Association between CYP3A5 Gene Polymorphism and Post-Transplant Acute Immune Rejection in Renal Transplant Recipients Receiving Tacrolimus Therapy: A Correlation Study.

BACKGROUND: Acute immune rejection remains a challenge in the post-transplant period, with approximately 7.8% of renal transplant recipients experiencing rejection episodes within the first year. Genetic polymorphisms in the CYP3A5 gene, which influences tacrolimus metabolism, have garnered interest regarding their association with clinical outcomes in renal transplantation. METHODS: This retrospective correlation study analysed clinical data from kidney transplant patients who received tacrolimus treatment at our hospital from June 2015 to June 2023. The presence of CYP3A5 gene polymorphisms, tacrolimus trough levels, and demographic and clinical data were collected and analysed. RESULTS: A total of 105 kidney transplant patients were included. Patients were divided into acute immune rejection (n = 56) and non-acute immune rejection (n = 49) groups. The distribution of CYP3A5 gene polymorphisms differed significantly between the acute rejection and non-acute rejection groups (p = 0.037). The acute rejection group exhibited a higher frequency of CYP3A5 *1/*1 or *1/*3 genotypes than the non-acute rejection group. No statistically significant differences were found in the tacrolimus trough levels between the two groups. Correlation analysis revealed a statistically significant correlation between CYP3A5 gene polymorphism and post-transplant acute immune rejection (r = 0.223, p < 0.05). CONCLUSIONS: This study demonstrated a significant association between CYP3A5 gene polymorphism and the risk of post-transplant acute immune rejection in renal transplant recipients receiving tacrolimus therapy. These findings highlighted the importance of genetic variability in tacrolimus metabolism when managing immunosuppressive therapy in transplant recipients.

AHR Dependent Toxicity by Retene Requires Metabolic Competence.

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds frequently detected in the environment with widely varying toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of genes, including xenobiotic metabolizing enzymes like Cytochrome P450s (CYPs); however, not all PAHs act via this mechanism. We screened several parent and substituted PAHs in in vitro AHR activation assays to classify their unique activity. Retene (1-methyl-7-isopropylphenanthrene) displays Ahr2 dependent teratogenicity in zebrafish, but did not activate human AHR or zebrafish Ahr2, suggesting a retene metabolite activates Ahr2 in zebrafish to induce developmental toxicity. To investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a, cyp1b1, and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. Cyp1a-null fish were generated using CRISPR-Cas9. Cyp1a-null fish showed increased sensitivity to retene toxicity, while Cyp1b1-null fish were less susceptible, and microbiome elimination had no significant effect. Zebrafish required exposure to retene between 24 and 48 hours post fertilization (hpf) to exhibit toxicity. After static exposure, retene concentrations in zebrafish embryos increased until 24 hpf, peaked between 24 and 36 hpf, and decreased rapidly thereafter. We detected retene metabolites at 36 and 48 hpf, indicating metabolic onset preceding toxicity. This study highlights the value of combining molecular and systems biology approaches with mechanistic and predictive toxicology to interrogate the role of biotransformation in AHR-dependent toxicity.

Unraveling the molecular significance of CYP1A2 (rs762551; c.-9-154 C>A) genetic variant on breast carcinoma susceptibility: Insights from case-control study and meta-analysis.

