Hydroxyl Radical Production and Human DNA Damage Induced by Ferric Nitrilotriacetate and Hydrogen Peroxide1

Reactivities of l-'r" chelates of aminopolycarboxylic acids with DNA were investigated by the DNA-sequencing technique using 32P 5'-endlabeled DNA fragments obtained from the human c-Ha-ras-1 protoon cogene, and the reaction mechanism was studied by electron spin reso nance spectroscopy. Ferric nitrilotriacetate (Fe3*-NTA) plus hydrogen peroxide caused strong DNA cleavage in the presence of albumin. No or little DNA cleavage was observed with ferric chloride or I-V" chelates of other aminopolycarboxylic acids tested in the presence of hydrogen peroxide. The DNA cleavage by Fe^-NTA plus hydrogen peroxide without piperidine treatment occurred at positions of every nucleotide although a specific cleavage was observed, whereas cleavages at the positions of guanine and thymine increased predominantly with piperidine treatment. Electron spin resonance studies using free radical traps dem onstrated that of Fe3* chelates of aminopolycarboxylic acids, Fe3*-NTA was the most effective catalyst in hydrogen peroxide-derived production of hydroxyl radicals under our conditions. The results suggest that Fe3*NTA catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radicals, which subsequently cause the strong base alterations of guanine and thymine, and deoxyribose-phosphate backbone breakages. The possibility that the Fe3*-NTA-induced DNA damage is the initiation and/or promotion of carcinogenesis by l-'c'*-\ I Vis discussed. INTRODUCTION VIA' is a chelating agent for metals and a potential substitute for phosphates in household detergents. NTA has been shown to be a potent carcinogen and promoter (1-6). It has been reported that long-term administration of NTA at high dose to rats induced tumors of the urinary bladder and kidney (1-3). Some papers also reported that NTA showed promoting effects on renal tubular cell tumors and urinary bladder carcinogenesis initiated by nitrosamine derivatives (4-6). Furthermore, Midorikawa et al. reported that renal cell carcinoma was observed in Fe3+-NTA-treated rats, while no tumors were formed in NTAor Al3+-NTA-treated rats (7). Another iron complex, for example, iron dextran, has also been shown to be carcinogenic to mice and rats, producing local tumors at the injection sites (8). Thus, it can be presumed that NTA, which itself has no carcinogenicity, binds with Fe3+ to form the complex partici pating in carcinogenesis. The idea that damage to DNA is a critical event not only in the initiation but also in the promotion phase of carcinogenesis has been proposed (9-12). In the latter phase the role of free oxygen radicals has been emphasized (13). We examined Fe3+NTA-induced DNA damage in the presence of hydrogen perReceived 6/5/87; revised 9/14/87; accepted 9/21/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by a research grant of the Fujiwara Foun dation of Kyoto University and Grant-in-Aid for Scientific Research No. 61570255 from the Ministry of Education. Science and Culture of Japan. 1To whom requests for reprints should be addressed. 3The abbreviations used are: NTA. nitrilotriacetic acid; DMPO, 5,5-dimethylpyrroline-jV-oxide; DMPO-OH, hydroxyl radical adduci of 5,5-dimethylpyrroline-/V-oxide; EGTA, ethylene glycol bis(0-aminoethylether)-/V,/VyV',Ar'tetraacetic acid; HEDTA. /V-(2-hydroxyethyl)ethylenediaminetriacetic acid; CDTA, 1,2-cyclohexylenediaminetetraacetic acid; DTP A, diethylenetriamine yvyv.JV'.JVJV-pentaacetic acid; SOD, Superoxide dismutase; ESR, electron-spin resonance. oxide by using 32P 5'-end-labeled DNA fragments of defined sequence obtained from the human c-Ha-ros-l protooncogene, comparing with the ferric complexes of other aminopolycar boxylic acids. Since iron compounds administered are incor porated in the various protein systems of the body, the experi ments estimating DNA damage were performed in the presence of albumin for a simple protein model. We also investigated chemical basis for the different behavior of these iron chelates with respect to radical production by using ESR spin-trapping techniques. Fe3+-NTA plus hydrogen peroxide was shown to cause strong DNA damage and to produce hydroxyl radicals to the most extent among Fe3+ chelates of aminopolycarboxylic