Cytochrome P450 Transcripts Research Articles

Scopoletin alleviates acetaminophen-induced hepatotoxicity through modulation of NLRP3 inflammasome activation and Nrf2/HMGB1/TLR4/NF-κB signaling pathway.

Scopoleitin (SP), a bioactive compound from many edible plants and fruits, exerts a wide range of biological activities, however the role and mechanism of SP in acetaminophen (APAP)-induced hepatotoxicity remains unclear. In this study, we verified the protective effect of SP on APAP-induced liver injury (AILI) hepatotoxicity and explore the underlying molecular mechanisms. Here, we showed that SP alleviated AILI by reducing serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, hepatic histopathological damage, inflammation, and liver cell apoptosis. In addition, SP attenuated the accumulation of malondialdehyde (MDA) and exhaustion of glutathione (GSH) levels and increased the superoxide dismutase (SOD) levels induced by APAP. Consistently, SP significantly reduced the gene transcription of cytochrome P450 (CYP)2E1, CYP1A2, and CYP3A11 in the livers of mice induced by APAP. Moreover, SP pretreatment effectively promoted the expression of Nrf2, Keap1, and its signal downstream HO-1, NQO1, GCLc, and GCLm, suggesting the activation of the Nrf2 signaling pathway. SP inhibited APAP-induced hepatocyte apoptosis by regulating the protein levels of apoptosis-related proteins (cytochrome C, Bax, Caspase-3, Bcl2, and PARP). SP suppressed APAP-induced expression of NLRP3 and reduced the levels of proinflammatory factors, including tumor necrosis factor-alpha (TNF-α), F4/80, Caspase-1, and interleukin (IL)-1 beta (IL-1β). Moreover, SP downregulated APAP-induced high-mobility group box 1 (HMGB1) and toll-like receptor 4 (TLR4) expression, inhibited nuclear factor kappa-B (NF-κB) and MAPK activation. Taken together, our study reveals the protective roles of SP against AILI through the downregulation of NLRP3 expression, and the inhibition of the Nrf2/HMGB1/TLR4/NF-κB signaling pathways.

Functional Roles of Five Cytochrome P450 Transcripts in the Susceptibility of the Yellow Fever Mosquito to Pyrethroids Revealed by RNAi Coupled With Insecticide Bioassay.

We evaluated the possible roles of five cytochrome P450 transcripts in the susceptibility of both adults and larvae of Aedes aegypti to three pyrethroids using RNA interference (RNAi) coupled with insecticide bioassays. RNAi by feeding larvae with chitosan/dsRNA nanoparticles led to reductions of CYP6AA5, CYP6AL1, CYP9J32, CYP4J16A, and CYP4J16B transcripts by 38.7%, 46.0%, 46.52%, 44.0%, and 41.0%, respectively, and increased larval mortality by 46.0% to permethrin when CYP9J32 was silenced and by 41.2% to cypermethrin when CYP6AA5 was silenced. RNAi by injecting dsRNA in adults led to reductions of CYP6AA5, CYP6AL1, and CY4J16A transcripts by 77.9%, 80.0%, and 87.1% (p < 0.05), respectively, at 96 h and reduction of CYP9J32 transcript by 46.5% at 24 h after injection. In contrast, CYP4J16B was repressed by 78.2% at 72 h after injection. Exposure of the adults injected with CYP6AA5 dsRNA resulted in 1.5- to 2.0-fold increased susceptibility to cypermethrin as compared with the control. Homology modeling of CYP6AA5 followed by ligand docking showed that distances between the heme iron and the putative aromatic hydroxylation site were 9.2, 7.2, and 9.1 Å for permethrin, cypermethrin, and deltamethrin, respectively. For the aliphatic hydroxylation site, these distances were 5.3, 4.9, and 3.1 Å. These results supported that CYP6AA5 may be able to metabolize cypermethrin preferentially by aliphatic hydroxylation as indicated by the close interaction with the heme iron. Our study also suggests that the detoxification roles of cytochrome P450 genes in A. aegypti may vary according to the mosquito developmental stages, cytochrome P450 genes, and insecticides.

