Mechanism for proliferation inhibition by various selenium compounds and selenium-enriched broccoli extract in rat glial cells Jan-Ying Yeh 1 , Bor-Rung Ou 2 , Yu-Chuan Liang 2 , Joel Burchfiel 3 , Judy A. Butler 3 , Neil E. Forsberg 4 & Philip D. Whanger 3 * 1 Department of Biotechnology and Bioinformatics, Asia University, Taichung, 413 Taiwan, R.O.C.; 2 Department of Animal Science and Biotechnology, Tunghai University, Taichung, 407 Taiwan, R.O.C.; 3 Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, USA; 4 Department of Animal Sciences, Oregon State University, Corvallis OR, USA; *Author for correspondence (Tel.: +1-541-737-1789; Fax:+1-541-737-0497; E-mail: phil.whanger@oregonstate.edu) Received 20 October 2005; Accepted 21 January 2006 Key words: apoptosis, broccoli extract, cellular glutathione peroxidase (cGPX), hydrogen peroxide (H 2 O 2 ), selenium Abstract The objective of this study was to investigate the differential effects of various selenium (Se) compounds and Se-enriched broccoli extracts on cell proliferation and the possible mechanism responsible for the Se- induced growth inhibition. C6 rat glial cells were incubated with graded concentrations up to 1000 nM of selenite, selenate, selenomethionine (SeM), Se-methyl-selenocysteine (SeMCys), high-Se broccoli (H-SeB) extract or low-Se broccoli (L-SeB) extract for 24 and 48 h. MTT results indicated that all Se sources and levels examined inhibited C6 cell proliferation at 48 h. The results from cell cycle progression and apoptosis analysis indicated that SeM, SeMCys, H-SeB or L-SeB treatments at the concentration of 1000 nM reduced the cell population in G 0 /G 1 phase, but induced G 2 /M phase arrest and increased apoptosis and secondary necrosis in C6 cells at 24 h. The populations of apoptotic cells and secondary necrotic cells were increased by all Se sources examined. The COMET assay indicated that there was no significant DNA single-strand break found for all Se treatments in C6 cells for 48 h. In addition, the Se-induced proliferation inhibition may involve a hydrogen peroxide (H 2 O 2 )-dependent mechanism with elevated cellular glutathione perox- idase (cGPX) activity. Both H-SeB and L-SeB inhibited C6 cell proliferation but H-SeB was less inhibitory than L-SeB. The proliferation inhibition by H-SeB in C6 cells is apparently related to the increased H 2 O 2 with the elevated cGPX activity, but the inhibition by L-SeB was H 2 O 2 -independent without change in cGPX activity. Introduction Selenium (Se) is an essential trace element for humans and animals and is known to suppress carcinogenesis nonspecifically in animals (Whanger 2004). After oral supplementation with 200 lg Se/ day as enriched yeast for 4.5 years the cancer incidence of lung, colon and prostate cancers was significantly reduced (Clark et al. 1996, 1998). This is consistent with other human trials that indicated that Se supplementation reduced the incidence of certain cancers (Whanger 2004). The form and the concentration of Se used are critical for cancer prevention (Sinha & El-Bayoumy 2004). Various Se compounds influence cellular responses differently in various tumor models (Tapiero et al. 2003). Most of the cancer preven- tion studies have used selenite or selenomethionine (SeM) as the primary Se compounds (Sinha & El- Bayoumy 2004). Se-methylselenocysteine (SeMCys) BioMetals (2006) Ó Springer 2006 DOI 10.1007/s10534-006-0007-y