BACKGROUND: The human cytochrome P450 (CYP) superfamily encompasses different categories of isoenzymes that contribute to multiple metabolic processes involving drug detoxification, cellular signaling, and the proliferation of malignant tissues. Using genetic technology, customized bioinformatic analysis, and meta-analysis design, the main goal of this study was to identify the association between the CYP1A2*rs762551 variant and the susceptibility to breast carcinoma (BRCA). METHODS: The case-control study was conducted based on 104 BRCA women and 102 healthy controls. Using the TaqMan allelic discrimination assay, the CYP1A2 (rs762551; c.-9-154 C>A) variant was genotyped. Bioinformatic frameworks and logistic regression analysis were used to assess the involvement of this genetic variant in BRCA development. A meta-analysis design was accomplished based on our case-control study and other previously published records. Publication bias, heterogeneity between studies, and trial sequential analysis (TSA) were analyzed. RESULTS: The CYP1A2*rs762551 variant conferred protection against BRCA development under allelic (OR = 0.48, p-value < 0.001), dominant (OR = 0.34, p-value < 0.001), and recessive (OR = 0.44, p-value = 0.011) models. However, this intronic variant was correlated with a decreased risk of BRCA among late-onset menopause women compared to other cases. Bioinformatic analysis confirmed that this genetic variant has a functional impact on the progression of tumorgenesis. Moreover, this meta-analysis design included 12922 BRCA women and 15603 healthy controls. Our findings disclosed the contribution of the CYP1A2*rs762551 variant with protection against cancer development among Caucasian females under allelic (OR = 0.75, p-value = 0.025), and dominant (OR = 0.58, p-value = 0.015) models. CONCLUSIONS: This case-control study confirmed the contribution of the CYP1A2*rs762551 variant with decreased risk of BRCA development among Egyptian subjects. Moreover, BRCA women with late-onset menopause conferred protection against cancer progression compared to other subjects. Our findings identified that this meta-analysis design achieved protection against BRCA development among Caucasian women compared to other ethnicities.

Lambda-cyhalothrin induces heart injury in chickens by regulating cytochrome P450 enzyme system and inhibiting Nrf2/HO-1 pathway.

Lambda-cyhalothrin (LCT) is a common pyrethroid insecticide widely used for ectoparasite control and hygiene pest prevention in poultry and this study aimed to investigate the mechanisms of LCT-induced cardiac injury in chickens. Low, medium, and high-dose LCT exposure models in chickens were established and hematoxylin and eosin (H&E) staining, dihydroethidium (DHE) staining, TUNEL staining, immunofluorescence, biochemical analysis, and gene expression analysis were used to study the effects of LCT exposure on the chicken heart. The results showed that LCT exposure increased the serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH), led to muscle fiber breakage and inflammatory cell infiltration and caused cardiac tissue damage. The DHE staining and biochemical analysis revealed that LCT exposure resulted in the excessive accumulation of ROS, decreased activities/levels of catalase (CAT), total superoxide dismutase (T-SOD), and glutathione (GSH), and increased levels of the oxidative damage marker malondialdehyde (MDA). The TUNEL staining indicated that LCT exposure increased apoptosis possibly through the elevated expression of pro-apoptotic genes in the mitochondrial pathway, the reduced expression of anti-apoptotic genes, the upregulation of pro-inflammatory factors and the downregulation of anti-inflammatory factors. Here, LCT exposure significantly inhibited the expression of genes in the Nrf2/HO-1 pathway and activated the expression of genes in the CYP450 enzyme system. Compared to the low-dose group, the high-dose LCT exposure group showed lower levels of apoptosis and inflammation, possibly related to the low oxidative stress levels mediated by the decreased expression of the CYP450 enzyme system. In conclusion, LCT exposure induces oxidative stress, apoptosis, and inflammation in chicken hearts, which may be associated with the inhibition of the Nrf2/HO-1 pathway and activation of the CYP450 enzyme system. This study provides a theoretical basis for the safer use of insecticides in poultry production.

Gal-1-mediated cytochrome p450 activation promotes fibroblast into myofibroblast differentiation in pulmonary fibrosis.