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells.

Acrylamide (AA) can be formed during the thermal processing of carbohydrate-rich foods. Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., contaminates many cereal-based products. In addition to potential co-exposure through a mixed diet, co-occurrence of AA and DON in thermally processed cereal-based products is also likely, posing the question of combinatory toxicological effects. In the present study, the effects of AA (0.001-3 mM) and DON (0.1-30 µM) on the cytotoxicity, gene transcription, and expression of major cytochrome P450 (CYP) enzymes were investigated in differentiated human hepatic HepaRG cells. In the chosen ratios of AA-DON (10:1; 100:1), cytotoxicity was clearly driven by DON and no overadditive effects were observed. Using quantitative real-time PCR, about twofold enhanced transcript levels of CYP1A1 were observed at low DON concentrations (0.3 and 1 µM), reflected by an increase in CYP1A activity in the EROD assay. In contrast, CYP2E1 and CYP3A4 gene transcription decreased in a concentration-dependent manner after incubation with DON (0.01-0.3 µM). Nevertheless, confocal microscopy showed comparably constant protein levels. The present study provided no indication of an induction of CYP2E1 as a critical step in AA bioactivation by co-occurrence with DON. Taken together, the combination of AA and DON showed no clear physiologically relevant interaction in HepaRG cells.

Genome sequencing of Elaeocarpus spp. stem blight pathogen Pseudocryphonectria elaeocarpicola reveals potential adaptations to colonize woody bark

BackgroundElaeocarpus spp. stem blight, caused by Pseudocryphonectria elaeocarpicola, is a destructive disease, which will significantly reduce the productivity and longevity of Elaeocarpus spp. plants, especially in the Guangdong Province of China. However, few information is available for P. elaeocarpicola. To unravel the potential adaptation mechanism of stem adaptation, the whole genome of P. elaeocarpicola was sequenced by using the DNBSEQ and PacBio platforms.ResultsP. elaeocarpicola harbors 44.49 Mb genome with 10,894 predicted coding genes. Genome analysis revealed that the P. elaeocarpicola genome encodes a plethora of pathogenicity-related genes. Analysis of carbohydrate-active enzymes (CAZymes) revealed a rich variety of enzymes participated in plant cell wall degradation, which could effectively degrade cellulose, hemicellulose and xyloglucans in the plant cell wall and promote the invasion of the host plant. There are 213 CAZyme families found in P. elaeocarpicola, among which glycoside hydrolase (GH) family has the largest number, far exceeding other tested fungi by 53%. Besides, P. elaeocarpicola has twice as many genes encoding chitin and cellulose degradation as Cryphonectria parasitica, which belong to the same family. The predicted typical secreted proteins of P. elaeocarpicola are numerous and functional, including many known virulence effector factors, indicating that P. elaeocarpicola has great potential to secrete virulence effectors to promote pathogenicity on host plants. AntiSMASH revealed that the genome encoded 61 secondary metabolic gene clusters including 86 secondary metabolic core genes which was much higher than C. parasitica (49). Among them, two gene cluster of P. elaeocarpicola, cluster12 and cluster52 showed 100% similarity with the mycotoxins synthesis clusters from Aspergillus steynii and Alternaria alternata, respectively. In addition, we annotated cytochrome P450 related enzymes, transporters, and transcription factors in P. elaeocarpicola, which are important virulence determinants of pathogenic fungi.ConclusionsTaken together, our study represents the first genome assembly for P. elaeocarpicola and reveals the key virulence factors in the pathogenic process of P. elaeocarpicola, which will promote our understanding of its pathogenic mechanism. The acquired knowledge lays a foundation for further exploration of molecular interactions with the host and provide target for management strategies in future research.

Placental cytochrome P450 methylomes in infants exposed to prenatal opioids: exploring the effects of neonatal opioid withdrawal syndrome on health horizons.

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights. Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications. Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers. Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances. Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment.

