P450 3As Research Articles

Regulatory variants in a novel distal enhancer regulate the expression of CYP3A4 and CYP3A5.

The cytochrome P450 3As (CYP3As) are abundantly expressed in the liver and metabolize many commonly prescribed medications. Their expression is highly variable between individuals with little known genetic cause. Despite extensive investigation, cis-acting genetic elements that control the expression of the CYP3As remain uncharacterized. Using chromatin conformation capture (4C assays), we detected reciprocal interaction between a distal regulatory region (DRR) and the CYP3A4 promoter. The DRR colocalizes with a variety of enhancer marks and was found to promote transcription in reporter assays. CRISPR-mediated deletion of the DRR decreased expression of CYP3A4, CYP3A5, and CYP3A7, supporting its role as a shared enhancer regulating the expression of three CYP3A genes. Using reporter gene assays, we identified two single-nucleotide polymorphisms (rs115025140 and rs776744/rs776742) that increased DRR-driven luciferase reporter expression. In a liver cohort (n=246), rs115025140 was associated with increased expression of CYP3A4 mRNA (1.8-fold) and protein (1.6-fold) and rs776744/rs776742 was associated with 1.39-fold increased expression of CYP3A5 mRNA. The rs115025140 is unique to the African population and in a clinical cohort of African Americans taking statins for lipid control rs115025140 carriers showed a trend toward reduced statin-mediated lipid reduction. In addition, using a published cohort of Chinese patients who underwent renal transplantation taking tacrolimus, rs776744/rs776742 carriers were associated with reduced tacrolimus concentration after adjusting for CYP3A5*3. Our results elucidate a complex regulatory network controlling expression of three CYP3A genes and identify two novel regulatory variants with potential clinical relevance for predicting CYP3A4 and CYP3A5 expression.

Coordinated Transcriptional Regulation of Cytochrome P450 3As by Nuclear Transcription Factor Y and Specificity Protein 1

The cytochrome P450 3A subfamily plays vital roles in the metabolism of endogenous chemicals and xenobiotics. Understanding the basal expression of CYP3A in humans and pigs is crucial for drug evaluation. In this study, we demonstrated that the basal transcriptional regulation of CYP3A genes in hepatocytes is evolutionarily conserved between humans and pigs. The basal expression of CYP3A genes is transactivated by two cis-acting elements, the CCAAT and GC boxes, located a constant distance apart in the proximal promoter region of six CYP3A genes. Mutation analysis of these two cis-acting elements suggested that they play important roles in mediating basal expression, but to different extents because of the nucleotide variations in the elements. Two transcription factors, nuclear transcription factor Y (NF-Y) and specificity protein 1 (Sp1), directly bind to these cis-acting elements in CYP3A proximal promoters in HepG2 cells and porcine hepatocytes. Furthermore, changing the distance between the NF-Y and Sp1 binding sites resulted in decreases in the promoter activity of CYP3A genes. Conclusively, our results show that human and porcine CYP3A genes are regulated by NF-Y and Sp1 in a coordinated manner, and that the distance between these two cis-acting elements is crucial for constitutive CYP3A expression.

Effect of the active ingredient of Kaempferia parviflora, 5,7-dimethoxyflavone, on the pharmacokinetics of midazolam.

5,7-Dimethoxyflavone (5,7-DMF), one of the major components of Kaempferia parviflora, has anti-obesity, anti-inflammatory, and antineoplastic effects. On the other hand, in vitro studies have reported that it directly inhibits the drug metabolizing enzyme family cytochrome P450 (CYP) 3As. In this study, its safety was evaluated from a pharmacokinetic point of view, based on daily ingestion of 5,7-DMF. Midazolam, a substrate of CYP3As, was orally administered to mice treated with 5,7-DMF for 10days, and its pharmacokinetic properties were investigated. In the group administered 5,7-DMF, the area under the curve (AUC) of midazolam increased by 130% and its biological half-life was extended by approximately 100min compared to the control group. Compared to the control group, 5,7-DMF markedly decreased the expression of CYP3A11 and CYP3A25 in the liver. These results suggest that continued ingestion of 5,7-DMF decreases the expression of CYP3As in the liver, consequently increasing the blood concentrations of drugs metabolized by CYP3As.

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p < 0.05) and highly (r = 0.82, p < 0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.4. Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.5. Recombinant marmoset P450 3As had high Vmax/Km values for C-8-hydroxylation, comparable to Km values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower Km values than those of liver microsomes.6. These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.

