Site-specific DNA damage by phenylhydrazine and phenelzine in the

Sep 24, 1991 - Phenylhydrazine cleaved isolated DNA inthe presence of Cu(II), Mn(III), hemin, Fe(III)-. EDTA, or peroxidase/H202, while phenelzine cle...
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Chem. Res. Toxicol. 1992,5, 440-446

Site-Specific DNA Damage by Phenylhydrazine and Phenelzine in the Presence of Cu(I1) Ion or Fe(II1) Complexes: Roles of Active Oxygen Species and Carbon Radicals Koji Yamamoto and Shosuke Kawanishi* Department of Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan Received September 24, 1991

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(II1)EDTA, or peroxidase/H202, while phenelzine cleaved in the presence of Cu(I1). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(II1)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical ('OH) on the DNA damage suggest the involvement of 'OH. On the other hand, Cu(I1)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(1)-specific chelator, but not by 'OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(1) plus H202but not to that induced by Cu(I1) plus H202,it is speculated that the copper-oxygen complex derived from the reaction of H202 with Cu(1) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(I1). A comparison between scavenger effects on the DNA damage and those on radical production deteded with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(11)-, hemin-, or Fe(II1)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, 'OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.

Introduction The biological significance of oxygen free radicals has recently attracted much interest ( 1 , 2 ) ,especially in connection with carcinogenesis ( 3 , 4 ) . It is well-known that hydroxyl free radical ('OH)' is generated from H202by Fenton reaction with reduced iron. In contrast, whether copper acta like iron or not remains to be clarified. It has been suggested that Hz02reacts with Cu(1) to give 'OH, which causes DNA damage (5-8). However, recent studies on the reaction of H202with Cu(1) have suggested that Cu(II1) or Cu(1) peroxide [Cu(I)OOH] is formed as a reactive intermediate (9,101. We have previously suggested that the main active species causing DNA damage induced by Cu(I1) plus H202are copper-oxygen complexes with similar reactivity to 'OH and/or singlet oxygen (11). The metabolism of hydrazine and its derivatives is thought to involve the production of free radical intermediates (12). Ortiz de Montellano et al. have extensively investigated the reaction of hydrazine derivatives with hemoproteins via carbon-centered radicals (13,14). The oxidation of phenylhydrazine initiated by Cu(I1) or oxyhemoglobin in buffered aqueous solution is a complex process involving H202,02-,and phenyl radical (15-17). Phenelzine [ (2-phenylethy1)hydrainelis a monoamine oxidase inhibitor and is used as an antidepressant. The metabolism of phenelzine by microsomes yields 2phenylethyl radical (18), which is speculated to be less reactive than phenyl radical ( 1 4 ) . Phenylhydrazine and phenelzine were reported to produce the tumors of blood vessel and lung in mice (19-21). Phenylhydrazine induced a significant DNA fragmentation in the liver and lung of mice (21). These two aromatic *To whom correspondence and reprint requests should be addressed.

hydrazines induced mutation in bacteria without addition of S-9 mixture (21). DNA damage and H202production were observed during autoxidation of phenelzine (22). Therefore, it is considered that phenylhydrazine and phenelzine exert their toxic action through a mechanism other than the drug-metabolizing enzyme system. Leite and August0 (23) reported that 2-phenylethyl radical, produced from phenelzine oxidation, could directly damage DNA. Relevantly, methyl radical was shown to have the ability to alkylate the 8-position of guanine residues (24). On the other hand, we have recently found that copperoxygen complex rather than the methyl radical plays a more important role in methylhydrazine plus Cu(I1)-induced DNA damage (25).In this study, we examine which is more important in phenylhydrazine- and phenelzineinduced DNA damage, carbon-centered radicals (phenyl radical, 2-phenylethyl radical) or oxygen-derived active species ('OH, copper-oxygen complex).

