Distinct mechanisms of oxidative DNA damage induced by carcinogenic nickel subsulfide and nickel oxides.

The U.S. National Toxicology Program has shown clear evidence of carcinogenicity of nickel subsulfide (Ni(3)S(2)) and some evidence of carcinogenicity of NiO (green) in rats. In the present study, DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. In cultured HeLa cells, Ni(3)S(2) induced a significant increase in 8-hydroxydeoxyguanosine (8-OH-dG) formation, whereas NiO (black), NiO (green), and NiSO(4) did not. On the other hand, in rats, intratracheal instillation of all these nickel compounds significantly increased 8-OH-dG content in the lungs. The disparities in DNA damage between cultured cells and animals could be accounted for by two different mechanisms for nickel-induced oxidative DNA damage in lungs of rats. One is direct oxidative DNA damage: Ni(II) enters the cells and then reacts with endogenous and/or nickel sulfide-produced hydrogen peroxide (H(2)O(2)) to give reactive oxygen species that cause DNA damage. This mechanism is supported by oxidative damage to isolated DNA treated with Ni(II) and H(2)O(2). The other mechanism is indirect oxidative DNA damage due to inflammation. This double mechanism for DNA damage may explain the relatively high carcinogenic risk associated with Ni(3)S(2).

A number of metals have carcinogenic and co-carcinogenic potentials. Regarding mechanisms of metal carcinogenesis, we reported that carcinogenic chromate (VI) reacts with hydrogen peroxide (H 2 O 2 ) to produce a hydroxyl free radical and singlet oxygen ( 1 O 2 ) that cause damage to isolated DNA (1). Furthermore, carcinogenic Fe(III) nitrilotriacetate, Co(II), and Ni(II) were shown to react with H 2 O 2 to produce a hydroxyl free radical, 1 O 2 , and metal-oxygen complexes that cause site-specific DNA damage (2)(3)(4). On the basis of these findings, we have emphasized the role of reactive oxygen species (ROS) in metal carcinogenesis (5).
The early epidemiologic data indicated different carcinogenic risks from inhalation of different nickel compounds (6). The International Agency for Research on Cancer (IARC) has classified nickel compounds such as NiSO 4 , NiO, nickel hydroxides, and crystalline nickel as agents carcinogenic to humans (Group 1) (7). Recently, the U.S. National Toxicology Program has shown clear evidence of carcinogenicity of Ni 3 S 2 and some evidence of carcinogenicity of NiO (green) in rats (8,9). However, no evidence for carcinogenicity of water-soluble NiSO 4 in rats has been provided (10), and this result has been inconsistent with the evaluation by IARC.
In this study, to clarify the mechanism of nickel carcinogenesis, we investigate the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in cultured cells and lungs of rats treated with nickel compounds by using electrochemical detector-coupled high-performance liquid chromatography (HPLC-ECD). A characteristic oxidative DNA lesion, 8-OH-dG, has attracted much attention in relation to mutagenesis and carcinogenesis (11,12). To clarify the participation of reactive oxygen species in nickel-induced DNA damage, we examined the DNA damage and site specificity of DNA cleavage induced by nickel compounds in the presence of H 2 O 2 , using 32 P-5´-end-labeled DNA fragments.

Analysis of 8-OH-dG Formation
Isolated DNA. Calf thymus DNA (100 µM) was incubated with 20 µM NiSO 4 and/or 100 µM H 2 O 2 in 200 µL of 4 mM phosphate buffer (pH 7.8) at 37°C. After ethanol precipitation, DNA was digested to the nucleosides with nuclease P1 and alkaline phosphatase and measured by HPLC-ECD as described previously (4).
DNA of cultured cells. After the addition of nickel compounds suspended or in solution (10 µg/mL), cells were incubated at 37°C for 24 hr and harvested by centrifugation. For isolation of DNA, nucleic acid extraction system (model 341; Gene Pure, Applied Biosystems, Foster City, CA, USA) was used. DNA isolation steps were performed under helium. The content of 8-OH-dG was determined after enzyme digestion of DNA using an HPLC-ECD (13).
DNA of lungs. After cutting off the trachea, we gently homogenized half the defrosted lungs in 3 mL of phosphate-buffered saline (PBS) solution with a motor-driven Teflon-glass tissue homogenizer for 30 sec. The nuclei were obtained by centrifugation at 1,750×g for 5 min. DNA was isolated from 1/20 volume of suspended nuclei fraction in PBS by the same extraction system used for DNA isolation from cultured cells, and 8-OH-dG was analyzed as described previously (4,13,14).

