Published: August 04, 2011 r2011 American Chemical Society 1586 dx.doi.org/10.1021/tx200291p | Chem. Res. Toxicol. 2011, 24, 1586–1596 ARTICLE pubs.acs.org/crt Comparative Toxicity of Arsenic Metabolites in Human Bladder Cancer EJ-1 Cells Hua Naranmandura,* ,†,‡ Michael W. Carew, § Shi Xu, † Jane Lee, || Elaine M. Leslie, ‡,§ Michael Weinfeld, || and X. Chris Le* ,‡ † Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China ‡ Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2G3, Canada § Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada ) Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada ’ INTRODUCTION Arsenic occurs naturally in the environment as an element of the earth’s crust, and chronic ingestion of arsenic-contaminated drinking water can cause skin, lung, and urinary bladder cancer in humans. 1,2 Although arsenic has been known as a human carcinogen for more than one hundred years, there is no conclusive understanding of the mechanism of action for this effect. In mammals, ingested inorganic arsenic is transformed metabolically into methylated metabolites by arsenic methyl- transferase (AS3MT) and is then excreted in the urine mostly in the form of methylated metabolites such as monomethylarsonic acid (MMA V ) and dimethylarsinic acid (DMA V ). 35 Recently, a few minor thiolated arsenic metabolites, dimethylmonothio- arsinic acid (DMMTA V ), dimethyldithioarsinic acid (DMDTA V ), and monomethylmonothioarsonic acid (MMMTA V ) have been found in human and animal urine, and some of them have also been detected in organs in vitro and in vivo. 68 However, little is known about the molecular mechanisms and toxicological and biological significance of thioarsenicals. Moreover, the arsenic species responsible for arsenic toxicity and carcinogenesis in the bladder remain to be clearly identified. The bladder is one of the identified target tissues for arsenic toxicity. 1,2 Wei et al. found that DMA V caused urinary bladder cancer in the rat after exposure to DMA V for two years and that the generation of ROS is likely to play an important role in the early stages of DMA V carcinogenesis, suggesting that DMA V exposure may be relevant to the carcinogenic risk of inorganic arsenic in humans. 9 Others have also detected several thioarse- nicals in the urine of DMA V -exposed rats and a minor quantity of thioarsenicals such as DMMTA V and MMMTA V in the urine of rats after exposure to iAs III . 6,10,11 They observed that DMMTA V Received: June 9, 2011 ABSTRACT: The human bladder is one of the primary target organs for arsenic-induced carcinogenicity, and arsenic metabolites in urine have been suspected to be directly involved in carcinogenesis. Thioarsenicals are commonly found in human and animal urine and are also considered to be highly toxic arsenic metabolites. The present study was performed to gain insight into the toxicity and accumulation of arsenic species found in urine, including arsenate (iAs V ), arsenite (iAs III ), monomethylarsonic acid (MMA V ), monomethylmonothioarsonic acid (MMMTA V ), di- methylarsinic acid (DMA V ), dimethylarsinous acid (DMA III ), dimethyl- monothioarsinic acid, (DMMTA V ), and dimethyldithioarsinic acid (DMDTA V ) in human bladder cancer EJ-1 cells. The order of cytotoxi- city of these arsenic compounds in EJ-1 human bladder cancer cells was DMA III , DMMTA V > iAs III . iAs V > MMMTA V > MMA V , DMA V , and DMDTA V , indicating that the sulfur-containing DMMTA V was among the most toxic arsenic compounds similar to trivalent DMA III . We further characterized the DNA damage, generation of highly reactive oxygen species (hROS), and expression of proteins p21 and p53 in cells after exposure to iAs III , DMA III , and DMMTA V . Cellular exposure to DMMTA V resulted in reduced protein expression of p53 and p21, increased DNA damage, and increased intracellular hROS (hydroxyl radical). In contrast, iAs III significantly increased the protein expression of p21 and p53 and did not increase the hROS at the IC 50 . Intracellular glutathione (GSH) was reduced by 60% after exposure to DMA III or DMMTA V , suggesting that DMMTA V causes cell death through oxidative stress. In contrast, GSH levels increased in cells exposed to iAs III , and hROS only increased after a long exposure to iAs III . Our findings demonstrate that DMMTA V may be one of the most toxicologically potent arsenic species, relevant to arsenic-induced carcinogenicity in the urinary bladder.