Experimental Procedures Materials. Phenelzine sulfate (purity >97%) was purchased from ICN Biomedicals, Inc., Cleveland, OH. Phenylhydrazinium sulfate (purity 98.6%), CuCl,, other metal chlorides, ethanol, and D-mannitOl were from Nacalai Tesque, Inc., Kyoto, Japan.

Caution: Phenylhydrazine may cause contact dermatitis and should be handled carefully. POBN was purchased from Aldrich, Milwaukee, WI. DMPO was from Labotec, Co., Ltd., Tokyo, Japan. Bathocuproinedisulfonic acid was from Dojin Chemicals Co., Kumamoto, Japan. Superoxide dismutase (3000units/mg from bovine erythrocytes) and catalase (45000unita/mg from Abbreviations: 'OH, hydroxyl free radical; 02-,superoxide;DTPA, diethylenetriaminepentaacetic acid; DMPO, 5,5-dimethyl-l-pyrroline N-oxide;DMPO-OH, hydroxyl radical adduct of 5,5dimethyl-l-pyrroline N-oxide; POBN, a-(l-oxy-4-pyridyl)-N-tert-butylnitrone; NTA, nitrilotriacetic acid.

0 1992 American Chemical Society

DNA Damage by Phenylhydrazine

A

B

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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Figure 1. Autoradiogram of q-labeled DNA fragments incubated with phenylhydrazine and phenelzine in the presence of metal. The reaction mixture contained the 32P-5'-end-labeled 337-base-pair fragment (PstI 2345-AuaI* 2681), 50 pM per base of sonicated calf thymus DNA, and 5 pM DTPA in 200 pL of 10 mM sodium phosphate buffer at pH 7.9. Where indicated, 0.1 mM phenylhydrazine (A), or 0.5 mM phenelzine (B), and 20 pM metal ion were added. After the incubation a t 37 "C for the indicated durations, followed by the piperidine treatment, the DNA fragments were electrophoresed on an 8% polyacrylamide, 8 M urea gel (12 cm X 16 cm),and the autoradiogram was obtained by exposing X-ray film to the gel. Lane 1,control (no metal, no hydrazines);lane 2, no metal, lane 3, CuC1,; lane 4, MnC1,; lane 5, Mn(II1) pyrophosphate; lane 6 FeC1,; lane 7, hemin; lane 8, Fe(II1)-EDTA; lane 9, (NH4)2Fe*(S04)z.Incubation time was 30 min except for lane A3 (5 min) and lane B3 (10 min). bovine liver) were from Sigma, St. Louis,MO. [y32P]ATP (so00 Ci/mmol) was purchased from Du Pont-New England Nuclear, Boston, MA. Restriction enzymes (AuaI, XbaI, PstI) and T4 polynucleotide kinase were purchased from New England Biolabs, Beverly, MA. Peroxidase (260 units/mg from horseradish) was from Toyobo Co., Osaka, Japan. Mn(II1) pyrophosphate was prepared according to Archibald and Fridovich (26). Analysis of Damage of Isolated DNA by Phenylhydrazine and Phenelzine. DNA fragments were prepared from plasmid pbcNI which carries a 6.6-kilobase BamHI chromosomal DNA segment containing human c-Ha-ras-1protooncogene (27). Singly labeled 341-base-pair fragment (XbaI 1906-AuaI* 2246), 98base-pair fragment (AuaI* 2247-PstI 2344), and 337-base-pair fragment (PstI 2345-AuaI* 2681) were obtained according to the method described previously (11). The asterisk indicates 32Plabeling and nucleotide numbering starts with the BamHI site (27). The standard reaction mixture in a microtube (Eppendorf) contained 0.5 mM phenylhydrazine or phenelzine, 20 pM metal ion, 50 pM per base of sonicated calf thymus DNA, [32P]DNA fragment, and 5 pM DTPA in 200 p L of 10 mM sodium phosphate buffer (PH 7.9). After incubation at 37 OC for indicated durations, the DNA fragments were heated a t 90 "C for 20 min in 1 M piperidine where indicated and electrophoresed as previously described (11). The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure (28) using a DNA sequencing sytem (LKB 2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) was used for measurement of the relative amounta of oligonucleotides from treated DNA fragments. ESR Spectra Measurements. ESR spectra were measured at room temperature using a JES-FE3XG (JEOL, Tokyo, Japan) spectrometer with 100-kHz field modulation according to the method described previously (25). Spectra were recorded with a microwave power of 16 mW and a modulation amplitude of 1.0 G. The magnetic fields were calculated by the splitting of Mn(II) in MgO (AH- = 86.9 G). DMPO and POBN were used as radical trapping reagents.