Pulmonary fibrosis (PF) results from excessive extracellular matrix (ECM) deposition and tissue remodeling after activation of fibroblasts into myofibroblasts. Abnormally deposited fibrotic ECM, in turn, promotes fibroblast activation and accelerates loss of lung structure and function. However, the molecular mediators and exact mechanisms by which fibrotic ECM promotes fibroblast activation are unclear. In a bleomycin-induced PF mouse model, we found Galectin-1 (Gal-1) expression was significantly increased in lung tissue, and overexpression of Gal-1 plasmid-transfected fibroblasts were activated into myofibroblasts. Using the decellularization technique to prepare decellularized fibrotic ECM and constructing a 3D in vitro co-culture system with fibroblasts, we found that decellularized fibrotic ECM induced a high expression of Gal-1 and promoted the activation of fibroblasts into myofibroblasts. Therefore, Gal-1 has been identified as a pivotal mediator in PF. Further, we found that decellularized fibrotic ECM delivered mechanical signals to cells through the Gal-1-mediated FAK-Src-P130Cas mechanical signalling pathway, while the CYP450 enzymes (mainly involved in CYP1A1, CYP24A1, CYP3A4, and CYP2D6 isoforms) acted as a chemical signalling pathway to receive mechanical signals transmitted from upstream Gal-1, thereby promoting fibroblast activation. The Gal-1 inhibitor OTX008 or the CYP1A1 inhibitor 7-Hydroxyflavone prevented PF in mice and inhibited the role of fibrotic ECM in promoting fibroblast activation into myofibroblasts, preventing PF. These results reveal novel molecular mechanisms of lung fibrosis formation and identify Gal-1 and its downstream CYP1A1 as potential therapeutic targets for PF disease treatmnts.

Systematic Review of Naturally Derived Substances That Act as Inhibitors of the Nicotine Metabolizing Enzyme Cytochrome P450 2A6.

Tobacco smoking has been highlighted as a major health challenge in modern societies. Despite not causing death directly, smoking has been associated with several health issues, such as cardiovascular diseases, respiratory disorders, and several cancer types. Moreover, exposure to nicotine during pregnancy has been associated with adverse neurological disorders in babies. Nicotine Replacement Therapy (NRT) is the most common strategy employed for smoking cessation, but despite its widespread use, NRT presents with low success and adherence rates. This is attributed partially to the rate of nicotine metabolism by cytochrome P450 2A6 (CYP2A6) in each individual. Nicotine addiction is correlated with the high rate of its metabolism, and thus, novel strategies need to be implemented in NRT protocols. Naturally derived products are a cost-efficient and rich source for potential inhibitors, with the main advantages being their abundance and ease of isolation. This systematic review aims to summarize the natural products that have been identified as CYP2A6 inhibitors, validated through in vitro and/or in vivo assays, and could be implemented as nicotine metabolism inhibitors. The scope is to present the different compounds and highlight their possible implementation in NRT strategies. Additionally, this information would provide valuable insight regarding CYP2A6 inhibitors, that can be utilized in drug development via the use of in silico methodologies and machine-learning models to identify new potential lead compounds for optimization and implementation in NRT regimes.

Identification and Expression Analysis of Cytochrome P450 Genes Probably Involved in Triterpenoid Saponins Biosynthesis in Astragalus mongholicus.

Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.

Identification and expression dynamics of CYPome across different developmental stages of Maconellicoccus hirsutus (Green).

Maconellicoccus hirsutus is a highly polyphagous insect pest, posing a substantial threat to various crop sp., especially in the tropical and sub-tropical regions of the world. While extensive physiological and biological studies have been conducted on this pest, the lack of genetic information has hindered our understanding of the molecular mechanisms underlying its growth, development, and xenobiotic metabolism. The Cytochrome P450 gene, a member of the CYP gene superfamily ubiquitous in living organisms is associated with growth, development, and the metabolism of both endogenous and exogenous substances, contributing to the insect's adaptability in diverse environments. To elucidate the specific role of the CYP450 gene family in M. hirsutus which has remained largely unexplored, a de novo transcriptome assembly of the pink mealybug was constructed. A total of 120 proteins were annotated as CYP450 genes through homology search of the predicted protein sequences across different databases. Phylogenetic studies resulted in categorizing 120 CYP450 genes into four CYP clans. A total of 22 CYP450 families and 30 subfamilies were categorized, with CYP6 forming the dominant family. The study also revealed five genes (Halloween genes) associated with the insect hormone biosynthesis pathway. Further, the expression of ten selected CYP450 genes was studied using qRT-PCR across crawler, nymph, and adult stages, and identified genes that were expressed at specific stages of the insects. Thus, the findings of this study reveal the expression dynamics and possible function of the CYP450 gene family in the growth, development, and adaptive strategies of M. hirsutus which can be further functionally validated.

