Elsevier

Toxicology in Vitro

Volume 17, Issues 5–6, October–December 2003, Pages 803-810
Toxicology in Vitro

Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes

https://doi.org/10.1016/S0887-2333(03)00123-1Get rights and content

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 CdCl2, HgCl2 or dichromate administration 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′,7′-dichlorofluorescin diacetate resulted in its rapid uptake and deacetylation by intracellular esterases to form 2′,7′-dichlorofluorescin. A marked increase in ROS formation occurred with LD50 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 LD50 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)]. Furthermore 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 H2O2 or generated by the intracellular redox cycling of nitrofurantoin was also inhibited by lysosomotropic agents and ferric chelators suggesting that lysosomal Fe(II) contributes to H2O2-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).

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. Anthropogenic 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 environmental pollutants in that they are neither created or destroyed by humans but are only transported and transformed into various products which in turn directly or indirectly affect the growth and longevity of aquatic or terrestrial animals. Defining the mechanisms of metal carcinogenecity 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 effectiveness of metals (at a cytotoxic dose) for inducing “ROS” formation was CuCl2>K2Cr2O7 >HgCl2>CdCl2 (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 CuCl2 or K2Cr2O7 induced hepatocyte cytotoxicity (Pourahmad et al., 2001b, Pourahmad, and O'Brien, 2001).

The possible cellular sources of “ROS” production include plasma membrane NADPH oxidase and intracellular cytosolic xanthine oxidase, peroxisomal oxidases, endoplasmic reticular oxidases, mitochondrial electron transport components and lysosomal pool of Fe2+/Cu+ which makes it susceptible for Haber–Weiss reaction with H2O2 generating agents. The two latter things are considered to be the major sources of “ROS” that have been implicated in a number of diseases and disorders (Skulachev, 1999).

The major objective of this study was to investigate the possible intracellular source of “ROS” formation for carcinogenic metals (CuII, CrVI, NiII, CoII, CdII, HgII, AsIII) which is likely one of their potential mechanisms at inducing carcinogenecity.

Section snippets

Chemicals

Rhodamine 123 were obtained from Aldrich Chemical Company (Milwaukee, WI, USA). Collagenase (from Clostridium histolyticum) and Hepes were purchased from Roche (Montreal, Canada). Trypan blue, CuCl2, CdCl2, HgCl2, CoCl2, NiCl2, potassium dichromate, sodium arsenite, d-mannitol, dimethyl sulfoxide, catalase, superoxide dismutase, chloroquine diphosphate, methylamine HCl, 3-methyl adenine, monensin sodium, thiobarbituric acid, trichloroacetic acid (TCA), sodium pentobarbital and heparin were

Results

As shown in Tables 1A and B however both redox active carcinogenic metals (CuII, CrVI, NiII, CoII) and non-redox active carcinogenic metals (CdII, HgII, AsIII) induced a rapid decline of mitochondrial membrane potential. But glycolytic ATP generators (Fructose / xylitol) or glutamine (a mitochondrial ATP generator) only prevented non-redox active carcinogenic metals (CdII, HgII, AsIII) induced cytotoxicity,”ROS” formation and the decline in membrane potential (Table 1A, Illustration 1).

Discussion

In this study we found that the ATP generators fructose and xylitol and l-glutamine (a mitochondrial ATP generator) prevented non-redox active metals induced cytotoxicity and “ROS” formation (Table 1A, Illustration 1) which indicates that the cell death may be a consequence of mitochondrial MPT pore opening and consequent ATP depletion. Lack of mitochondrial ATP results in intracellular acidosis and osmotic injury which leads to plasma membrane lysis (Pourahmad and O'Brien, 2000b).

Mitochondrial

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