Results Cleavage of 32P-LabeledDNA Fragments Induced by Phenylhydrazine and Phenelzine in the Presence

Chem. Res. Toxicol., Vol. 5, No. 3,1992 441

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Figure 2. Cu(I1)-mediated DNA cleavage by phenylhydrazine and phenelzine with or without the piperidine treatment. The reaction mixture contained the =P-5'-end-labeled 337-base-pair fragment, 50 p M per base of sonicated calfthymus DNA, indicated concentration of phenylhydrazine (A) or phenelzine (B), 20 pM CuC12,and 5 pM DTPA in 200 pL of 10 mM sodium phosphate buffer a t pH 7.9. Control lane, no hydrazines; lane 1,0.05 mM; lane 2,O.l mM; lane 3,0.2 mM; lane 4,0.5 mM; lane 5 , l mM; lane 6,0.05 mM; lane 7,O.l mM; lane 8,0.2 mM; lane 9,0.5 mM; lane 10,l mM. After the incubation at 37 "C for 10 min, followed by the piperidine treatment (control lane and lanes 1-5), or without the piperidine treatment (lanes6-10), the DNA fragments were analyzed by the method described in the Figure 1legend.

of Metal. The extent of damage to isolated DNA induced by phenylhydrazine or phenelzine in the presence of metal ions was estimated by gel electrophoretic analysis (Figure 1). No oligonucleotide was observed with phenylhydrazine alone (Figure lA, lane 2), showing that phenylhydrazine itself is not a DNA-damaging agent. The upper band and lower band in the control show single-stranded and double-stranded forms of intact DNA fragment, respectively. At a low concentration (0.1 mM), phenylhydrazine induced DNA damage in the presence of Cu(I1) (lane 3), Mn(II1) (lane 5), or Fe(II1)-EDTA (lane 8). With 0.02-0.2 mM phenylhydrazine, Mn(II1)-mediated DNA damage was observed, whereas with 1mM phenylhydrazine, the damage was not observed (data not shown). At a high concentration (1mM), phenylhydrazine induced DNA damage even in the presence of hemin (data not shown). None of these metal compounds induced DNA damage without phenylhydrazine. On the other hand, phenelzine caused DNA damage in the presence of Cu(I1) (Figure lB, lane 3). A very small quantity of DNA damage was also observed in the presence of hemin or Fe(II1)-EDTA (Figure lB, lanes 7 and 8). DNA Damage by Phenylhydrazineand Phenelzine in the Presence of Cu(I1). The DNA damage increased with phenylhydrazine concentrations up to 0.5 mM and did not increase over 0.5 mM phenylhydrazine (Figure 2A). DNA damage by phenelzine plus Cu(I1) (Figure 2B)was weaker than that by phenylhydrazine plus Cu(II). In either case, DNA cleavage was appreciable even without piperidine treatment, indicating the breakage of the deoxyribose phosphate backbone (lanes 6-10). The DNA cleavage increased with piperidine treatment (lanes 1-5), suggesting the involvement of base alteration and/or liberation. DNA Damage by Phenylhydrazineand Phenelzine in the Presence of Peroxidase or Ferricyanide. DNA damage was caused by phenylhydrazine in the presence of peroxidase/H202(Figure 3A, lane 6). The DNA damage was observed without the addition of H202(lanes 3-5). Phenylhydrazine plus heat-denatured peroxidase also caused DNA damage (data not shown). The results suggest that the peroxidase exerts catalysis in the DNA damage by its heme rather than by its enzymatic action.

