CYP3A Subfamily Research Articles

Diversifying selection detected in only a minority of xenobiotic-metabolizing CYP1-3 genes among primate species

1. Primates exhibit a high degree of among-species dietary diversity, which likely exposes them to varying levels of xenobiotic compounds. Here, we examined the evolution of primate CYP1-3 gene families, and we classified the 15 CYP1-3 gene subfamilies as either xenobiotic-metabolizing (XM) or endogenous-metabolizing (EM) based on sources in the P450 literature. 2. We predicted that XM P450s would show (1) greater variability in gene-copy number and (2) more evidence of diversifying selection and, especially on codons that encode the substrate-recognition sites (SRSs) for the final enzymes. 3. Counter to our first prediction, EM and XM P450s showed similar levels of variation in gene-copy number. We did find, however, that four XM P450 subfamilies (CYP2C, CYP2D, CYP2E, and CYP3A) showed evidence of diversifying selection while no EM subfamilies demonstrated any consistent signal of diversifying selection. Of these four, CYP2C, CYP2D, and CYP3A showed significant links between SRSs and diversifying selection. 4. These results reveal an amount of evolutionary dynamism that would not be expected when viewing P450 subfamilies along a simple binary EM/XM spectrum. We recommend that comparative studies of cytochrome P450 evolution should focus on the CYP2C, CYP2D, CYP2E, and CYP3A subfamilies.

CYP3A subfamily activity affects the equilibrium concentration of Phenazepam® in patients with anxiety disorders and comorbid alcohol use disorder.

Phenazepam® is prescribed to relieve anxiety and sleep disorders during alcohol withdrawal, although it is associated with undesirable side effects. Aim: To demonstrate changes in the safety and efficacy profiles of Phenazepam in patients with anxiety disorders and comorbid alcohol use disorder. Materials & methods: A total of 94 Russian patients with alcohol use disorder received 4.0mg of Phenazepam per day in tablets. We used a urinary 6-beta-hydroxycortisol/cortisol ratio to evaluate CYP3A activity. Results: A statistically significant inverse correlation between Phenazepam plasma concentration and CYP3A activity was found (r=-0.340 and p=0.017). Correlation between the concentration/dose ratio and phenotyping results was also statistically significant (r=0.301 and p=0.026). Conclusion: The safety and efficacy of Phenazepam depend on CYP3A genetic polymorphisms.

Comparison of steroid hormone hydroxylation mediated by cytochrome P450 3A subfamilies

Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

Piperine Is a Mechanism-Based Inactivator of CYP3A.

Piperine (PPR) is the representative alkaloid component of the piper species (family: Piperaceae). Our rapid screening study found PPR caused time-dependent inhibition of cytochrome P450s (CYP) 3A and 2D6, and CYP3A was inactivated the most. Further study demonstrated that PPR is a time-, concentration-, and NADPH-dependent inhibitor of CYP3A, and significant loss (49.5% ± 3.9%) of CYP3A activity was observed after 20minute incubations with 80 μM PPR at 37°C. The values of K I and k inact were 30.7 μM and 0.041 minutes-1, respectively. CYP3A competitive inhibitor ketoconazole showed protective effect against the enzyme inactivation. Superoxide dismutase/catalase and GSH displayed minor protection against the PPR-caused enzyme inactivation. Ferricyanide partially reduced the enzyme inhibition by PPR. Additionally, NADPH-dependent formation of reactive metabolites from PPR were found in human liver microsomal incubation mixtures. An ortho-quinone intermediate was trapped by NAC in microsomal incubations with PPR. DM-PPR, demethylene metabolite of PPR, showed weak enzyme inactivation relative to that caused by PPR. It appears that both carbene and ortho-quinone intermediates were involved in the inactivation of CYP3A caused by PPR. SIGNIFICANCE STATEMENT: CYP3A subfamily members (mainly CYP3A4 and CYP3A5) play a critical role in drug metabolism. Piperine (PPR), a methylenedioxyphenyl derivative combined with an unsaturated ketone, is the major active ingredient of pepper. PPR revealed time-, concentration-, and NADPH-dependent inhibitory effect on CYP3A. Carbene and quinone metabolites were both involved in the observed CYP3A inactivation by PPR. Apparently, the unsaturated ketone moiety did not participate in the enzyme inactivation. The present study sounds an alert of potential risk for food-drug interactions.

