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ALTERATIONS IN A11 TRANS RETINOIC ACID METABOLISM IN LIVER MICROSOMES FROM MICE TREATED WITH HEPATOTUMORIGENIC AND NON-HEPATOTUMORIGENIC CONAZOLES
Citation:
CHEN, P., W. PADGETT, T. MOORE, W. M. WINNIK, S. Y. THAI, S. D. HESTER, AND S. NESNOW. ALTERATIONS IN A11 TRANS RETINOIC ACID METABOLISM IN LIVER MICROSOMES FROM MICE TREATED WITH HEPATOTUMORIGENIC AND NON-HEPATOTUMORIGENIC CONAZOLES. Presented at North Carolina Society of Toxicology Fall Meeting - Metabolic Syndrome, Research Triangle Park, NC, October 12, 2007.
Impact/Purpose:
The purpose of this study was to examine the metabolism of atRA by liver microsomes from conazole-treated mice, and characterize the microsomes from conazole-treated mice, and characterize the associated hepatic cytochrome P450 (Cyp) enzyme(s) involved in atRA metabolism.
Description:
Conazoles are fungicides used in crop protection and as pharmaceuticals. Triadimefon and propiconazole are hepatotumorigenic in mice, while myclobutanil is not. Previous toxicogenomic studies suggest that alteration of the retinoic acid metabolism pathway may be a key event in conazole-induced hepatotumorigenesis. All trans-retinoic acid (atRA) has cancer-preventative properties by inhibiting cell proliferation and promoting cell differentiation and apoptosis. The purpose of this study was to examine the metabolism of atRA by liver microsomes from conazole-treated mice, and characterize the associated hepatic cytochrome P450 (Cyp) enzyme(s) involved in atRA metabolism. In vitro metabolism of atRA was assessed in liver microsornes from male CD-1 mice following four daily intraperitoneal (IP) injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). Total atRA metabolism activity (sum of 4-OH- and 4-oxo-atRA metabolites) and expression of atRA metabolism genes in the livers were quantified with high performance liquid chromatography (HPLC) and quantitative real time polymerise chain reaction (qRT-PCR). Formation of 4-OH-, 4-oxo-atRA. and total atRA metabolism were significantly increased by all three conazoles. In addition, propiconazole-induced microsomes possessed greater metabolizing activities based on total atRA metabolism and 4-OH-atRA formation compared to myclobutanil-induced microsomes (Figure 1). Cyp26a1, Cyp2b10(20), Cyp2c65 and Cyp1a2 genes were significantly over-expressed in the livers from propiconazole- or triadimefon-treated mice, while only Cyp2b10(20) and Cyp3a13 genes were over-expressed in the myclobutanil-treated mice. Total atRA metabolism was inhibited by a series of chemicals (listed in decreasing potency based on IC50 determinations): ketoconazole>metyrapone>ticlopidine>diethyldithiocarbamate>quercetin, while α-naphthoflavone did not inhibit atRA oxidation. Immuno-inhibition studies with antisera specific for Cyp26a1, Cyp3a or Cyp2b suggested all three Cyps were involved in atRA metabolism. The over-expression of Cyp26a1, Cyp2b10(20) and Cyp3a11/Cyp3a13 enzymes and increased atRA metabolism induced by conazoles are consistent with increased atRA oxidation in the liver. The implications of these changes with respect to reduced atRA level in hepatic tissues suggest that it might be a necessary event in conazole-induced hepatotumorigenesis.