442 Chem. Res. ToxicoZ., VoZ.5, No. 3,1992

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Yamamoto and Kawanishi

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Figure 3. DNA cleavage induced by phenylhydrazine and phenelzine in the presence of peroxidase H202or ferricyanide. The reaction mixture contained the P-5'-end-labeled 337base-pair fragment, 50 pM per base of sonicated calf thymus DNA, and 5 pM DTPA in 200 pL of 10 mM sodium phosphate buffer at pH 7.9. (A) Peroxidase, 1mM phenylhydrazine, and/or 2 mM H202were added where indicated. Lane 1, 200 units/mL peroxidase; lane 2,0.25 unib/mL peroxidase + phenylhydrazine; lane 3, 2.5 units/mL peroxidase + phenylhydrazine; lane 4, 25 units/mL peroxidase + phenylhydrazine; lane 5, 200 units/mL peroxidase + phenylhydrazine; lane 6,200 units/mL peroxidase + H202+ phenylhydrazine. (B) Potassium ferricyanide, phenylhydrazine, 1500 units/mL catalase, and/or 800 mM ethanol were added where indicated. Lane 1, control; lane 2, 30 mM K3Fe111(CN)6;lane 3, 10 mM phenylhydrazine; lane 4, 1 mM phenylhydrazine + 3 mM K3Fe111(CN)6; lane 5,lO mM phenylhydrazine + 30 " IK3Fem(CN6;lane 6,lO mM phenylhydrazine + 30 mM K3]i"en1(CN),,no piperidine treatment; lane 7,lO mM phenylhydrazme + 30 mM K3Fem(CN6+ calane 8 , l O mM phenylhydrazine + 30 mM K3Fe1'(CN), + heat-denatured catalase; lane 9,10 mM phenylhydrazine + 30 mM K3Fen1(CN), + 0.8 M ethanol. (C) Potassium ferricyanide and phenelzine were added where indicated. Lane 1 , l O mM phenelzine; lane 2 , l mM phenelzine and 3 mM K3Fem(cN),; lane 3,lO mM phenelzine and 30 mM K3Fen1(CN)@After the incubation a t 37 "C for 30 min (A) or 10 min (B, C), followed by the piperidine treatment except for lane B6, the DNA fragments were analyzed by the method described in the Figure 1 legend.

k

Phenelzine caused no or little DNA damage in the presence of peroxidase/H202(data not shown). Weak DNA damage was observed with 1mM phenylhydrazine plus 3 mM Fenr(CN)63-(Figure 3B, lane 4). At a high concentration [10 mM phenylhydrazine plus 30 mM Fe"'(CN)63-], the DNA damage was increased (lane 5). Since the effect of piperidine treatment was small, the breakage of the deoxyribose phosphate backbone seemed to occur more than base alteration and/or liberation (lane 5/lane 6). Catalase and 'OH scavenger (ethanol) did not inhibit the DNA damage (lanes 7 and 9), indicating no involvement of H202and 'OH. Slight DNA damage was observed With phenelzine plus Ffl(CN)6* (Figure 3c, lane 3) Effects of Scavengers on DNA Damage by Phenylhydrazine and Phenelzine in the Presence of Metal. Figure 4A shows the effects of scavengers on DNA damage by phenelzine plus Cu(I1). Catalase inhibited the DNA damage (lane 4), whereas 'OH scavengers (ethanol, mannitol) did not inhibit it (lanes 2 and 3). In the case of phenylhydrazine plus Cu(II), basically similar scavenger effects were observed. Ethanol and mannitol showed inhibitory effects on the DNA damage only to a small extent (Figure 4B, lanes 2 and 3), suggesting that 'OH radical participation is small, if any. The inhibition by catalase was not complete (Figure 4B, lane 4). It was reported that phenylhydrazine inhibited catalase activity (29). However, since catalase activity was not completely inhibited by phenylhydrazine under the conditions used, there remains a possibility of the minor involvement of an active species unrelated to H2OP In contrast, DNA damage by phe-