A non-lethal malarial infection results in reduced drug metabolizing enzyme expression and drug clearance in mice

BackgroundGiven the central importance of anti-malarial drugs in the treatment of malaria, there is a need to understand the effect of Plasmodium infection on the broad spectrum of drug metabolizing enzymes. Previous studies have shown reduced clearance of quinine, a treatment for Plasmodium infection, in individuals with malaria.MethodsThe hepatic expression of a large panel of drug metabolizing enzymes was studied in the livers of mice infected with the AS strain of Plasmodium chabaudi chabaudi, a nonlethal parasite in most strains of mice with several features that model human Plasmodium infections. C57BL/6J mice were infected with P. chabaudi by intraperitoneal injection of infected erythrocytes and sacrificed at different times after infection. Relative hepatic mRNA levels of various drug metabolizing enzymes, cytokines and acute phase proteins were measured by reverse transcriptase-real time PCR. Relative levels of cytochrome P450 proteins were measured by Western blotting with IR-dye labelled antibodies. Pharmacokinetics of 5 prototypic cytochrome P450 substrate drugs were measured by cassette dosing and high-resolution liquid chromatography-mass spectrometry. The results were analysed by MANOVA and post hoc univariate analysis of variance.ResultsThe great majority of enzyme mRNAs were down-regulated, with the greatest effects occurring at the peak of parasitaemia 8 days post infection. Protein levels of cytochrome P450 enzymes in the Cyp 2b, 2c, 2d, 2e, 3a and 4a subfamilies were also down-regulated. Several distinct groups differing in their temporal patterns of regulation were identified. The cassette dosing study revealed that at the peak of parasitaemia, the clearances of caffeine, bupropion, tolbutamide and midazolam were markedly reduced by 60–70%.ConclusionsThese findings in a model of uncomplicated human malaria suggest that changes in drug clearance in this condition may be of sufficient magnitude to cause significant alterations in exposure and response of anti-malarial drugs and co-medications.

Genotyping and phenotyping CYP3A4\CYP3A5: no association with antiplatelet effect of clopidogrel.

The objective of this study was to determine the impact of polymorphism of CYP3A subfamily isoenzymes (allelic variants of CYP3A4*22 and CYP3A5*3) on the efficacy clopidogrel in patients with an acute coronary syndrome (ACS), who have undergone percutaneous coronary intervention (PCI). Platelet activity was determined on a VerifyNow P2Y12 test system in 81 patients with ACS aged 37-91 who had PCI. The activity of CYP3A4/5 was expressed as the ratio of the concentrations of cortisol and 6β-hydroxycortisol was performed by using high performance liquid chromatography. Genotyping was performed by using real-time polymerase real-time chain reaction. The frequencies for the CYP3A5 gene, rs 776746, were identified as follows: 77 (95.1%)-CC, 4 (4.9%)-CT; the allele frequencies by loci for the CYP3A4, rs rs35599367, were as follows: 78 (96.3%)-GG, 3 (3.7%)-AG. There was no statistically significant genotype-dependent difference between the presence of a minor T and G alleles and the presence of clopidogrel resistance (OR 3.53; 95% CI 0.46-26.94; p = 0.233 and p = 0.443, respectively). The average level of the metabolic relationship (6β-hydroxycortisol/cortisol) between the clopidogrel-resistant group and the normal platelet reactivity group was not statistically significantly different: 3.3 ± 2.8 versus 3.2 ± 3.2; p = 0.947. So, the activity of CYP3A4/5 was not related to platelet aggregation rates in this model. Genotyping and phenotyping CYP3A4\CYP3A5 does not predict the antiplatelet effect of clopidogrel. More extensive research is required to establish their clinical relevance.