The Capsicum terpenoid biosynthetic module is affected by spider-mite herbivory

In response to herbivory, Capsicum annuum leaves adapt their specialized metabolome that may protect the plant against herbivore feeding either directly or indirectly through volatile metabolites acting as cues for natural enemies of the herbivore. The volatile blend of spider-mite infested leaves differs from non-challenged leaves predominantly by a higher contribution of mono- and sesquiterpenes. In addition to these terpenoids released into the headspace, the terpenoid composition of the leaves alters upon herbivory. All this suggests an important role for terpenoids and their biosynthetic machinery in the defence against herbivory. Here, we show that the C. annuum genome contains a terpene synthase (TPS) gene family of 103 putative members of which structural analysis revealed that 27 encode functional enzymes. Transcriptome analysis showed that several TPS loci were differentially expressed upon herbivory in leaves of two C. annuum genotypes, that differ in susceptibility towards spider mites. The relative expression of upstream biosynthetic genes from the mevalonate and the methylerythritol phosphate pathway also altered upon herbivory, revealing a shift in the metabolic flux through the terpene biosynthetic module. The expression of multiple genes potentially acting downstream of the TPSs, including cytochrome P450 monooxygenases, UDP-glucosyl transferases, and transcription factors strongly correlated with the herbivory-induced TPS genes. A selection of herbivory-induced TPS genes was functionally characterized through heterologous expression and the products that these enzymes catalysed matched with the volatile and non-volatile terpenoids induced in response to herbivory.

Comparative transcriptome analysis of Veratrum maackii and Veratrum nigrum reveals multiple candidate genes involved in steroidal alkaloid biosynthesis

Veratrum (Melanthiaceae; Liliales) is a genus of perennial herbs known for the production of unique bioactive steroidal alkaloids. However, the biosynthesis of these compounds is incompletely understood because many of the downstream enzymatic steps have yet to be resolved. RNA-Seq is a powerful method that can be used to identify candidate genes involved in metabolic pathways by comparing the transcriptomes of metabolically active tissues to controls lacking the pathway of interest. The root and leaf transcriptomes of wild Veratrum maackii and Veratrum nigrum plants were sequenced and 437,820 clean reads were assembled into 203,912 unigenes, 47.67% of which were annotated. We identified 235 differentially expressed unigenes potentially involved in the synthesis of steroidal alkaloids. Twenty unigenes, including new candidate cytochrome P450 monooxygenases and transcription factors, were selected for validation by quantitative real-time PCR. Most candidate genes were expressed at higher levels in roots than leaves but showed a consistent profile across both species. Among the 20 unigenes putatively involved in the synthesis of steroidal alkaloids, 14 were already known. We identified three new CYP450 candidates (CYP76A2, CYP76B6 and CYP76AH1) and three new transcription factor candidates (ERF1A, bHLH13 and bHLH66). We propose that ERF1A, CYP90G1-1 and CYP76AH1 are specifically involved in the key steps of steroidal alkaloid biosynthesis in V. maackii roots. Our data represent the first cross-species analysis of steroidal alkaloid biosynthesis in the genus Veratrum and indicate that the metabolic properties of V. maackii and V. nigrum are broadly conserved despite their distinct alkaloid profiles.

Mapping and validation of a new quantitative trait locus (QTL) for fruit size in watermelon (Citrullus lanatus)

