop., a troublesome weed in corn and other agricultural fields, has developed resistance to herbicides that inhibiting ALS (Acetolactate Synthase), such as nicosulfuron. Understanding the weed's resistance patterns and mechanisms is crucial. However, little is known of the non-target site resistance (NTSR) mechanisms of D. sanguinalis owing to a lack of relevant genome sequences and other materials. Therefore, in this study, a population of D.sanguinalis presenting multiple resistance was tested and found that its high level of resistance to ALS-inhibiting herbicides was not associated with target-related alterations.Administration of P450 inhibitors reversed the resistance to ALS-inhibiting herbicides. Following the application of ALS-inhibiting herbicides, the activities of NADPH-P450 reductase and p-nitroanisole O-demethylase (PNOD) were notably greater in the resistant population of D. sanguinalis than those in the susceptible population. The results suggested P450 enzyme familyplays a major role in the metabolic resistance mechanism, that increased P450 enzyme activity promote cross-resistance in D. sanguinalis to ALS-inhibiting herbicides. RNA-seq analysis showed that five genes from the P450 family (CYP709B2, CYP714C2, CYP71A1, CYP76C2, and CYP81E8) were upregulated in resistant D. sanguinalis. In conclusion, the upregulation of several P450 genes is responsible for establishing resistance to ALS-inhibiting herbicides in D. sanguinalis.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8–14 amino acids inhibited (63–99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46–95%) compared to wildtype when the linker was shortened by 8–14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PreambleThis manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Inflammation-mediated drug interactions of olokizumab and cytochrome P450 activities in patients with rheumatoid arthritis.

In patients with rheumatoid arthritis (RA), interleukin (IL)-6 affects the activity of cytochrome P450 (CYP) enzymes. Treatment with anti-IL-6 therapy can reverse the IL-6-mediated downregulation of CYP enzymes, resulting in changes in plasma levels of CYP substrates. The primary objective of this study was to evaluate the impact of the IL-6 inhibitor olokizumab on the pharmacokinetics of CYP probe substrates in subjects with active RA. Seventeen patients with active RA were orally administered a phenotyping cocktail of midazolam (CYP3A4 substrate), omeprazole (CYP2C19 substrate), warfarin (CYP2C9 substrate) and caffeine (CYP1A2 substrate) alone and 2weeks after a single subcutaneous injection of 128 mg olokizumab. The pharmacokinetic parameters of each substrate were calculated using noncompartmental analysis. Sixteen of 17 enrolled patients received the complete doses of the cocktail drugs and olokizumab and were eligible for the pharmacokinetic evaluations. After single-dose administration of olokizumab, the exposure of midazolam and omeprazole decreased by 30-33% and 26-32%, respectively, compared to when the substrates were administered along via cocktail. In the presence of olokizumab, caffeine exposure increased by 19-23% compared to caffeine administration alone. There were no significant changes in S-warfarin exposure. In patients with active RA, olokizumab potentially reverses the IL-6-mediated suppression of CYP3A4 and CYP2C19. According to FDA guidance, olokizumab is considered a weak inducer of CYP3A4 and CYP2C19.