In silico and in vitro analysis of genetic variants of the equine CYP3A94, CYP3A95 and CYP3A97 isoenzymes

Cytochrome P450 enzymes (CYPs) of the equine CYP3A subfamily are predominantly involved in drug metabolism. In this study, genetic variants of the equine CYP3A94, CYP3A95, and CYP3A97 were identified and characterized using in silico modeling and in vitro enzyme kinetics.The genomes of 81 horses were sequenced to obtain the genetic variants. Structural CYP modifications of the most frequent variants were analyzed in silico using the 3D-structures predicted by homology modeling. Enzyme kinetic analyses were performed using testosterone as substrate.Twenty genetic variants were found including five missense variants (CYP3A94:p.Asp217Asn, CYP3A95:p.Asp214His, CYP3A95:p.Ser392Thr, CYP3A97:p.Ile119Thr, CYP3A97:p.Met500Val) with a higher percentage of minor allele frequency (MAF) (range 0.2–0.4). A splice-site variant (c.798 + 1G > A) in CYP3A94, likely to generate a truncated protein, was found in 50% of the horses. CYP3A94:p.Asp217Asn and CYP3A95:p.Asp214His were localized on the CYP F-α-helix, an important region for the substrate interactions in the human CYP3A4. Testosterone 2β-hydroxylation was diminished in CYP3A94217Asn and CYP3A95392Thr. Ketoconazole inhibited 2β-hydroxylation differently in the five variants with the most pronounced inhibition obtained for CYP3A95392Thr.In vitro and in silico analyses of genetic variants allow unraveling structural features in equine CYPs that correlate with changes in the CYP activity.

MON-357 Recurrent Adrenal Crises in a Cytochrome P450 Ultra-Rapid Metabolizer

Background The cytochrome P450s (CYPs) are a superfamily of oxidative enzymes of which the CYP3A subfamily is responsible for the metabolism of approximately 50% of all drugs. This includes exogenous and endogenous steroids. Over 20 polymorphisms of CYP3A4 have been described with three metabolic phenotypes: ultra-rapid, extensive and poor metabolizers. The CYP3A4*1B has an allelic frequency of 2-9% in Caucasians and is associated with increased enzymatic activity. Clinical Case This is a 49-year-old Caucasian female with a known history of Addison’s disease for 2 years, Hashimoto’s thyroiditis, Osteoporosis, Celiac disease, Vitamin D deficiency and Pernicious Anemia, who presented to the ER after a syncopal episode following 3 days of nausea and vomiting. On arrival, she was hypothermic, hypotensive and tachycardic with a BP of 57/40 mmHg, pulse of 112 BPM. On exam she looked cachectic with tanned skin, dark palmar creases and pigmented oral mucosa. Initial labs showed normal CBC, low sodium (125, n 135 - 148 MEQ/L), hyperkalemia (5.8, n 3.5 - 5.3 MEQ/L), normal blood glucose and renal function. Further testing showed low AM cortisol <1.0 ug/dL (n 6.7-22.6 ug/dL), high ACTH (1010, n 6-50pg), TSH 7 (n 0.35 - 4.94 UIU/ML), Free T4 0.70 (n 0.70 - 1.48 NG/DL), TPO antibodies 759.06 (n<5.61 IU/ML), low aldosterone level <1.0 (n 3 - 16 ng/dL), and high renin activity 19.53 (n 0.25 - 5.82 ng/mL/h). After IV fluids resuscitation and IV steroids, she became hemodynamically stable and was transitioned to oral hydrocortisone 20 mg AM, 10 mg PM, and fludrocortisone 0.1 mg daily. Five days later, she returned to the hospital with adrenal crisis. Despite adherence to her medications, she had several crises per month. Home regimen was adjusted to 100 mg hydrocortisone PO every 6 hours without resolution. During one of her hospitalizations, plasma cortisol was undetectable one hour after 100 mg hydrocortisone PO dose. EGD showed normal mucosa, thus malabsorption from celiac disease unlikely. Her frequent adrenal crises and undetectable levels of serum cortisol despite compliance, prompted evaluation of Cytochrome P450 genotype which revealed CYP3A4 Genotype B, allele 22, not detected. Patient was diagnosed as a CYP3A4 ultra-rapid metabolizer. She is currently being treated with Hydrocortisone 100mg IM every 4 hours and fludrocortisone 0.2mg PO every 4 hours. Since these adjustments, her adrenal crises are less frequent. Conclusion Variability in the expression of CYP3A4 can cause unpredictable interindividual drug responses. Exogenous steroids are not exempt. In this patient with Addison’s disease, pharmacogenomic testing helped us individualize her maintenance dose to one that is intramuscular, and a higher dose and frequency, than what would be suggested in the current literature.

