Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes Jalal Pourahmad a, *, Peter J. O‘Brien b , Farzaneh Jokar a , Bahram Daraei a a Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, PO Box 14155-6153, Iran b Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ont. Canada M5S 2S2 Accepted 30 June 2003 Abstract Severe chronic liver disease results from the hepatic accumulation of copper nickel, cobalt or iron in humans and on the other hand cadmium, dichromate and arsenic may induce lung or kidney cancer. Acute or chronic CdCl 2 , HgCl 2 or dichromate admin- istration induces hepatic and nephrotoxicity in rodents. Oxidative stress is often cited as a possible cause but has not yet been measured. For the first time we have measured the reactive oxygen species (ROS) formation induced when cells are incubated with metals and determined its source. Hepatocytes incubated with 2 0 ,7 0 -dichlorofluorescin diacetate resulted in its rapid uptake and deacetylation by intracellular esterases to form 2 0 ,7 0 -dichlorofluorescin. A marked increase in ROS formation occurred with LD 50 concentrations of cadmium [Cd(II)], Hg(II) or arsenite [As(III)] which was released by proton ionophores that uncouple oxidative phosphorylation. Uncouplers or oxidative phosphorylation also inhibited ROS formation induced by these metals, which suggests that mitochondria are major contributors to endogenous ROS formation. Glycolytic substrates also inhibited Cd(II)/Hg(II)/ As(III)-induced ROS formation and confirms that mitochondria are the site of ROS formation. By contrast ROS formation by LD 50 concentrations of Cu(II), Ni(II), Co(II) or dichromate [Cr(VI)] were not affected by uncouplers or glycolytic substrates. However they were inhibited by lysosomotropic agents or endogenous inhibitors [in contrast to Hg(II), Cd(II) or As(III)]. Fur- thermore Cu(II), Ni(II), Co(II) or Cr(VI) accumulated in the lysosomes and the ROS formed caused a loss of lysosomal membrane integrity. The release of lysosomal proteases and phospholipases also contributed to hepatocyte cytotoxicity. ROS formation and cytotoxicity induced by added H 2 O 2 or generated by the intracellular redox cycling of nitrofurantoin was also inhibited by lysoso- motropic agents and ferric chelators suggesting that lysosomal Fe(II) contributes to H 2 O 2 -induced cytotoxicity. In conclusion, lysosomes are sites of cytotoxic ROS formation with redox transition metals (CuII, CrVI, NiII, CoII) whereas mitochondria are the ROS sites for non-redox or poor redox cycling transition metals (CdII, HgII, AsIII). # 2003 Published by Elsevier Ltd. 1. Introduction As a class of toxic agents, metals are a concern of the highest priority for human exposure. Metals have a vast array of remarkably adverse effects, including those of carcinogenicity and hepatotoxicity. Metals are also non- biodegradable and persist in the environment. Anthro- pogenic use has led to global dispersion of metals in the environment. Because of their wide distribution and extensive use in modern society, some human exposure to toxic metals is inevitable. Metals are also unique environ- mental pollutants in that they are neither created or destroyed by humans but are only transported and trans- formed into various products which in turn directly or indirectly affect the growth and longevity of aquatic or terrestrial animals. Defining the mechanisms of metal car- cinogenecity has been problematic because of the intricate nature of the interactions of metals with living systems. Various metals induced hepatocyte ‘‘ROS’’ formation before cytotoxicity ensued. The comparative effective- ness of metals (at a cytotoxic dose) for inducing ‘‘ROS’’ formation was CuCl 2 > K 2 Cr 2 O 7 > HgCl 2 > CdCl 2 (Pourahmad and O’Brien, 2000a; Pourahmad et al., 2001a; Pourahmad and O’Brien, 2001). Furthermore the cytotoxicity induced by these metals was prevented by the hydroxyl radical scavengers dimethyl sulfoxide or mannitol (Pourahmad and O’Brien, 2000a; Pourahmad et al., 2001a; Pourahmad and O’Brien, 2001). It was also demonstrated that lysosomal lipid peroxidation preceded CuCl 2 or K 2 Cr 2 O 7 induced hepatocyte cyto- 0887-2333/$ - see front matter # 2003 Published by Elsevier Ltd. doi:10.1016/S0887-2333(03)00123-1 Toxicology in Vitro 17 (2003) 803–810 www.elsevier.com/locate/toxinvit * Corresponding author. Tel.: +98-21-877-3521; fax: +98-21-879- 5008. E-mail address: j.porahmadjaktaji@utoronto.ca (J. Pourahmad).