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.

High Copper Selectively Alters Lipid Metabolism and Cell Cycle Machinery in the Mouse Model of Wilson Disease

“…Comparison of copper-induced mRNA changes with published mRNA profiles of liver responses to various stresses (32)(33)(34)(35) illustrates that the overall pattern of changes in response to copper accumulation (i.e. the combination of most altered pathways) is distinct.…”

Gene Expression Profiling of Rat Liver Treated With Serum Triglyceride-Decreasing Compounds

“…Although there have been various reports describing strategies to extract marker genes from the transcriptome data (Hibbs et al, 2004;Mutlib et al, 2006;Tan et al, 2006), the best way has not been established. In the present study, we have started to identify candidates of potential biomarker genes for interpretation of the fundamental mechanism(s) of plasma TG decrease, since our database contains several drugs that cause plasma TG decrease.…”

The Inactivation of Human CYP2E1 by Phenethyl Isothiocyanate, a Naturally Occurring Chemopreventive Agent, and Its Oxidative Bioactivation

“…Isocyanates are known to be highly reactive (Lee, 1992(Lee, , 1996, and toxicity has been observed with some isocyanates, such as methyl isocyanate, which causes severe pulmonary toxicity (Nemery et al, 1985), and naphthyl isocyanate, which has been shown to be mutagenic (Tamura et al, 1990). Other drugs and compounds that are metabolized to form isocyanates include tolbutamide (Guan et al, 1999), N-methylformamide (NMF) (Mutlib et al, 2006), and anticancer agents, such as nitrosoureas (Rice et al, 2005) and diarylsulfonylureas (Jochheim et al, 2002). Arguments have been made for the role of isocyanates in some of the biologic effects of these compounds.…”

“…Arguments have been made for the role of isocyanates in some of the biologic effects of these compounds. Methyl isocyanate formed from NMF is thought to be dmd.aspetjournals.org related to the hepatotoxicity induced by NMF (Mutlib et al, 2006). Alkyl isocyanates produced by the oxidative metabolism of tolbutamide and N,N9-bis(2-chloroethyl)-N-nitrosourea (BCNU) are known to inhibit glutathione reductase (Guan et al, 1999;Rice et al, 2005).…”