Transcriptomic analysis of Anabas testudineus and its defensive mechanisms in response to persistent organic pollutants exposure

The freshwater climbing perch (Anabas testudineus) can tolerate water environments contaminated with persistent organic pollutants (POPs). The mechanisms underlying this tolerance are unknown. We used de novo transcriptomic analysis to investigate the defensive mechanisms of A. testudineus against POPs based on its genetic features and biological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Our results revealed a specific expansion of cytochrome P450 (CYP) 3A subfamily, which may be involved in the elimination of certain POPs. In xenobiotic responses, the aryl-hydrocarbon receptor (AhR) pathway represents a critical signaling mechanism, and we characterized four AhR and two AhR nuclear translocator homologs and one AhR repressor (AhRR) gene in A. testudineus. TCDD-induced AhRR and CYP1A mRNA upregulation suggests that negative-feedback regulation of AhR signaling through AhRR helps avoid excessive xenobiotic responses. Furthermore, liver and gill transcriptomic profiles were markedly altered after TCDD exposure, with some of the altered genes being related to common defensive responses reported in other species. Based on the newly identified TCDD-altered genes, several A. testudineus-specific responses are proposed, such as enhanced fatty acid β-oxidation. The genetic features of CYP3A subfamily and AhR pathway and the TCDD-induced defensive biological processes elucidated here enhance our understanding of A. testudineus defensive responses against POPs.

Suppression of Hepatic CYP3A4 Expression and Activity by 3-Methylcholanthrene in Humanized PXR-CAR-CYP3A4/3A7 Mice.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that activate the aryl hydrocarbon receptor, thereby triggering a range of biologic responses, exemplified by the induction of CYP1A1 PAHs can also regulate the expression of members of the CYP3A subfamily, with reports of mainly suppressive effects on mouse hepatic Cyp3a11 expression, but paradoxically both inductive and suppressive effects on human hepatic CYP3A4 expression. Understanding the regulation of CYP3A4 expression by PAHs is important because of the widespread exposure of humans to these chemicals and the central role of the CYP3A4 enzyme in the metabolism of clinically important drugs and endogenous substances. The present study used 3-methylcholanthrene (MC) as a model PAH to characterize the in vivo regulation of CYP3A4 expression and activity in humanized pregnane X receptor-constitutive androstane receptor-CYP3A4/3A7 mice. Adult mice were treated by intraperitoneal injection with MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours later. As a positive control response, pronounced induction of hepatic Cyp1a1 by MC was confirmed at both time points in males and females at the mRNA, protein, and catalytic activity levels. Basal hepatic CYP3A4 expression and activity were significantly higher in female versus male mice. MC treatment suppressed hepatic CYP3A4 in female mice at 72 hours postdosing at the mRNA, protein, and catalytic activity levels. A similar response was observed in male mice, although the suppression of CYP3A4 protein levels did not achieve statistical significance. This mouse model will facilitate further studies of the mechanisms and consequences of CYP3A4 suppression by PAHs.

Development and Characterization of MDR1 (Mdr1a/b) CRISPR/Cas9 Knockout Rat Model

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new Mdr1a/b (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.

