TY - JOUR T1 - Trovafloxacin enhances LPS-stimulated production of TNF by macrophages: role of the DNA damage response JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.114.214189 SP - jpet.114.214189 AU - Kyle L Poulsen AU - Jesus Olivero Verbel AU - Kevin M Beggs AU - Patricia E Ganey AU - Robert A Roth Y1 - 2014/01/01 UR - http://jpet.aspetjournals.org/content/early/2014/05/09/jpet.114.214189.abstract N2 - 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-alpha (TNF) appearance in the plasma (Shaw et al., 2009ab). 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-alpha (TopIIa), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIa activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated H2A.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 prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor (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. ER -