title: Trovafloxacin enhances lipopolysaccharide-stimulated production of tumor necrosis factor-α by macrophages: role of the DNA damage response.
abstract: Trovafloxacin (TVX) is a drug that has caused idiosyncratic, drug-induced liver injury (IDILI) in humans. In a murine model of IDILI, otherwise nontoxic doses of TVX and the inflammagen lipopolysaccharide (LPS) interacted to produce pronounced hepatocellular injury. The liver injury depended on a TVX-induced, small but significant prolongation of tumor necrosis factor-α (TNF) appearance in the plasma. The enhancement of TNF expression by TVX was reproduced in vitro in RAW 264.7 murine macrophages (RAW cells) stimulated with LPS. The current study was designed to identify the molecular target of TVX responsible for this response in RAW cells. An in silico analysis suggested a favorable binding profile of TVX to eukaryotic topoisomerase II-α (TopIIα), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIα activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated histone 2A.X in RAW cells. Moreover, TVX induced activation of the DNA damage sensor kinases, ataxia telangiectasia mutated (ATM) and Rad3-related (ATR). The ATR inhibitor NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor [2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933)] was without effect. TVX prolonged TNF mRNA stability, and this effect was largely attenuated by NU6027. These results suggest that TVX can inhibit eukaryotic topoisomerase, leading to activation of ATR and potentiation of TNF release by macrophages, at least in part through increased mRNA stability. This off-target effect might contribute to the ability of TVX to precipitate IDILI in humans.
authors.affiliation: Department of Pharmacology & Toxicology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan (K.L.P., K.M.B., P.E.G., and R.A.R.); and Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia (J.O.-V.).
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Poulsen
authors.affiliation: Department of Pharmacology & Toxicology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan (K.L.P., K.M.B., P.E.G., and R.A.R.); and Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia (J.O.-V.).
authors.forename: Jesus
authors.identifier: http://orcid.org/0000-0003-3089-5872
authors.initials: J
authors.lastname: Olivero-Verbel
authors.affiliation: Department of Pharmacology & Toxicology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan (K.L.P., K.M.B., P.E.G., and R.A.R.); and Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia (J.O.-V.).
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Beggs
authors.affiliation: Department of Pharmacology & Toxicology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan (K.L.P., K.M.B., P.E.G., and R.A.R.); and Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia (J.O.-V.).
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Ganey
authors.affiliation: Department of Pharmacology & Toxicology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan (K.L.P., K.M.B., P.E.G., and R.A.R.); and Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia (J.O.-V.) rothr@msu.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Roth
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D024841:Fluoroquinolones / Q000037:antagonists & inhibitors / Q000633:toxicity*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009287:Naphthyridines / Q000037:antagonists & inhibitors / Q000633:toxicity*; D009603:Nitroso Compounds / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: C495818:2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; C475622:4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine; D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D024841:Fluoroquinolones; D008070:Lipopolysaccharides; D009025:Morpholines; D009287:Naphthyridines; D009603:Nitroso Compounds; D011743:Pyrimidines; D011753:Pyrones; D014409:Tumor Necrosis Factor-alpha; C080163:trovafloxacin; D004250:DNA Topoisomerases, Type II