Integrated Transcriptional and Proteomic Analysis with In Vitro Biochemical Assay Reveal the Important Role of CYP3A46 in T-2 Toxin Hydroxylation in Porcine Primary Hepatocytes

Both T-2 toxin and its metabolites are highly potent mycotoxins that can cause severe human and animal diseases upon exposure. Understanding the toxic mechanism and biotransformation process of T-2 toxin at a cellular level is essential for the development of counter-measures. We investigated the effect of T-2 toxin in porcine primary hepatocytes using porcine genome array and two-dimensional difference gel electrophoresis with matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. Integrated transcriptional and proteomic analysis demonstrated that T-2 toxin adversely affected porcine hepatocytes by initiating lipid metabolism disorder, oxidative stress response, and apoptosis. In addition, xenobiotic metabolism genes, including cytochrome P450 3As (CYP3A46 and CYP3A39), carboxylesterase 1Cs (CES1C4 and CES1C5), and epoxide hydrolase (EPHX1), increased in T-2 toxin treatment cells. Using HepG2 cells to over-express the recombinant xenobiotic metabolism genes above and rapid resolution liquid chromatography/tandem mass spectrometry to detect metabolites of T-2 toxin, we determined that porcine CYP3A46 mainly catalyzed T-2 to form 3'-hydroxy-T-2, which was further confirmed by purified CYP3A46 protein. However, recombinant porcine CES1C5 and EPHX1 did not enhance hydrolysis and de-epoxidation of T-2 implying that other esterases and epoxide hydrolases may play dominant roles in those reactions.

Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus

Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. Human liver microsomal fractions (HLMs) were prepared from diabetic (n= 12) and demographically matched nondiabetic (n= 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. Midazolam 1'- or 4-hydroxylation and testosterone 6β-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted.

CYP3As catalyze nifedipine oxidation in pig liver microsomes: Enzyme kinetics, inhibition and functional expression

Abstract Cytochrome P450 (CYP) 3As have received much attention because of their importance in drug metabolism. However, information on CYP3As in pigs is still largely incomplete. Herein, we focused on the involvement of CYP3As in nifedipine oxidation in pig liver microsomes. Enzyme kinetics, inhibition and immunochemical studies indicated that CYP3As might catalyze nifedipine oxidation and that ketoconazole could strongly inhibit this reaction in a noncompetitive and time-dependent manner. Furthermore, pig CYP3A46, showing the highest amino acid sequence identity to human CYP3A4, exhibited nifedipine oxidation activity. Collectively, these results provide strong evidence that CYP3As catalyze nifedipine oxidation in pig liver microsomes.

Pregnane X Receptor as a Therapeutic Target to Inhibit Androgen Activity

The androgen-androgen receptor signaling pathway plays an important role in the pathogenesis of prostate cancer. Accordingly, androgen deprivation has been the most effective endocrine therapy for hormone-dependent prostate cancer. Here, we report a novel pregnane X receptor (PXR)-mediated and metabolism-based mechanism to reduce androgenic tone. PXR is a nuclear receptor previously known as a xenobiotic receptor regulating the expression of drug metabolizing enzymes and transporters. We showed that genetic (using a PXR transgene) or pharmacological (using a PXR agonist) activation of PXR lowered androgenic activity and inhibited androgen-dependent prostate regeneration in castrated male mice that received daily injections of testosterone propionate by inducing the expression of cytochrome P450 (CYP)3As and hydroxysteroid sulfotransferase (SULT)2A1, which are enzymes important for the metabolic deactivation of androgens. In human prostate cancer cells, treatment with the PXR agonist rifampicin (RIF) inhibited androgen-dependent proliferation of LAPC-4 cells but had little effect on the growth of the androgen-independent isogenic LA99 cells. Down-regulation of PXR or SULT2A1 in LAPC-4 cells by short hairpin RNA or small interfering RNA abolished the RIF effect, indicating that the inhibitory effect of RIF on androgens was PXR and SULT2A1 dependent. In summary, we have uncovered a novel function of PXR in androgen homeostasis. PXR may represent a novel therapeutic target to lower androgen activity and may aid in the treatment and prevention of hormone-dependent prostate cancer.

