RESEARCH ARTICLE CYP450-mediated mitochondrial ROS production involved in arecoline N-oxide-induced oxidative damage in liver cell lines Tsu-Shing Wang 1,2 | Cheng-Ping Lin 1 | Yu-Pong Chen 1 | Mu-Rong Chao 3 | Chien-Chun Li 4 | Kai-Li Liu 4 1 Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan 2 Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan 3 Department of Occupational Safety and Health, Chung Shan Medical University, Taichung, Taiwan 4 Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan Correspondence Tsu-Shing Wang, PhD, Department of Biomedical Sciences, Chung Shan Medical University, No 110, Sec. 1, Chien-Kuo N. Rd., Taichung, Taiwan. Email: tsushing@csmu.edu.tw Funding information Ministry of Science and Technology, Taiwan, ROC, Grant/Award Number: MOST 104-2320-B-040-013 Abstract Background: IARC has classified the betel nut as a human environmental carcinogen. Previous studies have found that arecoline (AR) is the major alkaloid present in the saliva of betel quid chewers. Saliva contains a large content of AR which has been further shown to cause mutation of oral mucosa cells, resulting in oral cancer. Whereas, to date, there are only few studies reported the hepatotoxicity associated with arecoline and betel nut chewing. Therefore, the main purpose of this study was to determine the toxic effects of AR and its oxidative metabolite, arecoline N-oxide (ARNO), in normal liver cell lines. Methods: The cytotoxic, genotoxic, and mutagenic effects were detected by crystal violet staining, alkaline comet assay, and Salmonella mutagenicity test, respectively. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2-DCFDA assay. Results: Our results demonstrated that ARNO exerted higher cytotoxicity, DNA damage, and mutagenicity than its parent compound arecoline in liver cells. Antioxidants, such as N-acetyl- cysteine, Trolox, and penicillamine, strongly protected liver cells from ARNO-induced DNA damage and ROS production. Furthermore, co-treatment with Mito-TEMPO also effectively blocked ARNO-induced ROS production in liver cells. Besides antioxidants, co-treatment with 1-aminobenzotriazole and methimazole nearly completely suppressed ARNO-induced ROS production in liver cells. Conclusions: Our data suggest that arecoline ingested from the habit of chewing betel quid can be primarily oxidized to ARNO, thereby enhancing its toxicity through increased ROS produc- tion. Considering the excellent protective effects of both mitochondria-targeted antioxidant and CYP450 inhibitor on ARNO-induced ROS production in liver cells, mitochondria CYP450-mediated metabolism of ARNO may be a key mechanism. Collectively, our results provide novel cellular evidence for the positive connection between habitual betel quid chewing and the risk for liver damage. KEYWORDS arecoline, arecoline N-oxide, cytochrome P450, DNA strand break, liver cell lines, reactive oxygen species 1 | BACKGROUND Betel quid (BQ) is a combination of areca nut (Areca catechu L.), slaked lime, and Piper betle inflorescence or conditional folded in a Piper betle leaf. Chewing of BQ is a habit of great antiquity in South Asia, South- east Asia, and the Pacific Islands, with about 600 million users reported worldwide. 1,2 The areca nut in BQ has been recognized as a Group I carcinogen to humans by the International Agency for Research on Cancer 3 of the World Health Organization. Case-control studies from Asian countries have reported that BQ is a risk factor for oral cancer. 4-7 Besides oral cancer, BQ chewing is an independent risk Abbreviations: BQ, betel quid; DCFH-DA, 2 0 ,7 0 -dichlorofluorescein diacetate; LC-MS, liquid chromatography-mass spectrometry; NAC, N-acetylcysteine; ROS, reactive oxygen species. Received: 24 April 2018 Revised: 28 May 2018 Accepted: 31 May 2018 DOI: 10.1002/tox.22588 Environmental Toxicology. 2018;1–10. wileyonlinelibrary.com/journal/tox © 2018 Wiley Periodicals, Inc. 1