Novel Approaches for the Enhancement of Bioavailability of Drugs: An Updated Review.

In medicine, bioavailability is the percentage of a drug that enters the bloodstream and can be used to treat a patient. It has proven challenging throughout time to develop techniques that allow oral administration of most drugs, regardless of their properties, to achieve therapeutic systemic availability. This will be an impressive feat, considering that over 90% of pharmaceuticals are known to have limitations on their oral bioavailability. Improving bioavailability is crucial for optimizing the efficacy and safety of drugs. This review covers a wide range of techniques, including physical, chemical, and formulation approaches, highlighting their mechanisms, advantages, and limitations. Inhibitions of efflux pumps, inhibition of presystemic metabolism, and innovative drug delivery systems that capitalize on the gastrointestinal regionality of medicines are some of the new techniques that have drawn increased interest. Nanotechnology in pharmaceuticals is also being used in this field. We have collected the literature data from 2009 to 2024 using Science Direct, PubMed/Medline, Scopus, and Google Scholar.

Metabolism of 2,4-D in plants: comparative analysis of metabolic detoxification pathways in tolerant crops and resistant weeds.

The commercialization of 2,4-D (2,4-dichlorophenoxyacetic acid) latifolicide in 1945 marked the beginning of the selective herbicide market, with this active ingredient playing a pivotal role among commercial herbicides due to the natural tolerance of monocots compared with dicots. Due to its intricate mode of action, involving interactions within endogenous auxin signaling networks, 2,4-D was initially considered a low-risk herbicide to evolve weed resistance. However, the intensification of 2,4-D use has contributed to the emergence of 2,4-D-resistant broadleaf weeds, challenging earlier beliefs. This review explores 2,4-D tolerance in crops and evolved resistance in weeds, emphasizing an in-depth understanding of 2,4-D metabolic detoxification. Nine confirmed 2,4-D-resistant weed species, driven by rapid metabolism, highlight cytochrome P450 monooxygenases in Phase I and glycosyltransferases in Phase II as key enzymes. Resistance to 2,4-D may also involve impaired translocation associated with mutations in auxin/indole-3-acetic acid (Aux/IAA) co-receptor genes. Moreover, temperature variations affect 2,4-D efficacy, with high temperatures increasing herbicide metabolism rates and reducing weed control, while drought stress did not affect 2,4-D efficacy. Research on 2,4-D resistance has primarily focused on non-target-site resistance (NTSR) mechanisms, including 2,4-D metabolic detoxification, with limited exploration of the inheritance and genetic basis underlying these traits. Resistance to 2,4-D in weeds is typically governed by a single gene, either dominant or incompletely dominant, raising questions about gain-of-function or loss-of-function mutations that confer resistance. Future research should unravel the physiological and molecular-genetic basis of 2,4-D NTSR, exploring potential cross-resistance patterns and assessing fitness costs that may affect future evolution of auxin-resistant weeds. © 2024 Society of Chemical Industry.

Metabolic activation of WHO-congeners PCB28, 52, and 101 by human CYP2A6: evidence from in vitro and in vivo experiments.

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4'-trichlorobiphenyl (PCB28), 2,2',5,5'-tetrachlorobiphenyl (PCB52), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61 µg/L (5,89 nM) for PCB28, 316.98 µg/L (1,03 µM) for PCB52, and 151.1 µg/L (441 nM) for PCB101 from a 20 µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners.

Qualitative and quantitative status of cytochrome P450s after the administration of a liposomal platelet substitute in rat liver.

