Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1.

The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide

Cross-talk between nuclear receptors involved in the control of drug metabolism is being increasingly recognised as a source of drug side effects. Omeprazole is a well known activator of the aryl hydrocarbon receptor (AhR). We investigated the regulation of AhR by omeprazole-sulphide, a degradation metabolite of omeprazole, using CYP1A mRNA induction, reporter gene assay, receptor DNA binding, ligand binding, nuclear translocation, trypsin digests, and drug metabolism analysis in mouse Hepa-1c1c7, human HepG2 cells and primary human hepatocytes. Omeprazole-sulphide is a pure antagonist of AhR in Hepa-1c1c7 and HepG2 hepatoma cell lines. In Hepa-1c1c7 cells, omeprazole-sulphide is a ligand of AhR, inhibits AhR activation to a DNA-binding form, induces a specific pattern of AhR trypsin digestion and inhibits AhR nuclear translocation and subsequent degradation in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in highly differentiated primary human hepatocytes treated with rifampicin an agonist of the pregnane X receptor (PXR), omeprazole-sulphide behaves as an agonist of AhR. Inhibition of drug metabolizing enzymes by ketoconazole restores the antagonist effect of omeprazole-sulphide. Metabolic LC/MS analysis reveals that omeprazole-sulphide (AhR antagonist) is efficiently converted to omeprazole (AhR activator) by cytochrome P450 CYP3A4, a target gene of PXR, in primary human hepatocytes but not in hepatoma cells in which PXR is not expressed. This report provides the first evidence for a cross-talk between PXR/CYP3A4 and AhR. In addition, it clearly shows that conclusions drawn from experiments carried out in cell lines may lead to erroneous in vivo predictions in man.     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.     

The use of transgenic cell lines for evaluating toxic metabolites of carbamazepine

Human lymphoblastoid cell lines transgenic for human CYP450s were evaluated for the identification of toxic metabolites of the anticonvulsant drug carbamazepine (CBZ). Human CYP450 isoforms expressed by these cell lines included 1A1, 1A2, 2E1, 2A6 and 3A4. A dose-dependent inhibition of population growth from 50–200 g/ml CBZ was detected by measuring cell number and respiration. The inhibition increased with the growth rate of the various lines, which correlated inversely with the presence of CYP450s, and may have been caused by CBZ itself. Cytotoxicity was observed only at the highest dose and in the line lacking transfected CYP450s. Microsomal preparations from hCYP3A4/OR cells converted CBZ into its principal oxidative metabolite, carbamazepine-10,11-epoxide (CBZ-E), at a rate of 630 pmol/min per mg protein, confirming a major role of CYP3A4 in this reaction. However, no CBZ-E (or any metabolite) was recovered from any whole-cell incubation even though hCYP3A4 cells readily converted testosterone to 6ß-hydroxytestosterone. This suggests that differences exist between whole-cell and microsomal preparations of lymphoblastoid cells in their ability to metabolize CBZ.Abbreviations BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide - CBZ carbamazepine - CBZ-E carbamazepine-10, 11-epoxide - CYP450 cytochrome P450 - CYP3A4 cytochrome P450, isoform 3A4 - DMSO dimethyl sulfoxide - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl)tetrazolium - SIM selected-ion monitoring - TMS trimethylsilyl     

Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice

Metabolism of vitamin E is initiated by cytochrome P450 (CYP) enzymes usually involved in the metabolism of xenobiotics. Like other CYP substrates, vitamin E induced a reporter gene under the control of the pregnane X receptor (PXR) which regulates the expression of CYPs including CYP3A4. gamma-Tocotrienol, the most effective PXR activator, also induced endogenous CYP3A4 mRNA in HepG2 cells. Since these findings imply an interference of vitamin E with drug metabolism it was deemed necessary to investigate their in vivo relevance. Therefore, mice were grown for 3 months with alpha-tocopherol-deficient, -adequate, and -supranutritional diet, i.e. 2, 20 and 200 mg RRR-alpha-tocopheryl acetate/kg diet, respectively. Half of them received 250 microg gamma-tocotrienol/day for the last 7 days. After 3 months, hepatic levels of Cyp3a11 mRNA, the murine homolog to human CYP3A4, were about 2.5-fold higher in the 20 and 200 mg alpha-tocopherol groups than in the 2 mg group. After feeding 200 mg alpha-tocopherol for 9 months, Cyp3a11 mRNA was 1.7-fold higher than after 3 months. In contrast, gamma-tocotrienol did not induce Cyp3a11 mRNA. This could be explained by its high metabolism as demonstrated by the 20- to 25-fold increase in the urinary excretion of gamma-CEHC, the final metabolite of gamma-tocotrienol degradation. In conclusion, alpha-tocopherol maintains an adequate level of xenobiotic-metabolizing enzymes. If fed in supranutritional dosages, especially for longer times, alpha-tocopherol induces Cyp3a11 to levels which might interfere with drug metabolism.     

Rat pregnane X receptor: molecular cloning, tissue distribution, and xenobiotic regulation.

An orphan nuclear receptor, termed the pregnane X receptor (PXR), has recently been cloned from mouse and human and defines a novel steroid signaling pathway (Cell 92, 73-82, 1998; Proc. Natl. Acad. Sci. USA 95, 12208-122313, 1998). Transient cotransfection experiments demonstrate that the PXR responds to structurally dissimilar compounds and confers the induction of cytochrome P4503A (CYP3A), a subfamily of enzymes that involve the metabolism of two-thirds of drugs and other xenobiotics. In this report, we describe the molecular cloning, tissue distribution, and xenobiotic regulation of a rat PXR designated rPXR-1. rPXR-1 exhibits a 95% sequence identity with the mouse PXR, but only 79% identity with the human PXR, providing the molecular basis that rats and mice have a similar CYP3A induction profile but differ from humans. rPXR-1 gene was expressed abundantly in liver, intestine, and, to a lesser extent, kidney, lung, and stomach. The tissue distribution and the relative abundance of rPXR-1 mRNA among these tissues resemble those of CYP3A, suggesting that PXR is important not only for induction but also for constitutive expression of these enzymes. Xenobiotics known to induce liver microsomal enzymes showed differential effects on the rPXR-1 expression as determined by Northern blot analysis. Dexamethasone, for example, increased the accumulation of rPXR-1 mRNA, whereas troleandomycin slightly suppressed it. Compounds that increase PXR expression (inducers) and compounds that interact with PXR (ligands) likely have synergistic effects on CYP3A induction, which provides a novel molecular explanation for drug-drug interactions.