Watermelon (Citrullus lanatus) is one of the top ten fruit-bearing species in the world and is mainly grown for its edible fruit. However, the understanding of watermelon fruit size is still incomplete, with relatively few studies on the genetic and molecular mechanisms of this trait. In the present study, three segregation populations from one novel germplasm with small fruit and different inbred lines with medium fruit, big fruit and giant fruit, respectively, were constructed to locate and fine map one new quantitative trait locus (QTL) on chromosome 8 for fruit size in watermelon. Inheritance analysis of these three populations suggested that small fruit was controlled by a single recessive gene in watermelon. We located a new QTL for fruit size on chromosome 8, and developed markers to conduct fine mapping of the genomic region and to identify candidate genes. Specifically, we constructed a high-density genetic linkage map using restriction-site associated DNA (RAD)-based high-throughput genotyping data from a second-filial generation (F2) mapping family derived from novel germplasms with small fruit and big fruit. The total genetic map length was 1315.53 cM with an average inter-locus distance of 0.18 cM, which contained 7,272 high-quality single nucleotide polymorphism (SNP) markers distributed on 11 linkage groups corresponding to the number of chromosome pairs in watermelon. Genome wide QTL mapping revealed one significant QTL (fs-chr8) on chromosome 8 associated with fruit size. One major QTL (fs-B171) on chromosome 8 was also identified in another BC1 population derived from giant watermelon and small watermelon, which overlapped with fs-chr8. Moreover, based on the markers obtained from next-generation sequencing of the parental lines, we conducted fine mapping of the interval to a 304 kb region (from 26.83 to 27.14 Mb) on chromosome 8 using 7 polymerase chain reaction (PCR) based markers. The PCR based markers confirmed the accuracy of our mapping results for watermelon fruit size. Finally, candidate genes underlying fruit size were identified from the reference genome 97103v2, including genes related to cytochrome P450 family enzymes, gibberellin-regulated proteins, transcription factor and basic leucine zipper (bZIP) transcription factor family proteins. Our study provides fine mapping results for watermelon fruit size and will lay the foundation for the identification of candidate genes to elucidate the molecular mechanism underlying fruit size in this species.

CYP1A1, VEGFA and Adipokine Responses of Human Adipocytes Co-exposed to PCB126 and Hypoxia.

It is increasingly recognized that hypoxia may develop in adipose tissue as its mass expands. Adipose tissue is also the main reservoir of lipophilic pollutants, including polychlorinated biphenyls (PCBs). Both hypoxia and PCBs have been shown to alter adipose tissue functions. The signaling pathways induced by hypoxia and pollutants may crosstalk, as they share a common transcription factor: aryl hydrocarbon receptor nuclear translocator (ARNT). Whether hypoxia and PCBs crosstalk and affect adipokine secretion in human adipocytes remains to be explored. Using primary human adipocytes acutely co-exposed to different levels of hypoxia (24 h) and PCB126 (48 h), we observed that hypoxia significantly inhibits the PCB126 induction of cytochrome P450 (CYP1A1) transcription in a dose-response manner, and that Acriflavine (ACF)—an HIF1α inhibitor—partially restores the PCB126 induction of CYP1A1 under hypoxia. On the other hand, exposure to PCB126 did not affect the transcription of the vascular endothelial growth factor-A (VEGFA) under hypoxia. Exposure to hypoxia increased leptin and interleukin-6 (IL-6), and decreased adiponectin levels dose-dependently, while PCB126 increased IL-6 and IL-8 secretion in a dose-dependent manner. Co-exposure to PCB126 and hypoxia did not alter the adipokine secretion pattern observed under hypoxia and PCB126 exposure alone. In conclusion, our results indicate that (1) hypoxia inhibits PCB126-induced CYP1A1 expression at least partly through ARNT-dependent means, suggesting that hypoxia could affect PCB metabolism and toxicity in adipose tissue, and (2) hypoxia and PCB126 affect leptin, adiponectin, IL-6 and IL-8 secretion differently, with no apparent crosstalk between the two factors.

Overexpression of the global regulator FnVeA upregulates antitumor substances in endophytic Fusarium nematophilum.

