title: Identification of potential genomic biomarkers of hepatotoxicity caused by reactive metabolites of N-methylformamide: Application of stable isotope labeled compounds in toxicogenomic studies.
abstract: The inability to predict if a metabolically bioactivated compound will cause toxicity in later stages of drug development or post-marketing is of serious concern. One approach for improving the predictive success of compound toxicity has been to compare the gene expression profile in preclinical models dosed with novel compounds to a gene expression database generated from compounds with known toxicity. While this guilt-by-association approach can be useful, it is often difficult to elucidate gene expression changes that may be related to the generation of reactive metabolites. In an effort to address this issue, we compared the gene expression profiles obtained from animals treated with a soft-electrophile-producing hepatotoxic compound against corresponding deuterium labeled analogues resistant to metabolic processing. Our aim was to identify a subset of potential biomarker genes for hepatotoxicity caused by soft-electrophile-producing compounds. The current study utilized a known hepatotoxic compound N-methylformamide (NMF) and its two analogues labeled with deuterium at different positions to block metabolic oxidation at the formyl (d(1)) and methyl (d(3)) moieties. Groups of mice were dosed with each compound, and their livers were harvested at different time intervals. RNA was prepared and analyzed on Affymetrix GeneChip arrays. RNA transcripts showing statistically significant changes were identified, and selected changes were confirmed using TaqMan RT-PCR. Serum clinical chemistry and histopathologic evaluations were performed on selected samples as well. The data set generated from the different groups of animals enabled us to determine which gene expression changes were attributed to the bioactivating pathway. We were able to selectively modulate the metabolism of NMF by labeling various positions of the molecule with a stable isotope, allowing us to monitor gene changes specifically due to a particular metabolic pathway. Two groups of genes were identified, which were associated with the metabolism of a certain part of the NMF molecule. The metabolic pathway leading to the production of reactive methyl isocyanate resulted in distinct expression patterns that correlated with histopathologic findings. There was a clear correlation between the expression of certain genes involved in the cell cycle/apoptosis and inflammatory pathways and the presence of reactive metabolite. These genes may serve as potential genomic biomarkers of hepatotoxicity induced by soft-electrophile-producing compounds. However, the robustness of these potential genomic biomarkers will need to be validated using other hepatotoxicants (both soft- and hard-electrophile-producing agents) and compounds known to cause idiosyncratic liver toxicity before being adopted into the drug discovery screening process.
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA. mutliba@wyeth.com
authors.forename: Abdul
authors.initials: A
authors.lastname: Mutlib
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.forename: Jim
authors.initials: J
authors.lastname: Atherton
authors.forename: Leslie
authors.initials: L
authors.lastname: Obert
authors.forename: Seva
authors.initials: S
authors.lastname: Kostrubsky
authors.forename: Steven
authors.initials: S
authors.lastname: Madore
authors.forename: Sidney
authors.initials: S
authors.lastname: Nelson
mesh_terms: D000818:Animals; D015415:Biomarkers; D015536:Down-Regulation / Q000187:drug effects; D005559:Formamides / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D016678:Genome / Q000235:genetics*; D007553:Isotope Labeling; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D013997:Time Factors; D043922:Toxicogenetics; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D015415:Biomarkers; D005559:Formamides; C002950:methylformamide