Cytochrome P450 1A1 Enzyme Activity Research Articles

Inhibition of cytochrome P450 3A4 enzyme activity by alkaloid psilocin from magic mushrooms: potential for drug interactions

Inhibition of cytochrome P450 3A4 enzyme activity by alkaloid psilocin from magic mushrooms: potential for drug interactions

Delayed clearance of the pro-carcinogen benzo[a]pyrene in PLHC-1 cells when co-exposed to the antifungal drug clotrimazole and effects on the CYP1A biomarker

Cytochrome P450 1 A (CYP1A) is a key enzyme in the metabolism of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in animals, and a biomarker for environmental PAH exposure. The common antimycotic imidazole drug clotrimazole (CLO) has been detected in the aquatic environment and likely co-exists with BaP. Like BaP, CLO can bind to CYP1A enzymes and can act as a CYP1A inhibitor. Co-exposure of BaP with CLO significantly delayed BaP elimination in a fish liver cell line (PLHC-1). Intracellular BaP concentration was 2.4 times higher after 6 h in co-exposed cells, compared to cells exposed to BaP alone. Higher BaP concentrations in cells co-exposed to CLO positively correlated with CLO dose, indicating CLO-mediated delays in BaP clearance. After 24 h, BaP was undetectable irrespective of CLO co-exposure. In contrast, intracellular CLO concentrations remained constant over the 72 h experimental period. Co-exposure of BaP with CLO caused synergistic and time-dependent increases on the CYP1A biomarker both on CYP1A mRNA levels and on CYP1A enzyme activity, in accordance with an apparent delayed BaP elimination in the presence of CLO. These results indicate a toxicokinetic interaction between BaP and CLO on the CYP1A enzyme that delays metabolic clearance of BaP.

6-Formylindolo[3,2-b]carbazole Protects Against Angiotensin II-Induced Cellular Hypertrophy through the Induction of Cytochrome P450 1A1 and Its Associated 19(S)-HETE Metabolite In Vitro.

Aryl hydrocarbon receptor (AhR) is a multifunctional receptor that regulates cytochrome P450 1A1 (CYP1A1), an arachidonic acid (AA) metabolizing enzyme producing 19-hydroxyeicosatetraenoic acid (HETE). 6-formylindolo[3,2-b]carbazole (FICZ) demonstrates great affinity toward the AhR. Recently, we have shown that 19(S)-HETE is preferentially cardioprotective. This study investigates the role of FICZ on AhR and cytochrome P450 (CYP) 1A1-mediated AA metabolism and whether it attenuates angiotensin (Ang) II-induced cardiac hypertrophy. Adult human ventricular cardiomyocytes cell line treated with FICZ in the presence and absence of Ang II 10 μM. Protein levels of AhR and CYPs were determined by Western blot analysis and the mRNA expression of cardiac hypertrophic markers and CYPs were determined by real-time polymerase chain reaction. CYP1A1 enzyme activity and proteasomal degradation were determined by 7-ethoxyresorufin O-deethylase and proteasome 20S activity assays, respectively. Liquid chromatography tandem mass spectrometry was used to measure AA metabolites. Our results show that Ang II-induced cardiac hypertrophy modulates AA metabolites in an enantioselective manner, and that FICZ activates AhR in a time-dependent manner, inhibits AhR proteasomal degradation, induces CYP1A1, increases the concentration of 19(S)-HETE, and attenuates Ang II-induced cardiac hypertrophy by inhibiting the hypertrophic markers and decreasing cell surface area through midchain-HETE-dependent mechanism. In conclusion, the results demonstrate the ability of FICZ to protect against Ang II-induced cardiac hypertrophy by increasing the concentration of 19(S)-HETE through AhR regulated enzyme induction and inhibition of midchain-HETEs metabolites. SIGNIFICANCE STATEMENT: This study shows that 6-formylindolo[3,2-b]carbazole attenuate angiotensin II-induced cellular hypertrophy. The novel findings of our investigation are in characterizing the aryl hydrocarbon receptor involvement and the enantioselective differences in arachidonic acid metabolism in cardiac hypertrophy, which opens a new pathway to tackle and eventually treat heart failure.

CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.

Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes.

Furmonertinib (Alflutinib, AST2818), as a third-generation epidermal growth factor receptor inhibitor with an advanced efficacy and a relatively wide safety window, has been commercially launched in China recently. However, previous clinical studies demonstrated its time- and dose-dependent clearance in a multiple-dose regimen. In vitro drug metabolism and pharmacokinetic studies have suggested that furmonertinib is mainly metabolized by cytochrome P450 3A4 (CYP3A4) and can induce these enzymes via an increased mRNA expression. This study investigated two important evaluation criteria of CYP3A4 induction by furmonertinib through quantitative proteomics and probe metabolite formation: simultaneous (1) protein expression and (2) enzyme activity with sandwich-cultured primary human hepatocytes in the same well of cell culture plates. Results confirmed that furmonertinib was a potent CYP3A4 inducer comparable with rifampin and could be used as a positive model drug in in vitro studies to evaluate the induction potential of other drug candidates in preclinical studies. In addition, inconsistencies were observed between the protein expression and enzyme activities of CYP3A4 in cells induced by rifampin but not in groups treated with furmonertinib. As such, furmonertinib could be an ideal positive control in the evaluation of CYP3A4 induction. The cells treated with 10 µM rifampin expressed 20.16 ± 5.78 pmol/mg total protein, whereas the cells induced with 0.5 µM furmonertinib expressed 4.8 ± 0.66 pmol/mg protein compared with the vehicle (0.1% dimethyl sulfoxide), which contained 0.65 ± 0.45 pmol/mg protein. The fold change in the CYP3A4 enzyme activity in the cells treated with rifampin was 5.22 ± 1.13, which was similar to that of 0.5 µM furmonertinib (3.79 ± 0.52).

QSAR Analysis for the Inhibition of the Mutagenic Activity by Anthocyanin Derivatives

Flavonoid compounds modulate the cytochrome P450 3A4 enzyme activity and inhibit the mutagenic activity of mammalian cells, preventing carcinogen activation and cellular DNA damage. In this work, the quantitative structure-activity relationships (QSAR) theory is applied to predict the cytochrome P450 3A4 inhibition constant by anthocyanin derivatives. Different freely available software calculates 102,260 non-conformational molecular descriptors. A training set of 12 compounds is used to calibrate the best univariable linear regression models, while a test set of 4 compounds is used to explore their predictive capability. The present results are compared with previously reported ones by using 3D-QSAR, thus demonstrating that the proposed topological QSAR models achieve acceptable statistical quality. The proposed model provides a prospective QSAR guide for the search of new anthocyanin derivatives possessing high or low predicted mutagenicity.

Evaluation of the efficiency of chlorpyrifos-ethyl remediation by Methylobacterium radiotolerans and Microbacterium arthrosphaerae using response of some biochemical biomarkers.

This study reveals out detoxifying and antioxidant enzyme response of Gammarus pulex exposed/polluted to chlorpyrifos-ethyl insecticide before and after biodegradation/bioremediation by Methylobacterium radiotolerans and Microbacterium arthrosphaerae. Cytochrome P450 1A1, glutathione S-transferase, catalase, and superoxide dismutase activities in G. pulex exposed to chlorpyrifos-ethyl before and after bioremediation/biodegradation by these two bacteria during 24 and 96 h tested by using commercial ELISA kits. The activity of catalase enzyme was decreased depending on chlorpyrifos-ethyl before and after bioremediation/biodegradation the enzyme activity was increased repeatedly. Superoxide dismutase activity level increased after chlorpyrifos-ethyl exposure in 96 h (p > 0.05). Following bioremediation, superoxide dismutase enzyme activity decreased again during 24 h (p > 0.05) and increased during 96 h (p < 0.05). Statistical differences were not found in cytochrome P450 1A1 enzyme activity before and after the process (p > 0.05). No significant differences were determined during the activity of glutathione S-transferase in 24 h (p > 0.05). The activities of glutathione S-transferase were increased after exposure of chlorpyrifos-ethyl during 96 h. After bioremediation; the activity of glutathione S-transferase increased even more (p < 0.05). The results determined that activities of G. pulex at superoxide dismutase, catalase, and glutathione S-transferase are common biomarkers for revealing out the efficiency of bioremediation of chlorpyrifos-ethyl with these two types of soil bacteria. Graphical abstract.

