Active oxygen species in DNA damage induced by carcinogenic metal compounds.

Some carcinogenic metal compounds [chromate(VI), Fe(III) nitrilotriacetate, cobalt(II), and nickel(II)[ induced formation of various oxygen radical species in the presence of hydrogen peroxide. These oxygen radicals were suggested to give different kinds of site-specific DNA damage; 8-hydroxyl-2'-deoxyguanosine formation is included in the DNA damage. Using pulsed-field gel electrophoresis, nickel sulfide was shown to induce oxidative DNA cleavage in cultured cells. On the basis of these findings, we have emphasized the role of oxygen radicals in metal carcinogenesis. ImagesFigure 4.


Introduction
In 1986, we reported that carcinogenic chromate(VI) reacts with (H202) to produce hydroxyl free radicals (OH) and singlet oxygen (b2), which cause DNA damage (1) Since then, we have demonstrated that carcinogenic Fe(III) nitrilotriacetate, cobalt(II), and nickel(II) react with H202 to produce hydroxy radical, 102, and metal-oxygen complexes, which cause site-specific DNA damage (2)(3)(4)(5). On the basis of these findings, we have proposed that oxygen radicals may contribute to metal carcinogenesis (6). In 1989, Sugiyama et al. suggested that chromate(VI) induced DNA single-strand breaks in cultured cells via -OH formation (7,8). In addition, Kasprzak et al. reported 8-hydroxyl-2'-deoxyguanosine  formation in the kidney of rats treated with nickel acetate (9). Some recent studies in our laboratory of the important role of oxygen radicals in metal carcinogenesis are described here.

Materials and Methods
DNA damage was analyzed by the DNA sequencing technique using 32P 5'-endlabeled DNA fragments obtained from the human c-Ha-ras-1 protooncogene as previously described (3,10). DNA strand breaks in cultured cells were detected by using pulsed-field gel electrophoresis according to the method described previously (11). 8-OH-dG formation in DNA was analyzed by high-pressure liquid chromatographyelectron capture detector (HPLC-ECD) (12). Electron spin resonance (ESR) spectra were measured at room temperature using a JES-FE-3XG spectrometer (EOL, Tokyo, Japan) (10). Chemiluminescence was measured by using the Luminescence Reader (Aloka, Tokyo, Japan) with fluorescein.

Results and Discussion Chromium(V)
Carcinogenic chromium(VI) [Cr(VI)] has been reported to induce DNA lesions in vivo and in culture (13). We investigated reactivities of Cr compounds with DNA by the DNA sequencing technique using 32p 5'-end-labeled DNA fragments. Figure 1 shows piperidine-labile sites of the DNA fragment treated with sodium chromate(VI) plus H202. Cleavage occurred at every base residue but the cleavage at the guanine positions was more dominant than at the other three bases. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) and (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as -OH traps demonstrated that -OH is generated during the reaction of chromate(VI) with H202 (1). ESR studies using 2,2,6,6-tetramethyl-4piperidone demonstrated that 10 is also 2 generated during the reaction, and reacts specifically with deoxyguanosine monophosphate (dGMP) (1). These results indicate that sodium chromate(VI) reacts with H202 to produce -OH and 02; -OH causes every base alteration and deoxyribosephosphate backbone breakage, and 10 oxidizes the guanine residues resulting 2 in the formation of alkali-labile sites (1).

Fernc Nitrilotriacetate
In 1986, Ebina et al. reported the induction of the renal adenocarcinoma in rats by Fe-nitrilotriacetate (Fe-NTA) (16). We showed that Fe(III)-NTA catalyzes the decomposition of H202 to produce -OH, which subsequently causes DNA base alterations and backbone breakages. The DNA damage was without marked site specificity. Dizdaroglu et al. reported that Fe(III)-NTA plus H202 caused 8-OH-dG formation in isolated chromatin (17). Umemura et al. reported formation of 8-OH-dG in rat kidney DNA after IP administration of Fe-NTA (12). These studies suggest that Fe-NTA-induced  DNA damage in vivo is induced by oxygen radicals. Table 1 summarizes the activities of Fe(III)-chelates of aminopolycarboxylic acids for H202-dependent DNA damage and for OH formation from H202 (2). Fe(III)-NTA induced DNA cleavage in the presence of H202 , whereas Fe(III)-chelates of other aminopolycarboxylic acids did not induce that response under the conditions used. Fe(III)-HEDTA/ H202 system did not cause DNA damage, although it produced as much OH as the Fe(III)-NTA/ H202 system. These results may be interpreted by structural considerations. Fe(III)-NTA is supposed to approach the groove of the DNA double helix readily, whereas Fe(III)-HEDTA may not. Since OH is short-lived, it damages DNA only when produced in the vicinity of the DNA. We measured fluorescein-dependent chemiluminescence induced by Co(II) and H202. Figure 2 shows that the Co(II)induced chemiluminescence increased with increasing concentrations of H202. The intensity was enhanced about 3-fold in D20 in which the lifetime of singlet oxygen is at least 10 times that in H20. These results indicate that 102 is generated during the reaction of Co(II) with H202.