The special niche of endophytic fungi promotes their potential to produce antitumor compounds with novel structure and significant bioactivity for screening of new antitumor drugs. In our previous studies, we isolated a Fusarium strain from the roots of the medicinal plant Nothapodytes pittosporoides and identified it as Fusarium nematophilum. We found that the crude extract of F. nematophilum had significant antitumor activity on A549 cancer cells, and overexpressing the global regulatory factor FnVeA (the VeA gene of the fungus F. nematophilum) resulted in a significant increase in the antitumor activity, which was approximately fivefold higher than wild strain for relative inhibition rate. In FnVeAOE, the accumulation of indole, alkene, alkaloid, steroid, and flavonoid metabolites with potential antitumor activity was significantly upregulated compared with wild type via metabolomic analysis. Moreover, the transcriptome analysis showed that 134 differential genes were considered to be closely related to the biosynthesis of antitumor substances, of which 59 differential genes were considered as candidate key genes, and related to tryptophan dimethylallyltransferase, cytochrome P450 monooxygenase, polyketide synthases, and transcription factors. Taken together, we suggest that FnVeA may regulate the biosynthesis of antitumor substances by mediating the expression of genes related to secondary metabolic pathways in F. nematophilum.

Study on regulation of CYP450 enzyme system to reduce liver toxicity through compatibility of Aconiti Kusnezoffii Radix Cocta with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma

Aconiti Kusnezoffii Radix Cocta is one of the most commonly used medicinal materials in Mongolian medicine. Due to the strong toxicity of Aconiti Kusnezoffii Radix Cocta, Mongolian medicine often uses Chebulae Fructus, Glycyrrhizae Radix et Rhizoma to reduce the toxicity, so as to ensure the curative effect of Aconiti Kusnezoffii Radix Cocta while ensuring its clinical curative effect, but the mechanism is not clear. The aim of this study was to investigate the effects of Chebulae Fructus, Glycyrrhizae Radix et Rhizoma and Aconiti Kusnezoffii Radix Cocta on the mRNA transcription and protein translation of cytochrome P450(CYP450) in the liver of normal rats. Male SD rats were randomly divided into negative control(NC) group, phenobarbital(PB) group(0.08 g·kg~(-1)·d~(-1)), Chebulae Fructus group(0.254 2 g·kg~(-1)·d~(-1)), Glycyrrhizae Radix et Rhizoma group(0.254 2 g·kg~(-1)·d~(-1)), Aconiti Kusnezoffii Radix Cocta group(0.254 2 g·kg~(-1)·d~(-1))and compatibility group(0.254 2 g·kg~(-1)·d~(-1),taking Aconiti Kusnezoffii Radix Cocta as the standard). After continuous administration for 8 days, the activities of total bile acid(TBA), alkaline phosphatase(ALP), amino-transferase(ALT) and aspartate aminotransferase(AST)in serum were detected, the pathological changes of liver tissue were observed, and the mRNA and protein expression levels of CYP1 A2, CYP2 C11 and CYP3 A1 were observed. Compared with the NC group, the serum ALP, ALT and AST activities in the Aconiti Kusnezoffii Radix Cocta group were significantly increased, and the ALP, ALT and AST activities were decreased after compatibility. At the same time, compatibility could reduce the liver injury caused by Aconiti Kusnezoffii Radix Cocta. The results showed that Aconiti Kusnezoffii Radix Cocta could inhibit the expression of CYP1 A2, CYP2 C11 and CYP3 A1, and could up-regulate the expression of CYP1 A2, CYP2 C11 and CYP3 A1 when combined with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma. The level of translation was consistent with that of transcription. The compatibility of Chebulae Fructus and Glycyrrhizae Radix et Rhizoma with Aconiti Kusnezoffii Radix Cocta could up-regulate the expression of CYP450 enzyme, reduce the accumulation time of aconitine in vivo, and play a role in reducing toxicity, and this effect may start from gene transcription.

