title: The cytochrome P450 inhibitor SKF-525A disrupts autophagy in primary rat hepatocytes.
abstract: The cytochrome P450 (CYP) inhibitor SKF-525A is commonly used to study drug metabolism and toxicity, particularly hepatotoxicity. By using Western blot and immunofluorescence staining, we unexpectedly found that SKF-525A at 2-20 μM caused remarkable accumulation of microtubule-associated protein light chain 3 II (LC3-II) in primary rat hepatocytes at 1, 4 and 24 h, indicating that autophagy was disrupted. SKF-525A showed no effects on chloroquine induced LC3-II accumulation, suggesting that autophagic flux was blocked, which is further supported by the increased level of the p62 protein after SKF-525A treatment. SKF-525A did not affect proteasome activities or gene expression of LC3-II or p62. Immunofluorescence of green fluorescent protein fused lysosomal-associated membrane protein 1 (LAMP1, a specific protein marker for lysosomes) and LC3-II showed that co-localization of these two proteins was partially abolished by SKF-525A, indicating that autophagosome-lysosome fusion was blocked. The other five CYP inhibitors, metyrapone, 1-aminobenzotriazole, alpha-naphthoflavone, ticlopidine, and ketoconazole, showed no effects in parallel experiments. These findings provide novel insights into the mechanisms by which various CYP inhibitors differentially affect a same drug's toxicity in hepatocytes. The data also indicate that SKF-525A is not an ideal chemical inhibitor for probing the relation between CYP mediated metabolism and toxicity in primary hepatocytes.
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, US FDA, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, US FDA, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Yang
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, US FDA, 3900 NCTR Road, Jefferson, AR 72079, USA. Electronic address: qiang.shi@fda.hhs.gov.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shi
mesh_terms: D000818:Animals; D000071182:Autophagosomes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001343:Autophagy / Q000187:drug effects*; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011335:Proadifen / Q000633:toxicity*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D011335:Proadifen; D046988:Proteasome Endopeptidase Complex; C526109:Anxa3 protein, rat
keywords: Autophagy; Cytochrome P450; Hepatocyte; Hepatotoxicity; SKF-525A