In the process of the drug development, studies on the cytochrome P450 (CYP) profiles after its administration provided fundamental information regarding drug interactions with concomitantly administered drugs. Here, we evaluated the influence of the administration of H12-(ADP)-liposomes, a platelet substitute, on the mRNA and protein expression, and metabolic activity of CYPs, with focus on the CYP1A2, CYP2C11 and CYP3A2, in rat liver.At 24 h after administering saline or H12-(ADP)-liposomes (10 mg of lipids/kg), a quantitative RT-PCR and western blot analysis revealed that the mRNA and proteins expression of all of the target hepatic CYP isoforms were not different between the saline and H12-(ADP)-liposome groups. Furthermore, an ex vivo CYP metabolic activity assay showed that hepatic CYP metabolic activities in the H12-(ADP)-liposome group were comparable to the corresponding saline group. On the other hand, the area under the blood concentration-time curve for substitutes for CYP1A2 and CYP2C11 was higher in H12-(ADP)-liposome group than in saline group, but the degree of elevations was negligible levels.At a minimum, based on these results, we conclude that H12-(ADP)-liposomes have no quantitative and qualitative effect on the hepatic CYP isoforms, indicating that the drug interactions of H12-(ADP)-liposomes with CYP-metabolizing drugs would be negligible.

Cytochrome P450 F3 promotes colorectal cancer via inhibiting NRF2-mediated ferroptosis.

Cytochrome P450 F3 (CYP4F3) is recognized as a disease-associated immune response initiator that is involved in the synthesis of cholesterol, steroids, and lipids. This study identified the upregulation of CYP4F3 expression in colorectal cancer (CRC) and its association with poor patient prognosis through a comparative analysis between CRC tumor tissues with normal tissues from public databases. The overexpression of CYP4F3 in CT26.wt and SW620, promoted cell proliferation and migration, a reduction of cellular oxidative stress, an up-regulation of the oxidative stress-related pathway NRF2, and an inhibition of cellular ferroptosis. Additionally, inhibition of NRF2 activity stimulated cellular ferroptosis when CYP4F3 was overexpressed. Ferroptosis, characterized by iron-dependent lipid peroxidation, is a non-apoptotic way of cell death with a critical role in cancer development. When given a ferroptosis agonist to CYP4F3-overexpression CRC cells, NRF2 was activated, and cell proliferation and migration were reduced. Furthermore, the mice subcutaneously injected with CYP4F3-overexpression CT26.wt cells formed significantly larger tumors compared to the CYP4F3-vector CT26.wt cell group. This study systematically identified an important role of CYP4F3 in CRC development as a regulator of CRC cells to escape ferroptosis via NRF2, highlighting the significance of CYP4F3 as a potential therapeutic target for CRC.

Dietary oxyclozanide influences antioxidant enzyme activities and damages DNA in Galleria mellonella (Lepidoptera: Pyralidae).

Oxyclozanide (OXY) is an anthelmintic widely used in the treatment of flatworm infection and fasciolosis. It also has antiadenovirus, antibiofilm, antifungal, and antibacterial activities. Various chemicals have been suggested as alternative chemicals in insect pest management. Here, the oxidative and genotoxic effects of OXY on 7th instars, pupae and adults of the model organism Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) were examined. First-instar larvae were reared on 0.003, 0.03, 0.3, and 1.5 g OXY per 100 g artificial diets. Compared with all tested OXY concentrations and controls without OXY, dietary OXY led to increased antioxidant capacity and genotoxic effects. Concentrations of malondialdehyde, an oxidative stress marker, were significantly increased in adults of larvae reared on OXY-charged diets at 0.3 and 1.5 g/100 g compared to the adult control group. We also recorded a significant increase in the genotoxic test data (Tail length, Tail DNA %, Tail moment) at the same stages and concentrations. We recorded significant increases in glutathione-S-transferase, superoxide dismutase (SOD) and glutathione peroxidase activities in larvae fed high OXY concentrations. SOD and catalase activities were also significantly increased at the concentration of 0.03 g/100 g of OXY in the pupal and adult stages. Cytochrome P450 monooxygenase activity was significantly increased at the highest concentration of OXY in the larval and pupal stages. Also, our regression analysis indicates a correlation between the markers of oxidative stress, antioxidant enzymes and comet parameters. These data indicate that OXY induces oxidative stress and antioxidative enzyme response.