Cytochrome P450 3As Gene Expression and Testosterone 6.BETA.-Hydroxylase Activity in Human Fetal Membranes and Placenta at Full Term

Expression levels of cytochrome P450 (CYP) 3A4, CYP3A5 and CYP3A7 mRNAs in placentas and fetal membranes, which were split into amnion and chorion leave attached decidua (chorion/decidua), obtained from pregnant women with normal delivery (5 subjects) and Caesarean section (15 subjects) were determined. These CYP3A mRNAs were also expressed in amnion and chorion/decidua together with placenta, although the expression level of these mRNAs was strikingly different between subjects. The expression level of the CYP3A4 mRNA in the placenta was about 2-fold higher than those in amnion and chorion/decidua. On the other hand, the expression levels of CYP3A5 and CYP3A7 mRNAs were highest in chorion/decidua. The immunologically related protein(s) with CYP3A7 was detected in all tissues examined. Testosterone 6beta-hydroxylase activity in homogenate of human placenta, amnion and chorion/decidua were 26.6, 3.7 and 4.6 pmol/h/mg protein, respectively. These results suggest that CYP3As in fetal membranes have the metabolic function to protect the fetus from exposure to drugs.

Effects of synthetic polycyclic musks on estrogen receptor, vitellogenin, pregnane X receptor, and cytochrome P450 3A gene expression in the livers of male medaka ( Oryzias latipes)

This study demonstrates the effects of synthetic polycyclic musks such as Galaxolide (HHCB), Tonalide (AHTN), Traseolide (ATII), Celestolide (ADBI), Phantolide (AHMI) and Cashmeran (DPMI), both on the early life stage and on gene expression in the livers of male medaka ( Oryzias latipes). The toxicity ranking (the 96-h median lethal concentration) of the chemicals tested on 24-h-old medaka larvae descended in the order HHCB (0.95 mg/L) = ATII (0.95 mg/L) > AHTN (1.0 mg/L) > AHMI (1.2 mg/L) > ADBI (2.0 mg/L) ≫ DPMI (12 mg/L), indicating high acute toxicity of these compounds on the early life stages of medaka. Expression analysis of hepatic vitellogenin (VTG) protein showed potential estrogenic effects upon the addition of AHTN and HHCB, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two estrogen receptor (ER) subtypes (ERα and β), two VTGs (VTG I and II), pregnane X receptor (PXR), and two cytochromes P450 (CYP) 3As (CYP3A38 and 3A40) in the livers of male medaka treated with AHTN and HHCB at 5, 50 and 500 μg/L. Quantitative real-time PCR analyses revealed that hepatic ERα, VTG I, VTG II, and CYP3A40 mRNA responded to 500 μg/L of AHTN and/or HHCB after 3 days exposure, whereas no effects of these compounds on ERβ, PXR, and CYP3A38 mRNA transcription were observed. These results suggest that certain polycyclic musks, including AHTN and HHCB, induce the expression levels of hepatic ERα and VTG mRNA/protein and modulate expression levels of CYP3A40 mRNA in the livers of male medaka.

Discovery of Osmosensitive Transcriptional Regulation of Human Cytochrome P450 3As by the Tonicity-Responsive Enhancer Binding Protein (Nuclear Factor of Activated T Cells 5)

We report the discovery of an osmosensitive transcriptional control of human CYP3A4, CYP3A7, and CYP3A5. Ambient hypertonicity (350-450 mOsmol/kg) increased mRNA expressions of the CYP3A by approximately 10- to 20-fold in human-intestinal C(2)bbe1 cells, followed by an increase of CYP3A protein. Hypotonicity, on the other hand, suppressed CYP3A mRNA levels, indicating that physiological isotonic conditions may regulate the basal expression of CYP3A. Similar responses to ambient tonicity were observed in other human-derived cell lines (intestinal LS180 and hepatic HepG2) and human primary colonic cells. The 11-base pair tonicity-responsive enhancer (TonE) is an osmosensitive regulator that is activated by the transcription factor, the nuclear factor of activated T-cells 5 (NFAT5). Luciferase-based reporter assays of 13 consensus TonE motifs within +/-10 kilobases (kb) from the transcription start sites of CYP3A showed that only the CYP3A7 intron 2 region ( approximately 5 kb downstream from the transcription start site), which contains two TonE motifs (+5076/+5086 and + 5417/+5427), was responsive to hypertonicity stimuli. This observation was confirmed upon cotransfection with an NFAT5 expression vector, small interfering RNA, or dominant-negative NFAT5. Deletion and mutation analyses suggested that the TonE (+5417/+5427) is indispensable for the enhancer activity. NFAT5 binding to the CYP3A7 intron 2 TonE motif was demonstrated with electrophoretic mobility shift assay and in a native cell context by chromatin immunoprecipitation. We conclude that transcription of human CYP3A is influenced by ambient tonicity. The physiological significance of the tonic regulation of CYP3A enzymes remains to be determined.

