DNA Repair 4 (2005) 493–502 Alleviation of benzo[a]pyrene-diolepoxide-DNA damage in human lung carcinoma by glutathione S-transferase M2 Mao-Wen Weng a , Yi-Min Hsiao b , Hui-Ling Chiou e , Shun-Fa Yang c , Yih-Shou Hsieh c , Ya-Wen Cheng d , Chieh-Hsiang Yang a , Jiunn-Liang Ko a,∗ a Institute of Medical and Molecular Toxicology, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan 40203, ROC b Center for Research and Development, Chungtai Institute of Health Sciences and Technology, No. 11 Pu-tzu Lane, Pei-tun District 406, Taichung, Taiwan, ROC c Institute of Biochemistry and Biotechnology, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan 40203, ROC d Institute of Medicine, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan 40203, ROC e School of Medical Technology, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan 40203, ROC Received 12 August 2004; accepted 13 December 2004 Available online 19 January 2005 Abstract Cellular detoxification is important for the routine removal of environmental and dietary carcinogens. Glutathione S-transferases (GST) are major cellular phase II detoxification enzymes. MRC-5 cells have been found to exhibit significantly higher GST activity than human H1355 cells. This study investigates whether GST-M2 activity acts as a critical determinant of the target dose of carcinogenic benzo[a]pyrene- diolepoxide (BPDE) and whether it has an effect on MDM2 splicing in the two cell lines. We used RT-PCR to clone Mu-class GST cDNA. Two forms of GST coming from the cell lines were characterized as GST-M2 (from MRC-5 cells) and GST-M4 (from H1355 cells). Nested-PCR showed that BPDE-induced MDM2 splicing had occurred in the H1355 cell line but not in normal MRC-5 cells. Furthermore, using nested- PCR and competitive ELISA, we found that in H1355 cells modified to stably overexpress GST-M2, splicing was abolished and BPDE adducts appeared in low abundance. In conclusion, exogenously overexpressed GST-M2 was effective in reducing BPDE-induced DNA damage in H1355 cells. The catalytic activity of GST-M2 may play an important future role in lowering the incidence of BPDE-induced DNA damage. © 2004 Elsevier B.V. All rights reserved. Keywords: MDM2 splicing; BPDE adduct; GST-M2; DNA damage 1. Introduction In vivo metabolic activation of benzo[a]pyrene (B(a)P) produces the highly reactive metabolite benzo[a]pyrene- diolepoxide (BPDE), a possible causative agent for lung can- cer [1]. Covalent interaction of BPDE with DNA can form DNA adducts and play a critical role in B(a)P-induced tumori- genesis [2]. In previous studies of tobacco-related carcino- gen challenge, the reported levels of BPDE-induced DNA damage have varied widely, possibly reflecting individual ∗ Corresponding author. Tel.: +886 4 24730022x11694; fax: +886 4 24751101. E-mail address: jlko@csmu.edu.tw (J.-L. Ko). variation in susceptibility [3]. Therefore, BPDE is a rele- vant challenge mutagen to the study of susceptibility to to- bacco carcinogenesis. Given the significant variability, ge- netically determined host factors may modify the extent o f DNA damage in smokers’ lungs, and, therefore, modulate cancer risk. The human MDM2 gene is amplified to high copy numbers in approximately one-third of all human sarcomas [4]. Multiple spliced forms of MDM2 transcripts also have been observed in human lung cancer [5]. Alternative splic- ing of MDM2 mRNA has found the malignant phenotype in non-small-cell lung cancer [6]. In human tissue, several different mechanisms can hinder BPDEs alteration of DNA [7–9]. Glutathione S-transferases (GST) are major cellular phase II detoxification enzymes. 1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.12.006