CYP3C gene regulation by the aryl hydrocarbon and estrogen receptors in zebrafish

Cytochrome P450 (CYPs) enzymes are critical for the metabolism of exogenous and endogenous compounds. In mammals, the CYP3s are arguably the most important xenobiotic metabolizing enzymes and are all contained within the CYP3A subfamily. In fish, CYP3s include CYP3A and multiple subfamilies unique to the teleost lineage. The goal of this study was to provide insight on the regulation of genes in the CYP3C subfamily. Zebrafish, which have 4 CYP3C genes, were exposed to 17β-estradiol (E2; 0.001-10 μM) or β-naphthoflavone (βNF; 0.005-1 μM), prototypical ligands of the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR), respectively. Gene expression was measured in the liver, intestine and gonads using quantitative PCR. CYP1A and vitellogenin (VTG) gene expression were used as positive controls for AhR and ER regulation, respectively. Exposure to βNF resulted in the dose-dependant induction of CYP1A and CYP3C genes in the female intestine but not in the liver. E2 exposure resulted in the induction of all CYP3Cs in the male intestine and in the female liver. VTG was induced in both female and male livers. CYP3C3 and CYP3C4 were induced in the testis; CYP3C1 and CYP3C4 were slightly induced in the ovary. The time-course of gene induction was investigated in the liver and intestine after exposure to βNF (0.5 μM) and E2 (0.1 μM). Inducible genes were up-regulated within 12 h after exposure. These data support a role for the AhR and ER in the regulation of CYP3Cs. Overall, the induction of CYP3Cs by AhR and ER ligands is different from mammalian CYP3A and may suggest a functional role for CYP3Cs that involves planar aromatic hydrocarbons and steroids.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 234. Phytochemicals combat the detrimental effects of cigarette smoke on osteogenic differentiation

BACKGROUND CONTEXT Cigarette smoking has been shown to inhibit bone healing and increase the risk of pseudarthrosis after spinal fusion. We previously reported that activation of the Aryl Hydrocarbon Receptor (Ahr) by dioxin, a prototype activator of the Ahr, inhibits bone regeneration and spine fusion in rat model. We also reported that the dioxin-mediated inhibition of osteogenic differentiation is mitigated by co-treatment with Ahr antagonists in vitro. PURPOSE Since dozens of dioxin-like Ahr ligands are present in cigarette smoke, we are investigating the downstream mechanisms of cigarette smoke action on osteogenic differentiation to identify potential therapeutic options to mitigate the effects of cigarette smoke on bone. METHODS Particulate phase extract (PPE) was prepared by passing CSE through a 0.1μm PTFE filter and eluting in the DMSO to yield a final concentration of 40mg/mL. Primary rat bone marrow stromal cells (BMSC) were purchased from Cell Biologics (Chicago, IL) and cultured under basal standard or osteogenic media. BMSC were subsequently exposed to either the vehicle control (DMSO) or dioxin or PPE. Some cells received co-treatment with Ahr antagonists, including: a-naphthoflavone (ANF, a synthetic antagonist); resveratrol (Res, a stilbenoid found in grapes and present in red wine), and 3,3’-diindolylmethane (DIM, a breakdown product of the indole-3-carbinol, which is present in cruciferous vegetables). EROD assays were used to evaluate Ahr-mediated CYP1 family induction, and ALP and mineralization assays were used to quantify effects on osteogenic differentiation. Cell proliferation assays and Wnt signaling assays were also performed. RESULTS PPE-treated cells showed increased activity of CYP1A subfamily proteins, demonstrating Ahr activation. PPE treatment inhibited ALP activity, an effect which was at least partially mitigated by co-treatment with each of the Ahr antagonists. A similar response was seen in and mineralization and cell proliferation assays. RNA expression studies showed that PPE down-regulated numerous pro-osteogenic genes, such as ALP, RunX2, OCN, and Phex, whereas co-treatment with Ahr antagonists prevented PPE-mediated inhibition of those RNA. Interestingly, PPE decreased the ratio of active:inactive β-Catenin, an effect which was recapitulated by treatment with dioxin (TCDD). CONCLUSIONS Cigarette smokers are a historically difficult patient population for spine surgeons to treat due to increased rates of pseudarthrosis and complications following spinal fusion procedures. Our results suggest that Ahr hyper-activation may play an important role in the adverse effects of cigarette smoke on bone healing—potentially by perturbation of canonical Wnt signaling—and that Ahr antagonists may be protective in combating cigarette smoke-mediated inhibition of bone regeneration and healing.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.