Inhibitory Effect of Lygodium Root on the Cytochrome P450 3A Enzyme in vitro and in vivo.

PurposeThe aim of the present study was to investigate the interactions of the main components of Lygodium root (ie, p-coumaric acid, acacetin, apigenin, buddleoside and Diosmetin-7-O-β-D-glucopyranoside) with cytochrome P450 3A enzyme activity both in vitro and in vivo.MethodsIn vitro inhibition of drugs was assessed by incubating rat liver microsomes (RLMs) with a typical P450 3A enzyme substrate, midazolam, to determine their 50% inhibitory concentration (IC50) values. For the in vivo study, healthy male Sprague Dawley rats were consecutively administered acacetin or apigenin for 7 days at the dosage of 5 mg/kg after being randomly divided into 3 groups: Group A (control group), Group B (acacetin group) and Group C (apigenin group).ResultsAmong the five main components of Lygodium root, only acacetin and apigenin showed inhibitory effects on the cytochrome P450 3A enzyme in vitro. The IC50 values of acacetin and apigenin were 58.46 μM and 8.20 μM, respectively. Additionally, the in vivo analysis results revealed that acacetin and apigenin could systemically inhibit midazolam metabolism in rats. The Tmax, AUC(0-t) and Cmax of midazolam in group B and group C were significantly increased (P<0.05), accompanied by a significant decrease in Vz/F and CLz/F (P<0.05).ConclusionAcacetin and apigenin could inhibit the activity of the cytochrome P450 3A enzyme in vitro and in vivo, indicating that herbal drug interactions might occur when taking Lygodium root and midazolam synchronously.

Effects of short-term exposure to environmentally-relevant concentrations of benzo(a)pyrene-sorbed polystyrene to White seabass (Atractoscion nobilis)☆

Plastic marine debris hyper-concentrates hydrophobic contaminants such as polycyclic aromatic hydrocarbons (PAHs) and can transfer these sorbed contaminants to biota following ingestion. PAHs are known to induce cardiotoxicity and visual toxicity at sublethal doses. Juvenile White seabass (Atractoscion nobilis) fish were fed environmentally relevant concentrations of either virgin polystyrene or benzo(a)pyrene (BaP)-sorbed polystyrene for 5 days and were monitored for changes in phototactic response, swimming behavior, and hepatic cytochrome p450 1A (CYP1A) enzyme activity. No significant differences in the monitored endpoints were recorded in fish that ingested either polystyrene or BaP-sorbed polystyrene relative to control fish following the short-term exposure. However, fish exposed to 252 μg/L BaP alone as a positive control had significantly elevated CYP1A enzyme activity (p = 0.046) and impaired phototactic response (p = 0.020), though no altered swimming behavior was observed (p = 0.843) relative to control fish. These results demonstrate that pelagic fish ingesting environmentally relevant concentrations of BaP-sorbed polystyrene for a short, 5-day duration do not demonstrate measurable changes in vision, swimming activity, nor CYP1A activity. High variability within enzyme activity and behavioral responses suggest that lack of significant effects may be due to low sample size.

GSH/GSSG redox couple plays central role in aryl hydrocarbon receptor-dependent modulation of cytochrome P450 1A1.

