Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

S-nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation

Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the alpha- and the beta-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO*. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biologically specific in converting NO signals to zinc signals. This could bear importantly on the physiological action of MT-III, whose biological activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.     

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults

Background

Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.

Methods

A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.

Results

Purified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo.

General significance

Transduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.     

Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species

Islet transplantation is a promising therapy for Type 1 diabetes, but many attempts have failed due to early graft hypoxia or immune rejection, which generate reactive oxygen species (ROS). In the current study, we determined that transgenic overexpression of the antioxidant metallothionein (MT) in pancreatic beta cells provided broad resistance to oxidative stress by scavenging most kinds of ROS including H2O2, peroxynitrite radical released from streptozotocin, 3-morpholinosydnonimine (SIN-1), and superoxide radical produced by xanthine/xanthine oxidase. MT also reduced nitric oxide-induced beta cell death. A direct test of hypoxia/reperfusion sensitivity was made by exposing FVB and MT islets to hypoxia (1% O2). MT markedly reduced ROS production and improved islet cell survival. Because MT protected beta cells from a broad spectrum of ROS and from hypoxia, we considered it to be an ideal candidate for improving islet transplantation. We first tested syngeneic transplantation by implanting islets under the kidney capsule of the same strain, FVB mice, thereby eliminating the immune rejection component. Under these conditions, MT islets maintained much greater insulin content than control islets. Allotransplantation was then tested. MT transgenic and normal FVB islets were implanted under the kidney capsule of BALB/c mice that were previously treated with streptozotocin to induce diabetes. We found that MT islets extended the duration of euglycemia 2-fold longer than nontransgenic islets. The benefit of MT was due to protection from ROS since nitrotyrosine staining, an indicator of free radical damage, was much lower in MT grafts than in FVB grafts. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. These data demonstrate that treatment with a broad spectrum antioxidant protects islets from ROS damage such as that produced during the early phase of islet transplantation.     

DNA damage by oxygen radicals and excited state species: a comparative study using enzymatic probes in vitro

Repair enzyme-containing extracts from a variety of cell types are used to analyse and compare DNA damage induced by oxygen radicals and excited molecules. The differing potentials of these extracts for recognising DNA damage leads to characteristic DNA damage profiles after treatment with superoxide (xanthine/xanthine oxidase), gamma-rays, chemically generated singlet oxygen, photosensitizers (rose bengal, methylene blue), UV254 and a 1,2-dioxetane. Three different types of damage profiles are distinguished and assigned to the predominant action of hydroxyl radicals, singlet oxygen or to the photoexcitation of thymine residues. The method applied in this study allows the analysis of DNA damage and the identification or exclusion of the participation of different ultimate reactive species without chemical identification of the lesions.     

Metallothionein-III prevents glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.     

Free radical scavenging actions of hippocampal metallothionein isoforms and of antimetallothioneins: an electron spin resonance spectroscopic study.

The high concentration of zinc in the hippocampal mossy fiber axon boutons is localized in the vesicles and is mobilized by exocytosis of the zinc-laden vesicles. Furthermore, the mammalian hippocampi contain metallothionein (MT) isoforms which regulate the steady state concentration of zinc, an important antioxidant. Indeed, zinc deprivation leads to an increased lipid peroxidation, reduces the activity of Cu++-Zn++ superoxide dismutase, and protect against oxidative stress such as exposure to ultraviolet A irradiation. By employing electron spin resonance (ESR) spectroscopy, we have demonstrated that rat hippocampal MT isoforms 1 and 2 were able to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), hydroxyl radicals (*OH) generated in a Fenton reaction, and superoxide anions (O2*-) generated by the hypoxanthine and xanthine oxidase system. In addition, MT-1 isoform protected the isolated hepatocytes from lipid peroxidation as determined by thiobarbituric acid bound malondialdehyde. MT antibodies scavenged DPPH radicals, hydroxyl radicals and reactive oxygen species but not superoxide anions. The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-1 appears to be a superior scavenger of superoxide anions and 1,1-diphenyl-2-picrylhydrazyl radicals. Moreover, antibodies formed against MT isoform retain some, but not all, free radical scavenging actions exhibited by MT-1 and MT-2.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.     