Discovery of Major Quantitative Trait Loci and Candidate Genes for Fresh Seed Dormancy in Groundnut

Spanish bunch groundnut varieties occupy most of the cultivated area in Asia and Africa, and these varieties lack required 2-3 weeks of fresh seed dormancy (FSD) hampering kernel quality. Genomic breeding can help to improve commercial groundnut cultivars for FSD in a shorter time with greater precision. In this regard, a recombinant inbred line (RIL) population from the cross ICGV 02266 (non-dormant) × ICGV 97045 (dormant) was developed and genotyped with a 5 K mid-density genotyping assay. A linkage map was constructed with 325 SNP loci spanning a total map length of 2335.3 cM and five major QTLs were identified on chromosomes Ah01, Ah11, Ah06, Ah16 and Ah17. Based on differential gene expression using transcriptomic information from dormant (Tifrunner) and non-dormant (ICGV 91114) genotypes, histone deacetylases, histone-lysine N-methyltransferase, cytochrome P450, protein kinases, and ethylene-responsive transcription factor were identified as key regulators involved in the hormonal regulation of dormancy. Six Kompetitive Allele Specific PCR (KASP) markers were successfully validated in the diverse panel including selected RILs of the same population and germplasm lines. These validated KASP markers could facilitate faster breeding of new varieties with desired dormancy using marker-assisted early generation selection.

Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

Phylogeny of Leptographium qinlingensis cytochrome P450 genes and transcription levels of six CYPs in response to different nutrition media or terpenoids.

The ascomycete Leptographium qinlingensis is one of the major externally living fungal associates of Dendroctonus armandi, which can kill host trees when it invades Chinese white pine (Pinus armandii). We identified and phylogenetically analysed the cytochrome P450 (CYP) genes in the transcriptome of L. qinlingensis. Furthermore, the expression profiles of six CYPs in the mycelium of L. qinlingensis grown on different media or treated with terpenoids were detected, as well as their growth rates on different nutritional media and inhibition by terpenoids. The CYP evolution predicted that most of the CYPs occurred in a putative common ancestor shared between L. qinlingensis and Grosmannia clavigera. The growth rates and inhibition test result shows that L. qinlingensis has more similarity with G. clavigera, which can retrieve nutrition from pine wood and utilize monoterpenes as the sole carbon source. CYP582C and CYP52Z4 of L. qinlingensis might be involved in the metabolism of fatty acids and detoxification of terpenes and phenolics after the analysis of their transcription levels with different treatments.

Organophosphorus pesticide quinalphos (Ekalux 25 E.C.) reduces sperm functional competence and decreases the fertilisation potential in Swiss albino mice.

Quinalphos (QP)is one of the most commonly used organophosphate pesticide for agriculture. In this study,adultSwiss albino male mice were orally administered with 0.25, 0.5 and 1.0mg/kg of QP (Ekalux 25 E.C.) for ten consecutive days and the reproductive function was assessed at 35 and 70days after QP treatment. At highest dose (1.0mg/kg), QP exposure resulted in significant decrease in motility and increase in sperm head defects and DNA damage. Pharmacokinetic data showed a threefold increase in concentration of QP in the testis as compared to serum. QP was detectable in testes even after 24hr of administration indicating slow clearance from tissue. In addition, high oestradiol,low testosterone levelwith a parallel increase in aromatase and cytochrome P450 transcript levelswas observed. Significant decrease in fertilisation, lowerblastocyst rateand poor blastocyst quality was observed when spermatozoa collected from QP exposed mice were subjected to in vitro fertilisation. In conclusion, exposure of QP to male mice decreases the sperm functional competence and fertilising ability, whichappears to bemediated through elevated oxidative stress and altered steroidogenesis in testes.

A novel epigenetic mechanism unravels hsa-miR-148a-3p-mediated CYP2B6 downregulation in alcoholic hepatitis disease