The redox regulation of aryl hydrocarbon receptor (AHR) target genes such as the best characterized, cytochrome P450 1A1 (CYP1A1) has not been known. Therefore the aim of this study was to explore how cellular redox state can influence on AHR-dependent modulation of CYP1A1 transcription and enzyme activities. Male BALB/c albino mice, HepG2 cells, and human hepatoma cell line (HepG2-XRE-Luc) carrying CYP1A1 response elements were exposed to suggested endogenous ligand of AHR,6-formylindolo[3,2-b] carbazole (FICZ) alone or in combination with, buthionine-(S,R)-sulfoximine (BSO) or N-acetyl-l-cysteine (NAC). A clear link between CYP1A1 transcription and enzyme activity and changes in the glutathione/oxidised glutathione (GSH/GSSG) redox couple was shown. In vivo and in vitro findings demonstrated that the time course of AHR activation/inhibition is characterized by an increase/decrease in the GSH/GSSG ratio. Based on these findings, we propose that many environmental pollutants and oxidants by alteration in the intracellular redox potential may interfere with the normal function of AHR target genes.

Biochemical responses of Gammarus pulex to malachite green solutions decolorized by Coriolus versicolor as a biosorbent under batch adsorption conditions optimized with response surface methodology

The current study was aimed to investigate the detoxifying and antioxidant enzyme response of Gammarus pulex exposed to malachite green (MG) after decolorization by Coriolus versicolor. Response surface methodology (RSM) was utilized to optimize the decolorization conditions of MG synthetic solutions by C. versicolor. Glutathione (GSH), malondialdehyde (MDA) levels and glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), cytochrome P450 1A1 (CYP1A1) activities in G. pulex exposed to undecolorized (A1) and decolorized (A2) MG synthetic solution during 24 and 96 h were tested by using ELISA method. SOD and GPX enzyme activity was increased after decolorization (p > 0.05). CAT enzyme activity was increased in A2 group during 24 h (p > 0.05) but decreased during 96 h (p < 0.05). GSH levels were increased in A2 group during 24 and 96 h (p < 0.05). GST, CYP1A1 enzyme activity and MDA levels were decreased after decolorization during 96 h (p < 0.05). In this study, GSH levels, CAT, GST and CYP1A1 activities in G. pulex approved the capability of C. versicolor in MG decolorization, optimized with RSM.

Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.

Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.

Effect of Age and Allele Variants of CYP3A5, CYP3A4, and POR Genes on the Pharmacokinetics of Cyclosporin A in Pediatric Renal Transplant Recipients From Serbia.

The interindividual variability of cyclosporin A (CsA) pharmacokinetics might be explained by heterogeneity in the cytochrome P450 3A (CYP3A) subfamily. Altered CYP3A enzyme activity was associated with variant allele of P450 oxidoreductase gene (POR*28). The aim of this study was to assess the impact of age, CYP3A5*3, CYP3A4*22, and POR*28 alleles on CsA pharmacokinetics in pediatric renal transplant recipients. Renal transplant patients receiving CsA (n = 47) were genotyped for CYP3A5*3, CYP3A4*22, and POR*28. CYP3A5 nonexpressers had higher overall dose-adjusted predose concentration (C0/dose; ng/mL per mg/kg) compared with expressers (31.48 ± 12.75 versus 22.44 ± 7.12, P = 0.01). CYP3A5 nonexpressers carrying POR*28 allele had a lower overall dose-adjusted concentration (C2/dose) than those with POR*1/*1 genotype (165.54 ± 70.40 versus 210.55 ± 79.98, P = 0.02), with age as covariate. Children aged 6 years and younger had a lower overall C0/dose (18.82 ± 4.72 versus 34.19 ± 11.89, P = 0.001) and C2/dose (106.75 ± 26.99 versus 209.20 ± 71.57, P < 0.001) compared with older children. Carriers of CYP3A5*3 allele aged ≤6 years required higher dose of CsA and achieved lower C0/dose and C2/dose, at most time points, than older carriers of this allele. Carriers of POR*28 allele aged ≤6 years required higher doses of CsA, whereas they achieved lower C0/dose and C2/dose, at most time points, in comparison to older carriers of this allele. The significant effect of age (P < 0.002) and CYP3A5 variation (P < 0.02) was shown for overall C0/dose, whereas age (P < 0.00001) and POR variation (P = 0.05) showed significant effect on C2/dose. Regression summary for overall C2/dose in patients aged 6 years younger showed a significant effect of both CYP3A5 and POR variations (P < 0.016). Younger age, POR*28 allele, and CYP3A5*3 allele were associated with higher CsA dosing requirements and lower concentration/dose ratio. Pretransplant screening of relevant polymorphisms in accordance with age should be considered to adjust therapy.

