Toxicology 269 (2010) 67–72 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol A murine experimental anthracycline extravasation model: Pathology and study of the involvement of topoisomerase II alpha and iron in the mechanism of tissue damage Annemette V. Thougaard a,∗ , Seppo W. Langer b , Bo Hainau c , Morten Grauslund d , Birgitte Ravn Juhl e , Peter Buhl Jensen a,b , Maxwell Sehested a,e a TopoTarget A/S, Symbion Science Park, Fruebjergvej 3, 2100 Copenhagen, Denmark b Department of Oncology 5073, The Finsen Center, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark c Department of Pathology, Roskilde Sygehus, Køgevej 7-13, 4000 Roskilde, Denmark d Bartholin Institute, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark e Department of Pathology, Centre of Diagnostic Investigation, Rigshospitalet, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark article info Article history: Received 16 November 2009 Received in revised form 6 January 2010 Accepted 7 January 2010 Available online 15 January 2010 Keywords: Anthracycline extravasation Dexrazoxane Pathology Toxicity mechanism Topoisomerase II Iron chelation abstract The bisdioxopiperazine topoisomerase II catalytic inhibitor dexrazoxane has successfully been intro- duced into the clinic as an antidote to accidental anthracycline extravasation based on our preclinical mouse studies. The histology of this mouse extravasation model was investigated and found to be similar to findings in humans: massive necrosis in the subcutis, dermis and epidermis followed by sequestra- tion and healing with granulation tissue, and a graft-versus-host-like reaction with hyperkeratotic and acanthotic keratinocytes, occasional apoptoses, epidermal invasion by lymphocytes and healing with dense dermal connective tissue. The extension of this fibrosis was quantified, and dexrazoxane inter- vention resulted in a statistically significant decrease in fibrosis extension, as also observed in the clinic. Several mechanisms have been proposed in anthracycline extravasation cytotoxicity, and we tested two major hypotheses: (1) interaction with topoisomerase II alpha and (2) the formation of tissue damaging reactive oxygen species following redox cycling of an anthracycline Fe 2+ complex. Dexrazoxane could minimise skin damage via both mechanisms, as it stops the catalytic activity of topoisomerase II alpha and thereby prevents access of anthracycline to the enzyme and thus cytotoxicity, and also acts as a strong iron chelator following opening of its two bisdioxopiperazine rings. Using the model of extrava- sation in a dexrazoxane-resistant transgenic mouse with a heterozygous mutation in the topoisomerase II alpha gene (Top2a Y165S/+ ), we found that dexrazoxane provided a protection against anthracycline- induced skin wounds that was indistinguishable from that found in wildtype mice. Thus, interaction with topoisomerase II alpha is not central in the pathogenesis of anthracycline-induced skin damage. In contrast to dexrazoxane, the iron-chelating bisdioxopiperazine ICRF-161 do not inhibit the catalytic cycle of topoisomerase II alpha. This compound was used to isolate and test the importance of iron in the wound pathogenesis. ICRF-161 was found ineffective in the treatment of anthracycline-induced skin damage, suggesting that iron does not play a dominant role in the genesis of wounds. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Accidental extravasation of anthracyclines (e.g. doxorubicin and daunorubicin) occurs in approximately 0.1% of all infusions, and has been a feared event since the introduction of this class of can- cer therapeutic compounds in the clinic in 1964. Depending on the extent of the extravasation, a severe and even life threaten- ∗ Corresponding author. Present address: H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark. Tel.: +45 36433795; fax: +45 36438313. E-mail address: avth@lundbeck.com (A.V. Thougaard). ing necrosis can arise requiring extensive reconstructive surgery, and entailing long term pain, disfigurement and lack of function. The use of doxorubicin in the clinic is limited by its cardiotoxic effect, and preclinical and clinical studies have shown dexrazox- ane to be an efficient antidote to this toxicity (Imondi, 1998; van Dalen et al., 2008). Doxorubicin–iron complexes can redox cycle and thereby contribute to generation of reactive oxygen species (ROS), and this mechanism is thought to be involved in the observed cardiotoxicity. Ring-opened dexrazoxane is a strong iron chela- tor, which can complex bind Fe 2+ , thereby reducing ROS-induced doxorubicin-associated cell damage (Hasinoff et al., 2003; Hasinoff and Herman, 2007). Anthracyclines are believed to exert their anti- 0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.01.007