Metabolomics, is an ideal approach for analysis of the metabolites present in fluids, tissues or cells, and is evolving as a useful tool in drug research and development. Various analytical platforms [UPLC-ESI-QTOFMS, gas GC-MS and nuclear magnetic resonance] and multivariate data analysis [such as principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA)] have been applied in metabolomic-based xenobiotic metabolism studies. Using UPLC-ESI-QTOFMS based metabolomics, the metabolic maps have been determined for acetaminophen, aminoflavin, arecoline, melatonin, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and ifosfamide/cyclophosphamide, thioTEPA, procainamide, and noscarpine. Metabolomics has proven to be a powerful method for study of drug and carcinogen metabolism and to search for biomarkers of drug toxicity and efficacy. This methodology is especially valuable when combined with the use of P450 knockout and humanized mice. This technique is also being used to search for disease biomarkers that can be employed for diagnosis of cancer and monitoring of cancer therapy. Most of our screening studies are carried out using mouse cancer models to identify the biomarkers. Once identified, the biomarkers will be validated using samples from human patients.The metabolism of noscapine, a promising anti-tumor agent, was examined by metabolomics. A metabolic map was generated and the bioactivation of noscapine investigated. UPLC-ESI-QTOFMS was used to analyze in vitro incubation mixtures, urine and feces samples from mice treated with noscapine. Recombinant drug-metabolizing enzymes were employed to identify those involved in noscapine metabolism. Hepatic reduced glutathione (GSH) levels and serum biochemistry were also carried out to determine reactive metabolites of noscapine. Several novel phase I metabolites of noscapine were detected after oral gavage of mice, including an N-demethylated metabolite, two hydroxylated metabolites, one metabolite undergoing both demethylation and cleavage of the methylenedioxy group, and a bis-demethylated metabolite. Additionally, several novel glucuronides were detected, and their structures elucidated through MS/MS fragmentology. Recombinant enzymes screening showed the involvement of several cytochromes P450, flavin-containing monooxygenase 1 and the UDP-glucuronosyltransferases UGT1A1, UGT1A3, UGT1A9 and UGT2B7, in noscapine metabolism. In vitro glutathione trapping revealed the existence of an ortho-quinone reactive intermediate formed through further oxidation of a catechol metabolite. However, this bioactivation process of noscapine does not occur in vivo. Similar to this result, altered glutathione levels in liver and serum biochemistry revealed no evidence for hepatic damage thus indicating that, at least in mice, noscapine did not induce hepatotoxicity through bioactivation. A comprehensive metabolic map and bioactivation evaluation provides important information for the development of noscapine as an anti-tumor drug.Metabolomics was also used to determine the metabolism of procainamide, a type I antiarrhythmic agent, used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25-30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using UPLC-ESI-QTOFMS was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice aswell as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.Cytochromes P450 are also expressed in fetal livers of humans and may have a role of fetal toxicity. CYP3A7 is the predominant cytochrome P450 expressed in human fetal liver, accounting for 30-50% of the total CYP in fetal liver and 87-100% of total fetal hepatic CYP3A content. However, the lack of a rodent model limits the investigation of CYP3A7 regulation and function. Hence, double-transgenic mice expressing human PXR and CYP3A4/7 (Tg3A4/7-hPXR) were used to investigate the regulation and function of CYP3A7. Expression of CYP3A7 was monitored in mice that ranged in age from 14.5-d-old embryos to 8.5-d-old newborns;expression of CYP3A7 mRNA was increased before birth in the embryos and decreased after birth in the newborns. This is consistent with the observed developmental regulation of CYP3A7 protein levels and CYP3A7-mediated dehydroepiandrosterone 16alpha-hydroxylase activities. This developmental flux is also in agreement with previous studies that have investigated the expression of CYP3A7 in developing human liver. The regulation of CYP3A7 was further studied using hepatoblasts from the Tg3A4/7-hPXR mice. Glucocorticoids, including dexamethasone, cortisol, corticosterone, and cortisone all induced the expression of CYP3A7 mRNA, whereas rifampicin, an activator of PXR and an inducer of CYP3A4 in adult liver, had no effect on CYP3A7 expression. Cell-based promoter luciferase and chromatin immunoprecipitation assays further confirmed glucocorticoid receptor-mediated control of the CYP3A7 promoter. These findings indicate that CYP3A7 is developmentally regulated in mouse liver primarily by glucocorticoids through the glucocorticoid receptor. The Tg3A4/7-hPXR mouse model could therefore potentially serve as a tool for investigating CYP3A7 regulation and function.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005562-25
Application #
8552578
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2012
Total Cost
$1,094,579
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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