Original Contribution Heme oxygenase-1 attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation Hyung-Jin Kim a,b,1 , Hong-Seob So a,1 , Jeong-Han Lee a , Jae-Hyung Lee a , Channy Park a , Sung-Yeol Park a , Yun-Ha Kim a , Myung-Ja Youn a , Se-Jin Kim a , Sang-Young Chung c , Kang-Min Lee b , Raekil Park a, ⁎ a Vestibulocochlear Research Center (VCRC) and Department of Microbiology, Wonkwang University School of Medicine, 344-2 Shinyong-dong Iksan, Jeonbuk 570-749, South Korea b Division of Biological Sciences, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea c Department of Surgery, Chonnam National University Medical School, Kwangju 560-182, South Korea Received 12 May 2005; revised 10 January 2006; accepted 13 January 2006 Available online 8 February 2006 Abstract Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme catabolism, is known to modulate various cellular functions, including cytokine production, cell proliferation, and apoptosis, in stress-related conditions. However, the role of HO-1 in the auditory system remains elusive. Herein, we demonstrate that pharmacologic induction of HO-1 along with catalytic activation significantly suppressed apoptosis of HEI-OC1 cells induced by cisplatin. Studies of ectopic expression of pcDNA3-HO-1 and siRNA of HO-1 further revealed the protective role of HO-1 against cisplatin in HEI-OC1 cells. Among the catabolic metabolites of HO-1, both carbon monoxide (CO) and bilirubin were directly involved in the protective role of HO-1 against cisplatin through inhibition of reactive oxygen species generation. Furthermore, pharmacological induction of HO- 1 completely prevented the destruction of outer hair cell arrays by cisplatin through a CO-dependent mechanism in organotrophic culture of the rat primary organ of Corti explants. These results suggest that HO-1 may serve as a safeguard of auditory sensory hair cells against a variety of challenges of oxidative stress, including noise trauma, presbycusis, and ototoxic drugs, respectively. © 2006 Elsevier Inc. All rights reserved. Keywords: Ototoxicity; Cisplatin; Heme oxygenase-1; ROS; Free radical Introduction Cisplatin (cis-diamminedichloroplatinum II; CDDP) is an extensively used anticancer chemotherapeutic agent, which has serious side effects including ototoxicity and nephrotoxicity [1,2]. Ototoxicity is reported to be bilateral, symmetrical, progressive, irreversible, and often more severe and frequent in patients receiving higher and repeated doses of cisplatin [3,4]. Among the key cytotoxic mechanisms of cisplatin, which include mitochondrial dysfunction and inhibition of transcrip- tion/translation by forming DNA adducts, we were interested in the generation of reactive oxygen species (ROS), which damage many cellular functions through direct and/or indirect mechan- isms, including lipid peroxidation in auditory hair cells. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism, which leads to the generation of biliverdin, free iron, and carbon monoxide (CO) [5]. Three mammalian HO isoforms have been identified, one of which, HO-1, is a stress- responsive protein induced by various oxidative agents including heme [5], hyperoxia [6], hypoxia [7], and electro- philes [8] through transcriptional activations of AP-1, STAT, Free Radical Biology & Medicine 40 (2006) 1810 – 1819 www.elsevier.com/locate/freeradbiomed Abbreviations: CDDP, cis-diamminedichloroplatinum II; ROS, reactive oxygen species; HO, heme oxygenase; CO, carbon monoxide; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Hb, hemoglobin; CoPPIX, cobalt protoporphyrin IX; ZnPPIX, zinc protoporphyrin IX; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified essential medium; FBS, fetal bovine serum; OHC, outer hair cell; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; DCF, 2′,7′-dichlorofluorescein; SDS, sodium dodecyl sulfate; RT- PCR, reverse transcriptase–polymerase chain reaction. ⁎ Corresponding author. Fax: +82 63 852 0220. E-mail address: rkpark@wonkwang.ac.kr (R. Park). 1 These authors contributed equally to this work. 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.01.018