Nickel(II)
Nickel compounds have been shown to have seriously toxic and carcinogenic effects on humans (20). Costa and Mollen-hauer reported that carcinogenic activity of particulate nickel compounds was proportional to their cellular uptake (21). Nickel salts have been shown to cause DNA single strand breaks in cultured cells. Our experiments (4) with isolated DNA showed that nickel(II) [Ni(II)] ion caused extensive sitespecific damage (C-T-G>A) in the presence of H202' The incubation of calf thymus DNA with Ni(II) plus H202 for 6 hr increased the 8-OH-dG level about 10-fold. The result shows that Ni(II) ion reacts with H202 to produce active species causing oxidative DNA damage. Kasprzak and Hernandez (22) reported that addition of Ni(II) doubled the 8-OH-dG formation from double-stranded DNA by H2O2 in the presence of ascorbic acid.
We examined by ESR spectroscopy whether activated oxygen species are produced by the reaction of Ni(II) oligopeptides with H202. Figure 3 shows that the -OH adduct of DMPO was formed by the decomposition of H202 in the presence of Ni(II) GlyGlyHis. Adducts of DMPO were not observed with either H202 (Figure 3) or Ni(II) GlyGlyHis (data not shown). It is known that 0OH reacts with ethanol and formate to produce x-hydroxyethyl radicals and .CO radicals, respectively. However, in the case of Ni(II) oligopeptides and H202, the spin adducts of a-hydroxyethyl radicals and .CO-radicals were scarcely observed although ethanol and formate had inhibitory effects ( Figure 3). With mannitol, the spin adducts of mannitol-derived radicals were not observed. In contrast, with sulfur compounds (methional), which can scavenge active species with less reactivity than OH, the spin adducts of methional-derived radi- Raji cells were treated with 3-AT or DMTU for 1 hr and then exposed to nickel sulfide (10 pg/ml) in RPMI 1640 containing 6% fetal calf serum. After incubation at 37°C for 24 hr, the medium was removed and the cells were washed three times with PBS and prepared into agarose plugs and lysed. Electrophoresis was performed in TBE buffer, pH 8.3, by pulsed field (CHEF-DRII DNA megabase electrophoresis system, Bio-Rad) at 200 volts at 14°C. Switch time was 60 sec for 15 hr followed by a 90-sec switch time for 9 hr. The DNA in the gels was visualized in ethidium bromide.  cals were observed. This result led us to the idea that the -OH adduct is formed in the reaction of nickel-oxygen complex and DMPO (5).
In recent years, pulsed-field gel electrophoresis has emerged as a powerful tool for detection of DNA strand breaks in cultured cells. Figure 4 reveals that nickel sulfide induced cellular DNA cleavage. To clarify whether H202 participates in cellular DNA damage, we examined the effects of 3-aminotriazol (3-AT, a catalase inhibitor) and dimethylthiourea (DMTU, a highly permeable scavenger of H202) on the DNA double-strand breaks. 3-AT enhanced nickel sulfide-induced DNA damage whereas DMTU inhibited it ( Figure 4). These results suggest that H202 participates in DNA damage induced by nickel sulfide in vivo.
In summary, Table 2 outlines the DNA damage induced by some carcinogenic metal compounds via active oxygen species formation in vitro and in vivo. The metal compounds produced various types of oxygen radicals from H202 These oxygen radicals seem to be responsible for the metal carcinogenesis.