Studies on the Unusual 1β-Hydroxylated Bile Acid Biosynthesis in Infants.

Unusual bile acids (1β-hydroxylated bile acids), particularly 1β-hydroxyl-cholic acid (CA-1β-ol) and 1β-hydroxyl-chenodeoxycholic acid (CDCA-1β-ol), have been detected in the urine of infants. These acids are conjugated with amino acids, such as taurine, and are then excreted mainly via the urine. CA-1β-ol and CDCA-1β-ol are the predominant bile acids during infancy and are present in relatively large amounts in the urine. However, the biosynthetic pathway of 1β-hydroxylated bile acids in infants remains unclear. To investigate the biosynthetic pathway of 1β-hydroxylated bile acids during infancy, we performed a metabolic reaction using infant hepatocytes at 3 months after delivery. Glyco- and tauro-CA-1β-ol were identified by LC/tandem mass spectrometry (MS/MS) analysis of the extracted culture medium incubated with cholic acids (CAs). Further, we identified that ketoconazole suppressed CA 1β-hydroxylation and that the CYP3A subfamily was the primary group of enzymes responsible for CA-1β-ol formation. The present study provides new information about the biosynthetic pathway of 1β-hydroxylated bile acids during infancy.

Population Pharmacokinetics of Sirolimus in Healthy Chinese Subjects

Sirolimus is metabolized by the CYP3A subfamily and is a substrate of the P-glycoprotein. CYP3A5 and MDR1 gene polymorphisms have been reported to influence plasma concentrations of this drug and dose requirements, but some recent studies reported conflicting results. The aim of this study was to develop population pharmacokinetics to identify the potential influencing factors that explain pharmacokinetic variability in healthy Chinese adults. Twenty seven healthy Chinese subjects were enrolled into this study and were genotyped for CYP3A5 (6986A>G), MDR1 (3435C>T). Blood samples at different time point were collected from each subject and blood concentrations were measured. The population pharmacokinetic model was established using the non-linear mixed-effects model method, CYP3A5, MDR1, gender and other factors were evaluated in the model as covariates. The results indicated that CYP3A5 polymorphisms significantly influenced the clearance, the central compartment distribution volume was larger in male than that in female subjects, body surface area showed remarkable impact on peripheral compartment volume. All parameters were estimated with acceptable precision. Visual predictive check and bootstrap indicated that the population model adequately captured the observed profiles. This model may be useful in guiding clinicians to adjust drug dosing regimen.

Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus)

Cytochrome P450s (CYPs) play critical role in oxidative metabolism of numerous xenobiotics and endogenous compounds. The first CYP3A subfamily member in saltwater crocodile has been cloned and modelled for three-dimensional (3D) structure. The full-length cDNA was obtained employing reverse transcription polymerase chain reaction (RT-PCR) strategy and rapid amplification of cDNA ends (RACE). The cDNA sequence of 1659 nucleotides includes 132 nucleotides from 5′ untranslated region (UTR), an open reading frame of 1527 nucleotides encoding 509 amino acids designated as CYP3A163. The alignment of CYP3A163 sequence with CYP3A subfamily across the lineages exhibit the loss of 1 residue in birds and 7 residues in mammals in comparison to reptiles suggesting the adaptation processes during evolution. The amino acid identity of CYP3A163 with Alligator mississippiensis CYP3A77 and Homo sapiens CYP3A4 is 91% and 62% respectively. The 3D structure of CYP3A163 modelled using human CYP3A4 structure as a template with Phyre2 software, represents high similarity with its functionally important motifs and catalytic domain. Both sequence and structure of CYP3A163 display the common and conserved features of CYP3A subfamily. Overall, this study provides primary molecular and structural data of CYP3A163 required to investigate the xenobiotic metabolism in saltwater crocodiles.