Generation of specific antibodies and their use to characterize sex differences in four rat P450 3A enzymes following vehicle and pregnenolone 16α-carbonitrile treatment

The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16α-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 ≫ 3A18 > 3A9 ≅ 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively.

Ethnic differences between Japanese and Caucasians in the expression levels of mRNAs for CYP3A4, CYP3A5 and CYP3A7: lack of co-regulation of the expression of CYP3A in Japanese livers

Using a newly developed real-time reverse transcriptase-polymerase chain reaction method, mRNAs were quantitated for CYP3A4, CYP3A5 and CYP3A7 in adult livers from 24 Japanese and 24 Caucasian subjects to elucidate the potential ethnic differences in the expression levels of human cytochrome P450 (CYP) 3As. The expression level of CYP3A4 mRNA in Japanese livers (n = 24) was approximately three times higher than that in Caucasian livers (n = 24, p < 0.001). The mean level of CYP3A5 mRNA was approximately twice higher in Japanese (n = 9) than in Caucasians (n = 5) heterozygous for the CYP3A5 *1 allele (p = 0.057). The CYP3A7 mRNA level was twice higher in Japanese (n = 24) than in Caucasians (n = 22) carrying the CYP3A7 *1A/ *1A genotype (p = 0.042). The level of CYP3A4 mRNA did not correlate with those of CYP3A5 (r = 0.044, n = 24) or CYP3A7 (r = 0.21, n = 24) mRNAs in Japanese livers in contrast to co-regulatory expression of CYP3A4, CYP3A5 and CYP3A7 in Caucasian livers. The results indicate that there are ethnic differences in the expression levels of adult liver CYP3A mRNAs between Japanese and Caucasians, and that the mechanism(s) regulating the hepatic CYP3A expression may be different between these ethnic groups.

Effects of cytochrome b5 on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5 with CYP3A4 but not CYP3A7

Effects of cytochrome b 5 ( b 5) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b 5 in approximately one third of the substrates employed in this study. Such enhancement by b 5 was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b 5 with any substrates employed. V max values for midazolam 1′-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b 5, which was also seen with CYP3A4, although the extent of the effects of b 5 on S 50 ( K m ) and Hill coefficient differed dependent on substrates used. In contrast, b 5 did not alter any of these kinetic parameters of CYP3A7. The effects of b 5 on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b 5 in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.

Particular ability of cytochromes P450 3A to form inhibitory P450-iron-metabolite complexes upon metabolic oxidation of aminodrugs

The ability of 21 drugs containing an amine function to form inhibitory P450-iron-metabolite complexes absorbing around 455 nm was studied on liver microsomes from rats treated with various P450 inducers. These drugs belong to different chemical and therapeutic series and exhibit very different structures. In the case of eight compounds (diltiazem, lidocaine, imipramine, SKF 525A, fluoxetine, l-α-acetylmethadol, methadol and desmethyltamoxifen) whose oxidation by microsomes from rats treated with several inducers was studied, only dexamethasone (DEX)-treated rat microsomes and, to a lesser extent, phenobarbital (PB)-treated rat microsomes, were able to give significant amounts of 455 nm absorbing complexes. Ten of the 21 compounds studied gave such complexes with DEX-treated rat microsomes, while only three compounds gave complexes (in low amounts) with PB-treated rat microsomes only. For all compounds leading to complexes both with DEX- and PB-treated rat microsomes, much higher amounts of complexes were obtained with DEX-treated rat microsomes. DEX-treated rat microsomes also led to the most intense type I spectral interactions with most of the compounds studied, and very often exhibited the highest N-dealkylation activities towards the tertiary or secondary amine function of the drugs used. A few exceptions aside, there generally exists a qualitative relationship between the ability of P450 3As, induced by DEX, to bind and N-dealkylate amino compounds and their propensity to lead to 455 nm absorbing complexes. This was confirmed by in vivo experiments showing that rats treated with diltiazem, tamoxifen or imipramine accumulated large amounts of 455 nm absorbing complexes in their liver only after pretreatment with DEX and, to a lesser extent, with PB. This particular ability of P450 3As to oxidize amino drugs with formation of inhibitory P450-metabolite complexes could be of great importance for the appearance of drug interactions in man.