The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas

Alloxan and streptozotocin are widely used to induce experimental diabetes in animals. The mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood. The cytotoxic action of both these diabetogenic agents is mediated by reactive oxygen species, however, the source of their generation is different in the case of alloxan and streptozotocin. Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed by the Fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

Cytoprotective effects of catechin 7-O-β-D glucopyranoside against mitochondrial dysfunction damaged by streptozotocin in RINm5F cells

The protective effects of catechin 7-O-β-D glucopyranoside (C7G) against streptozotocin (STZ)-induced mitochondrial damage in rat pancreatic β-cells (RINm5F) were investigated. A marked increase in mitochondrial reactive oxygen species (ROS) was observed in STZ-treated cells; this increase was restricted by C7G treatment. C7G also scavenged superoxide anions and hydroxyl radicals generated by xanthine/xanthine oxidase (xanthine/XO) and the Fenton reaction (FeSO(4) + H(2) O(2)), respectively. C7G restored activity and expression of both mitochondrial manganese superoxide dismutase (MnSOD) and catalase (CAT), which were suppressed by STZ treatment. In addition, C7G prevented STZ-induced mitochondrial lipid peroxidation, protein carbonyl, and DNA base modification. C7G restored the loss of mitochondrial membrane potential (Δψ) that was disrupted by STZ treatment, and prevented cell death via inhibition of apoptosis. These results suggest that C7G has a protective effect against STZ-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction.     

Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage

The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases.     

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation. © 1996 Wiley-Liss, Inc.     

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells.

The objective of this study is to investigate if 8-methoxy-psoralen (8-MOP) plus ultraviolet A (UVA) radiation (PUVA) induces oxidative DNA damage. When calf thymus DNA was incubated with 8-MOP and irradiated with UVA (335-400 nm), the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was substantially increased by approximately 6-fold. Formation of 8-OHdG proportionally correlated with both UVA fluence and 8-MOP concentrations. Human epidermoid carcinoma cells were incubated with 10 microg 8-MOP per milliliter, followed by irradiation of 25 kJ/m2 UVA. The level of 8-OHdG increased by nearly 3-fold in PUVA-treated cells compared to 8-MOP and UVA controls. The formation of 8-OHdG correlated with DNA fragmentation as determined by spectrofluorometry. To investigate the reactive oxygen species (ROS) involved in PUVA-induced oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution prior to PUVA treatment. The results showed that only sodium azide and genistein significantly quenched PUVA-induced 8-OHdG, whereas catalase, superoxide dismutase, and mannitol exhibited no effect. The quencher study with cultured cells indicated that N-acetyl-cysteine and genistein protected oxidative DNA damage as well as DNA fragmentation by PUVA treatment. Our studies show that PUVA treatment is able to induce the formation of 8-OHdG in purified DNA and cultured cells and suggest that singlet oxygen is the principle reactive oxygen species involved in oxidative DNA damage by PUVA treatment.     

Mechanism of metal-mediated DNA damage induced by metabolites of carcinogenic 2-nitropropane

2-Nitropropane (2-NP), a widely used industrial solvent, is carcinogenic to rats. To clarify the mechanism of carcinogenesis by 2-NP, we investigated DNA damage by 2-NP metabolites, N-isopropylhydroxylamine (IPHA) and hydroxylamine-O-sulfonic acid (HAS), using 32P-5'-end-labelled DNA fragments obtained from genes that are relevant to human cancer. In the presence of Fe(III) EDTA, both IPHA and HAS caused DNA damage at every nucleotide position without marked site preference. The damage was inhibited by free hydroxyl radical (-*OH) scavengers, catalase and deferoxamine mesilate, an iron chelating agent. These results suggest that the DNA damage was caused by -*OH generated via H(2)O(2) by both IPHA and HAS. In contrast, in the presence of Cu(II), IPHA frequently caused DNA damage at thymine. The Cu(II)-mediated DNA damage caused by IPHA was inhibited by catalase, methional and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that the DNA damage induced by IPHA in the presence of Cu(II) was caused by a reactive oxygen species like the Cu(I)-hydroperoxo complex. On the other hand, HAS most frequently induced DNA damage at 5'-TG-3', 5'-GG-3' and 5'-GGG-3' sequences. Catalase and methional only partly inhibited the Cu(II)-mediated DNA damage caused by HAS, suggesting that the reactive oxygen species and another reactive species participate in this process. Formation of 8-oxodG by IPHA or HAS increased in the presence of metal ions. This study suggests that metal-mediated DNA damage caused by 2-NP metabolites plays an important role in the mutagenicity and the carcinogenicity of 2-NP.