Effects of Berberis vulgaris, and its active constituent berberine on cytochrome P450: a review.

The cytochrome P450 (CYP450) family is crucial for metabolizing drugs and natural substances. Numerous compounds, such as pharmaceuticals and dietary items, can influence CYP activity by either enhancing or inhibiting these enzymes, potentially leading to interactions between drugs or between drugs and food. This research explores the impact of barberry and its primary component "berberine" on key human CYP450 enzymes. The text discusses the effects of this plant on the 12 primary human CYP450 enzymes, with summarized data presented in tables. Berberine exerts an influence on the function of various CYP450 isoforms, including CYP3A4/5, CYP2D6, CYP2C9, CYP2E1, CYP1A1/2, and most isoforms within the CYP2B subfamily. Given the significant role of these CYP450 isoforms in metabolizing commonly used drugs and endogenous substances, as well as activating procarcinogens into carcinogenic metabolites, the influence of barberry and its active constituent on these enzymes may impact the pharmacokinetics and toxicity profiles of various compounds. More specifically, regarding the crucial role of CYP2D6 and CYP3A4 in metabolizing clinically used drugs, and the inhibitory effects of berberine on these two CYP450 isoforms, it seems that the most important drug interaction of berberine that should be considered is related to its inhibitory effect on CYP2D6 and CYP3A4. In conclusion, due to the impact of barberry on multiple CYP450 isoforms, healthcare providers should conduct thorough consultations and investigations to ensure patient safety and prevent any potential adverse interactions before recommending the consumption of these herbs. Additional research, particularly clinical trials is crucial for preventing any potentially adverse interactions in patients who consume this herb.

Metabolic characterization of the new benzimidazole synthetic opioids - nitazenes.

The recent re-emergence and the increasing popularity of nitazenes, a group of new synthetic opioids (NSO) that belong to the benzimidazole chemical class, has raised public health concerns. As a class of potential opioid analgesic agents whose development was discontinued in the 1960s due to their high potential for abuse, very little is known about their metabolism and physiologic disposition. In the current study, three nitazenes-butonitazene, isotonitazene and protonitaze were incubated in human liver microsomes (HLM), human S9 (HS9) fractions and recombinant cytochrome P450 enzymes. All three nitazenes were rapidly metabolized in both HLM and HS9 with over 95% depletion within 60 min. In HLM, butonitazene, isotonitazene and protonitazene had in vitro intrinsic clearance (CLint) (µL/min/mg protein) values of 309, 221 and 216 respectively compared to 150 of verapamil, the positive control. In HS9, CLint values were 217, 139, and 150 for butonitazene, isotonitazene and protonitazene respectively compared to only 35 for testosterone, the control probe substrate. Putative metabolite identified from this study include products of hydroxylation, desethylation, dealkylation, desethylation followed by dealkylation, and desethylation followed by hydroxylation. The metabolic phenotyping showed CYP2D6, CYP2B6 and CYP2C8 and the major hepatic enzymes responsible for the metabolism of nitazenes. Within 30 min of incubation, CYP2D6 depleted butonitazene (99%), isotonitazene (72%) and butonitazene (100%) significantly. The rapid metabolism of nitazenes may be an important factor in accurate and timely detections and quantitation of the unchanged drugs in human matrices following intoxication or in forensic analysis. The involvement of multiple polymorphic CYPs in their metabolism may play important roles in the susceptibility to intoxication and/or addiction, depending on the activity of the metabolites.

Brassica napus cytochrome P450 superfamily: Origin from parental species and involvement in diseases resistance, abiotic stresses tolerance, and seed quality traits.