Cytochrome P450 (CYP) enzymes play critical roles in drug transformation, and the total CYPs are markedly decreased in alcoholic hepatitis (AH), a fatal alcoholic liver disease. miRNAs are endogenous small noncoding RNAs that regulate many essential biological processes. Knowledge concerning miRNA regulation of CYPs in AH disease is limited. Here we presented the changes of key CYPs in liver samples of AH patients retrieved from GEO database, performed in silico prediction of miRNAs potentially targeting the dysregulated CYP transcripts, and deciphered a novel mechanism underlying miRNA mediated CYPs expression in liver cells. Nine miRNAs were predicted to regulate CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A4, among which hsa-miR-148a-3p was selected as a case study. Biochemical and molecular evidences demonstrated that miR-148a promoted CYP2B6 expression by increasing mRNA stability via directly binding to the 3′UTR sequence, and that this positive posttranscriptional regulation was AGO1/2-dependent. Further, luciferase reporter gene assay and RNA secondary structure analysis illustrated that the seedless target site, not the seed target site, controlled miR-148a-mediated CYP2B6 upregulation. Moreover, we identified HNF4A as a liver-specific transcription factor of MIR-148A through EMSA and chromatin immunoprecipitation experiments. In conclusion, ethanol downregulated miR-148a in hepatocytes through HNF4A regulation, which eventually decreased CYP2B6 expression. Our finding will benefit the understanding of dysregulated drug metabolism in AH patients and highlight an unconventional mechanism for epigenetic regulation of CYP gene expression.

Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis.

Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

Expression of cytochrome P450 isozyme transcripts and activities in human livers

Individual differences in cytochrome P450 (CYP) enzymes contribute to responses to drugs and environmental chemicals. The expression of CYPs is influenced by sex, age, and ethnicity. Human CYP studies are often conducted with human liver microsomes and liver cells to evaluate chemical induction and drug interactions. However, the basal or constitutive expression of CYP transcripts and enzyme activities in the intact liver are also important in our understanding of individual variation in CYPs. This study utilised 100 human liver samples to profile the constitutive expression of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A11 enzyme activity and transcript levels. The mRNA expression of the CYPs and xenobiotic receptors AhR, CAR, and PXR was examined via qPCR. Results showed that there was greater inter-individual variation in mRNA expression than in enzyme activities, except for CYP2C19. Females had higher CYP3A4 activity than males. Children had lower CYP4A14 activity, while elderly had lower P450 oxidoreductase activity. Compared to Caucasians, Hispanics had higher CYP2C8 activity and higher CYP2B6, CYP2C9, and CYP2C19 mRNA expression, whereas African Americans had lower CYP2D6 mRNA expression. These results add to our understanding of individual variations in xenobiotic metabolism and toxicology.

Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish.

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4′-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4’-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Transcriptome Analysis of Pyrethroid-Resistant Chrysodeixis includens (Lepidoptera: Noctuidae) Reveals Overexpression of Metabolic Detoxification Genes.

Chrysodeixis includens (Walker, [1858]) is one of the most important defoliator of soybean in Brazil because of its extensive geographical distribution and high tolerance to insecticides compared with other species of caterpillars. Because of this, we conducted bioassays to evaluate the efficacy of pyrethroid λ-cyhalothrin on a C. includens resistant strain (MS) and a susceptible (LAB) laboratory strain. High throughput RNA sequencing (RNA-seq) of larval head and body tissues were performed to identify potential molecular mechanisms underlying pyrethroid resistance. Insecticide bioassays showed that MS larvae exhibit 28.9-fold resistance to pyrethroid λ-cyhalothrin relative to LAB larvae. RNA-seq identified evidence of metabolic resistance in the head and body tissues: 15 cytochrome P450 transcripts of Cyp6, Cyp9, Cyp4, Cyp304, Cyp307, Cyp337, Cyp321 families, 7 glutathione-S-transferase (Gst) genes, 7 α-esterase genes from intracellular and secreted catalytic classes, and 8 UDP-glucuronosyltransferase (Ugt) were overexpressed in MS as compared with LAB larvae. We also identified overexpression of GPCR genes (CiGPCR64-like and CiGPCRMth2) in the head tissue. To validate RNA-seq results, we performed RT-qPCR to assay selected metabolic genes and confirmed their expression profiles. Specifically, CiCYP9a101v1, CiCYP6ae149, CiCYP6ae106v2, CiGSTe13, CiCOE47, and CiUGT33F21 exhibited significant overexpression in resistant MS larvae. In summary, our findings detailed potential mechanisms of metabolic detoxification underlying pyrethroid resistance in C. includens.