Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes Yueh-Chiao Yeh a , Hui-Chin Lai a,b , Chih-Tai Ting a,b , Wen-Lieng Lee a,b , Li-Chuan Wang a , Kuo-Yang Wang a,c , Hui-Chun Lai d , Tsun-Jui Liu a,b, * a Cardiovascular Center, Taichung Veterans General Hospital, Taichung, Taiwan b Departments of Medicine, Surgery, and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan c Department of Medicine, Chuan-Shan Medical University, Taichung, Taiwan d Chang-Gung University College of Medicine, Tao-Yuan and Chang-Gung Memorial Hospital, Lin-Koh Taipei, Taiwan 1. Introduction Male germ cell development, or spermatogenesis, demands complex chemical, physical, and genetic interactions in the testicular tissue [1]. The obligatory requirement of definite DNA integrity and the brisk activity of cell proliferation render this process very prone to disruption by extrinsic insults, particularly whichever inflict oxidative stress, DNA damage, and apoptosis. Examples include a number of anti-neoplastic agents that, though could suppress the growth of tumor cells biochemical pharmacology 74 (2007) 969–980 article info Article history: Received 17 April 2007 Accepted 19 June 2007 Keywords: Doxycycline Doxorubicin Testis Oxidative stress Apoptosis abstract Spermatogenic cells constitute one of the body tissues that are susceptible to doxorubicin- induced oxidative stress and apoptosis. To explore whether doxorubicin toxicity to these male germ cells could be prevented by adjuvant medication, this study was designed to examine the possible ameliorating action of doxycycline, an antibiotic with anti-oxidant property, on doxorubicin-induced oxidative and apoptotic effects in mouse testes. Male mice at 5-week of age were treated with vehicles, doxorubicin alone (3 mg/kg, i.p. every other day for 3 doses), doxycycline alone (2.5 mg/kg, i.p. every other day for 3 doses), or doxycycline plus doxorubicin (each dose given 1 day post-doxycycline). After 28 days, mice treated with doxorubicin alone displayed smaller body and testicular weights, reduced sperm counts, impaired spermatogenic capability (scarcer spermatids and spermatocytes), increased oxidative stress (malondialdehyde levels), decreased anti-oxidant activity (super- oxide dismutase and glutathione peroxidase), and elevated apoptotic indexes (upregulation of Bax and Bad, downregulation of Bcl-2 and Bcl-xL, release of cytochrome c from mito- chondria to cytosol, activation of caspase-3, and increase of cleaved caspase-3 abundance and TUNEL positive cells), while doxycycline pretreatment could effectively prevent nearly all of these abnormalities. These results provide firm evidence that doxycycline pretreat- ment would offset the oxidative and apoptotic impact imposed by doxorubicin, and imply doxycycline to be a promising adjuvant agent that may attenuate the toxicity of doxorubicin on testicular tissues in clinical practice. # 2007 Elsevier Inc. All rights reserved. * Corresponding author at: 160, Sec 3, Taichungkang Rd., Taichung 406, Taiwan. Tel.: +886 9 68950269; fax: +886 4 23599257. E-mail address: Tsun-JuiLiu ab *854k@vghtc.gov.tw">Tsun-JuiLiu ab *854k@vghtc.gov.tw (). Abbreviations: Dox, doxorubicin; Dc, doxycycline; DTT, DL-dithiothreitol; DAPI, 4 0 ,6 0 diamidino-2-phynylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2007.06.031