The highly bioactive molecule and signal substance 6-formylindolo[3,2-b]carbazole (FICZ) plays bi-functional roles in cell growth and apoptosis in vitro.

The maintenance of cellular homeostasis is a complex process that is governed by the receipt of prototypical growth and death signals. The endogenous functions of aryl hydrocarbon receptor (AHR) in cellular homeostasis are not well understood. We aimed to establish whether the disturbance of endogenously activated AHR can influence cell growth, and if so, what mechanism(s) are involved. Cell growth was measured in mouse hepatoma Hepa-1 wild-type and cytochrome P4501A1 (CYP1A1)-deficient c37 cells. In other sets of experiments, HepG2 cells were exposed to different doses of FICZ (0.01nM-1µM) alone or in combination with 50nM of the CYP1A1 inhibitor 3'methoxy-4'nitro-flavone (MNF). CYP1A1 enzyme activity, cell viability, oxidative stress, and several endpoints of apoptosis were measured. FICZ treatment at a high concentration or in combination with MNF induced sustained CYP1A1 activity and led to oxidative stress and activation of apoptosis via a mitochondrial-dependent pathway. In comparison with the wild-type Hepa-1 cells, c37 cells lacking CYP1A1 activity proliferated faster in normal medium which contains trace levels of FICZ. Besides, in HepG2 cells, FICZ stimulated cell growth at low concentrations but inhibited cell growth at high concentrations. Based on these findings, we propose that CYP1A1 inhibitors, by increasing the levels of the endogenous ligand FICZ, change the cell growth kinetics and trigger cell death and apoptosis through a mitochondrial-dependent pathway. Since AHR controls multiple cellular functions, a wide range of toxicity can be expected by disturbing its endogenous functions.

NADPH Oxidase-Dependent Mechanism Explains How Arsenic and Other Oxidants Can Activate Aryl Hydrocarbon Receptor Signaling.

The mechanisms explaining arsenic toxicity are not well understood, but physiological consequences of stimulated aryl hydrocarbon receptor (AHR) signaling both directly and through cross-talk with other pathways have been indicated. The aim of this study was to establish how arsenic interacts with AHR-mediated transcription. The human hepatoma cell line (HepG2-XRE-Luc) carrying a luciferase reporter under the control of two AHR response elements (AHREs) and immortalized human keratinocytes (HaCaT) were exposed to sodium arsenite (NaAsO2; As(3+)), alone or in combination with the endogenous high affinity AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Luciferase activity, cytochrome P4501A1 (CYP1A1) activity, oxidative stress-related responses, metabolic clearance of FICZ, and NADPH oxidase (NOX) activity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression were measured. Arsenic inhibited CYP1A1 enzyme activity and reduced the metabolic clearance of FICZ. Arsenic also led to activated CYP1A1 transcription but only in cells grown in medium containing trace amounts of the endogenous ligand FICZ, pointing to an indirect mechanism of activation. Initially, arsenic caused dose-dependent inhibition of FICZ-activated AHR signaling, disturbed intracellular GSH status, and increased expression of oxidative stress-related genes. Silencing of NOX4, addition of N-acetylcystein, or pretreatment with arsenic itself attenuated the initial dose-dependent inhibition of AHR signaling. Arsenic pretreatment led to elevated GSH levels and sensitized the cells to ligand-dependent AHR signaling, while silencing of Nrf2 significantly reduced arsenic-mediated activation of the AHR. In addition, influence of NOX on AHR activation was also observed in cells treated with the SH-reactive metals cadmium, mercury, and nickel. Together, the results suggest that SH-reactive agents via a new and possibly general NOX/H2O2-dependent mechanism can interfere with the endogenous regulation of the AHR.