Novel Interactions between Gut Microbiome and Host Drug-Processing Genes Modify the Hepatic Metabolism of the Environmental Chemicals Polybrominated Diphenyl Ethers.

The gut microbiome is a novel frontier in xenobiotic metabolism. Polybrominated diphenyl ethers (PBDEs), especially BDE-47 (2, 2', 4, 4'-tetrabromodiphenyl ether) and BDE-99 (2, 2', 4, 4',5-pentabromodiphenyl ether), are among the most abundant and persistent environmental contaminants that produce a variety of toxicities. Little is known about how the gut microbiome affects the hepatic metabolism of PBDEs and the PBDE-mediated regulation of drug-processing genes (DPGs) in vivo. The goal of this study was to determine the role of gut microbiome in modulating the hepatic biotransformation of PBDEs. Nine-week-old male C57BL/6J conventional (CV) or germ-free (GF) mice were treated with vehicle, BDE-47 or BDE-99 (100 μmol/kg) for 4 days. Following BDE-47 treatment, GF mice had higher levels of 5-OH-BDE-47 but lower levels of four other metabolites in liver than CV mice; whereas following BDE-99 treatment GF mice had lower levels of four minor metabolites in liver than CV mice. RNA sequencing demonstrated that the hepatic expression of DPGs was regulated by both PBDEs and enterotypes. Under basal conditions, the lack of gut microbiome upregulated the Cyp2c subfamily but downregulated the Cyp3a subfamily. Following PBDE exposure, certain DPGs were differentially regulated by PBDEs in a gut microbiome-dependent manner. Interestingly, the lack of gut microbiome augmented PBDE-mediated upregulation of many DPGs, such as Cyp1a2 and Cyp3a11 in mouse liver, which was further confirmed by targeted metabolomics. The lack of gut microbiome also augmented the Cyp3a enzyme activity in liver. In conclusion, our study has unveiled a novel interaction between gut microbiome and the hepatic biotransformation of PBDEs.

AHR and CYP1A expression link historical contamination events to modern day developmental effects in the American alligator

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that initiates a transcriptional pathway responsible for the expression of CYP1A subfamily members, key to the metabolism of xenobiotic compounds. Toxic planar halogenated aromatic hydrocarbons, including dioxin and PCBs, are capable of activating the AHR, and while dioxin and PCB inputs into the environment have been dramatically curbed following strict regulatory efforts in the United States, they persist in the environment and exposures remain relevant today. Little is known regarding the effects that long-term chronic exposures to dioxin or dioxin-like compounds might have on the development and subsequent health of offspring from exposed individuals, nor is much known regarding AHR expression in reptilians. Here, we characterize AHR and CYP1A gene expression in embryonic and juvenile specimen of a long-lived, apex predator, the American alligator (Alligator mississippiensis), and investigate variation in gene expression profiles in offspring collected from sites conveying differential exposures to environmental contaminants. Both age- and tissue-dependent patterning of AHR isoform expression are detected. We characterize two downstream transcriptional targets of the AHR, CYP1A1 and CYP1A2, and describe conserved elements of their genomic architecture. When comparisons across different sites are made, hepatic expression of CYP1A2, a direct target of the AHR, appears elevated in embryos from a site associated with a dioxin point source and previously characterized PCB contamination. Elevated CYP1A2 expression is not persistent, as site-specific variation was absent in juveniles originating from field-collected eggs but reared under lab conditions. Our results illustrate the patterning of AHR gene expression in a long-lived environmental model species, and indicate a potential contemporary influence of historical contamination. This research presents a novel opportunity to link contamination events to critical genetic pathways during embryonic development, and carries significant potential to inform our understanding of potential health effects in wildlife and humans.