Cytochromes P450 monooxygenases (CYP450s) constitute the largest enzymic protein family that is widely present in plants, animals, and microorganisms, participate in numerous metabolic pathways, and play diverse roles in development, metabolism, and defense. Rapeseed (Brassica napus) is an important oil crop worldwide and have many versions of reference genome. However, there is no systemically comparative genome-wide analysis of CYP450 family genes in rapeseed and its parental species B. rapa and B. oleracea. In this study, we identified 765, 293 and 437 CYP450 genes in B. napus, B. rapa and B. oleracea, respectively, which were unevenly located in A01-A10 and/or C01-C09 chromosomes in corresponding species. Phylogenetic relationship analysis indicated that 1745 CYP450 proteins from three Brassica species and Arabidopsis were divided into 4 groups. Whole genome duplication (WGD) or segmental duplication resulted in gene expansion of CYP450 family in three Brassica species. There were 33-83 SSR loci in CYP450 genes of three Brassica species, and numerous transcription factor binding sites were identified in their promoters. A total of 459-777 miRNAs were predicted to target 174-426 CYP450 genes in three Brassica species. Based on transcriptome data, BnCYP450s, BrCYP450s and BoCYP450s were differentially expressed in various tissues. There existed numerous BnCYP450 DEGs in response to pathogens and abiotic stresses. Besides, many BnCYP450 DEGs were involved in the regulation of important traits, such as seed germination, seed ALA content, and yellow-seed. The qRT-PCR experiment confirmed the transcriptome analysis results by validating two representative Sclerotinia-responsive BnCYP450 DEGs as an example. Three BnCYP450s genes (CYP707A1, CYP81F1, CYP81H1) might be regulated by seed-specific transcription factors BnTT1 and BnbZIP67 to participate in the development and metabolism of seed coat and embryo by undertaking related metabolic reactions.

An ancient role for CYP73 monooxygenases in phenylpropanoid biosynthesis and embryophyte development.

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species-the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis-consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology.

Intraplantar aminoglutethimide, a P450scc inhibitor, reduced the induction of mechanical allodynia in a rat model of thrombus-induced ischemic pain.

Neuroactive steroids (NASs) directly affect neuronal excitability. Despite their role in the nervous system is intimately linked to pain control, knowledge is currently limited. This study investigates the peripheral involvement of NASs in chronic ischemic pain by targeting the cytochrome P450 side-chain cleavage enzyme (P450scc). Using a rat model of hind limb thrombus-induced ischemic pain (TIIP), we observed an increase in P450scc expression in the ischemic hind paw skin. Inhibiting P450scc with intraplantar aminoglutethimide (AMG) administration from post-operative day 0 to 3 significantly reduced the development of mechanical allodynia. However, AMG administration from post-operative day 3 to 6 did not affect established mechanical allodynia. In addition, we explored the role of the peripheral sigma-1 receptor (Sig-1R) by co-administering PRE-084 (PRE), a Sig-1R agonist, with AMG. PRE reversed the analgesic effects of AMG during the induction phase. These findings indicate that inhibiting steroidogenesis with AMG alleviates peripheral ischemic pain during the induction phase via Sig-1Rs.

[Research progress in biosynthesis-related enzymes of coumarin compounds and their bioactivities].

Coumarins are natural products with benzopyran ring as the parent nucleus. Numerous coumarin derivatives exhibit a variety of pharmacological activities, including antibacterial, anti-inflammatory, antitumor, anti-coagulant, anti-osteoporotic, and insecticidal activities. Therefore, they play an important role in both medicine and agriculture. The development and utilization of coumarin derivatives have attracted increasing attention. The advancement of gene sequencing technology and the rapid progress in synthetic bio-logy have led to significant advancement in the biosynthesis of coumarin derivatives, and has received increasing attention from global researchers. This paper presents a comprehensive overview of the key biosynthesis-related enzymes of coumarin derivatives, such as cytochrome P450 enzyme(CYP450), prenyltransferase(PT), UDP-glucosyltransferase(UGT). Additionally, the pharmacological activities of these enzymes, including anti-tumor, anti-inflammatory, antioxidant, and antibacterial activities, are systematically summarized. This review aims to provide a valuable reference for the biosynthesis of coumarin derivatives and further exploration of their medicinal potential.