Maternal metabolizable protein restriction during late gestation on uterine and umbilical blood flows and maternal and fetal amino acid concentrations near term in sheep

To examine the effects of maternal metabolizable protein (MP) restriction during late gestation on uterine and umbilical blood flows, conceptus size, and amino acid concentrations in the uterine and umbilical vessels, 11 ewes with singleton pregnancies were assigned to one of three isocaloric diets formulated to provide 60% of MP (MP60), 80% of MP (MP80), or 100% of MP (MP100) requirements from days 100 to 130 of gestation. On day 130 of gestation, intraoperative uterine and umbilical blood flows were obtained as well as serum samples from the uterine artery, uterine vein, umbilical artery, and umbilical vein. Ewes on the MP60 diet had lighter (P=0.04) and smaller (P≤0.05) fetuses, but increased (P=0.02) uterine blood flow relative to fetal weight compared with MP100 ewes, with MP80 being intermediate. Umbilical blood flow was similar (P=0.70) across treatments. Glutamine, glycine, isoleucine, leucine, ornithine, serine, and valine concentrations were impacted (P≤0.02) by maternal treatment. While uterine flux of total serum nitrites was greater (P=0.03) in MP60 and MP80 ewes compared with MP100 ewes, fetal flux did not differ. Decreased maternal protein intake resulted in less (P<0.01) maternal cytochrome P450 1A enzyme activity. There were minimal impacts of maternal diet on steroid concentrations. Maternal dietary protein may alter fetal growth by impacting placental vasculature function and nutrient absorptive capabilities.

Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells.

Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.

Developmental toxicity, EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin-like PCB126.

Dioxin-like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L(-1) from 3-h post-fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non-inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126-induced developmental toxicity, we conducted ethoxyresorufin-O-deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real-time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L(-1) concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L(-1) doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L(-1) at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose-dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR.

Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp (Ctenopharyngodon idellus)

Cytochrome P450 3A(CYP3A) is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin(RIF) on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp(Ctenopharyngodon idellus).Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame(ORF) of 1 542 bp,a stop codon,a 3'-untranslated regions(3' UTR),and a polyA signal is present in the 3'UTR region.The ORF encodes a 513 amino acid(aa) protein,which has a signal peptide(29aa),two transmembrane helixes(23aa and 23aa),a heme binding domain(HBD,21aa),and six substrate recognition sites(SRS1-6).Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells(CIK) was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase(ERND) activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group(P0.05).The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.

Effect of borneol on cytochrome P450 3A enzyme and midazolam pharmacokinetics in rats

Borneol is a commonly used herbal medication in China and Japan. Previous studies have indicated that borneol could reduce the plasma concentrations of oneself and concomitant drugs, and its first-pass metabolism could be catalyzed by the cytochrome P450 3A (CYP3A) enzyme as well. The impact of borneol on CYP3A activity and efficacy in influencing the pharmacokinetics of co-administrated drugs is currently unknown. Therefore, the purpose of the current study is to investigate the effect of borneol on CYP3A enzyme in vivo. After treatment with borneol twice daily for 3days, rat liver microsomes were exposed to probe substrates to determine CYP3A enzyme activity, protein, and RNA harvested using microsomal testosterone 6β-hydroxylation as a marker of enzyme activity. To verify the result, the effect of borneol on the pharmacokinetics of the CYP3A model substrate midazolam was further examined. The results showed that borneol treatment had increased CYP3A expression at the mRNA, protein, and activity (testosterone 6β hydroxylase activity) level in rat liver microsomes. In addition, borneol accelerated the metabolism of midazolam, which was consistent with the enhancement in CYP3A metabolic capacity. The hepatic clearance (Cl) of midazolam injected via the caudal vein in rats following borneol co-administration was higher; however, the area under the curve (AUC0-∞) was lower than the solvent. Hence, it was proposed that borneol could increase the metabolic activity of the CYP3A enzyme, which might cause drug-drug interactions in humans when using Chinese herbal or Western medicine with borneol.