Analysis of DNA Damage Induced by Ni(II) plus H 2 O 2
Reaction mixtures containing the 32 P-5´end-labeled DNA fragment, 20 µM/base of calf thymus DNA, 2.5 mM H 2 O 2 , and 50 µM NiCl 2 in 10 mM phosphate buffer (pH 7.8) containing 5 µM diethylenetriamine-N,N,N´,N´,N´-pentaacetic acid were incubated at 37°C for 60 min. The DNA fragments were treated with piperidine and electrophoresed on a polyacrylamide gel.

Electron Spin Resonance Measurements
The spin adducts were measured using 5,5dimethylpyrroline-N-oxide as hydroxyl radical traps as previously described (15). Typical reaction mixture contained oligopeptides, NiCl 2 , H 2 O 2 , and 292 mM 5,5-dimethyl-1pyrroline N-oxide (DMPO) in 20 mM sodium phosphate buffer at pH 7.9. Spectra were recorded with a microwave power of 16 mW and a modulation amplitude of 1.0 G. Electron spin resonance (ESR) measurements were made at 25°C using a JES-FE-3XG spectrometer (JEOL, Tokyo, Japan) with 100 kHz field modulation.

Effects of Scavengers on DNA Damage Induced by Ni(II) plus H 2 O 2
To investigate whether active oxygen species were involved in the DNA damage, the effects of scavengers were examined. In experiments with hydroxyl radical scavengers, To estimate the site specificity of the DNA damage, 32 P-5´-end-labeled DNA fragments treated with Ni(II) plus H 2 O 2 , followed by the piperidine treatment, were electrophoresed and the autoradiogram was obtained. Autoradiograms were scanned with a laser densitometer. The DNA cleavage sites were determined by using the Maxam-Gilbert procedure (16). Ni(II) plus H 2 O 2 induced piperidine-labile sites frequently at cytosine, thymine, and guanine residues and rarely at adenine residue.

ESR Spectra of the Spin Adducts of DMPO Generated during the Reaction of Ni(II) GlyGlyHis with H 2 O 2
The hydroxyl radical adduct of DMPO was observed with 50 µM Ni(II) GlyGlyHis

Discussion
The present study and our previous data (4)  To examine what kind of ROS cause DNA damage, effects of various scavengers on DNA damage by Ni(II) plus H 2 O 2 were examined. Among hydroxyl radical scavengers, DMSO, methionine, methional, and sodium formate inhibited DNA cleavage, whereas ethanol and mannitol did not. These observations suggest a possibility that hydroxyl free radicals are generated in close association with DNA such that ethanol and mannitol cannot remove them. Furthermore, ESR studies using spin traps revealed that hydroxyl radical adducts are produced by the decomposition of H 2 O 2 in the presence of Ni(II) oligopeptides (15). These results support the speculation that reactive nickel-oxygen complexes participate in the DNA damage.
Treatment of cultured HeLa cells with Ni 3 S 2 (10 µg/mL) induced large increases in 8-OH-dG, whereas NiO (black), NiO (green), and NiSO 4 did not enhance the production of 8-OH-dG. However, high concentrations of nickel in the lungs of rats via intratracheal instillation of Ni 3 S 2 , NiO (black), NiO (green), and NiSO 4 induced increases in 8-OH-dG. Also, light microscopy demonstrated that in addition to Ni 3 S 2 , NiO (black), NiO (green), and NiSO 4 induced inflammatory reactions. It is reported that some carcinogenic metal sulfide particles were found to stimulate polymorphonuclear leukocytes to produce ROS (18). Therefore, it is considered that the nickel compounds used in this study can induce indirect damage through inflammation.
Disparities in DNA damage between cultured cells and animals could be accounted for by two different mechanisms for nickelinduced oxidative DNA damage in lungs of rats ( Figure 1). One is the direct oxidative DNA damage; that is, Ni(II) that has entered the cells reacts with endogenous and/or nickel sulfide-produced H 2 O 2 to give ROS that cause DNA damage, as was seen in cultured cells. The reactive species may be an oxo-Ni(IV) complex or an Ni(III)-peroxide complex, which can release hydroxyl radicals. This idea is supported by our previous study and the studies of others (15,19,20).
The other mechanism is indirect oxidative DNA damage due to inflammation. The important sources of endogenous oxygen radicals are phagocytic cells such as neutrophils and macrophages (21). It has been proposed that ROS including nitrogen oxide generated in inflamed tissues can cause injury to target cells and also damage DNA, which contributes to carcinogenic processes (22)(23)(24).
Based on the above overall model, two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds tested induced indirect damage through inflammation, and Ni 3 S 2 also showed direct oxidative DNA damage Metals Toxicity • Kawanishi et al.