Figure 4. Effects of 'OH scavengers and bathomprohe on DNA cleavage induced by phenelzine and phenylhydrazine in the p e n c e of Cu(I1) or hemin. The reaction mixture contained the 2P-5'-end-labeled 337-base-pair fragment, 50 pM per base of sonicated calf thymus DNA, phenelzine or phenylhydrazine, 20 pM metal ion, scavenger, and 5 pM DTPA in 200 pL of 10 mM sodium phosphate buffer a t pH 7.9. After the incubation a t 37 "C for indicated durations, followed by the piperidine treatment, the DNA fragments were analyzed by the method described in the Figure 1legend. (A) 0.5 mM phenelzine + 20 pM CuC12, 10 miry (B) 1mM phenylhydrazine + 20 pM CuC12,5 min, (C) 1mM phenylhydrazine + 20 pM hemin, 30 min. Lanes 1,no scavenger; lanes 2,0.8 M ethanol; lanes 3,0.2 M mannitol; lanes 4, catalase (1500 units/mL); lanes 5, heated denatured catalase (1500 units/mL); lanes 6,50 pM bathocuproine.

nylhydrazine plus hemin was inhibited by 'OH scavengers (Figure 4C, lanes 2 and 3). The DNA damage was partially inhibited by catalase (lane 4). In the case of Fe(II1)EDTA, similar scavenger effects were observed. DNA damage by phenylhydrazine plus Mn(1II) was also inhibited by 'OH scavengers (data not shown). The addition of bathocuproine, a Cu(1)-specificchelator (Figure 4A,B, lanes 6), completely inhibited DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II), suggesting the involvement of Cu(1) in the DNA damage. Site Specificity of DNA Cleavage Induced by Phenylhydrazine and Phenelzine in the Presence of Metal. 32P-5'-End-labeledDNA fragments treated with phenylhydrazine and phenelzihe in the presence of Cu(I1) ion or Fe(II1) complexes were electrophoresed, and autoradiograms were obtained. For the measurement of relative intensity of DNA cleavage, the autoradiograms were scanned with a laser densitometer. The DNA cleavage sites were determined by reference to the cleavage position produced by the Maxam-Gilbert procedure (28). In the presence of Cu(II), phenylhydrazine (Figures 5A and 6A) and phenelzine (Figures 5B and 5C) induced DNA cleavage of the same site specificity; the predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. To clarify what active species participate in the DNA cleavage, the pattem of DNA cleavage induced by phenylhydrazine plus Cu(I1) was compared with that by H202in the presence of Cu(1) or Cu(I1). Phenylhydrazine plus Cu(I1) showed a cleavage pattem similar to that by Cu(1) plus H202 (Figure (Figure 6B) but not to that by Cu(I1) plus H202 6C). On the other hand, DNA cleavage induced by phenylhydrazine in the presence of hemin occurred at positions of every nucleotide without marked site specificity (Figure 7A). Phenylhydrazine plus Fe(II1)-EDTA gave a similar pattern of DNA cleavage (Figure 7B). DNA cleavage induced by phenylhydrazine in the presence of Fem(CN)6*occurred at positions of every nucleotide (data not shown). Production of Carbon-CenteredRadical Adducts during Copper-Catalyzed Autoxidation of Phenylhydrazine and Phenelzine. Figure 8A-1 shows an ESR

Chem. Res. Toxicol., Vol. 5, No. 3, 1992 443

DNA Damage by Phenylhydrazine

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Nucleotide Number Figure 5. Site specificity of Cu(I1)-mediated DNA cleavage induced by phenylhydrazine and phenelzine. 32P-5’-End-labeled fragment in 200 p L of 10 mM sodium phosphate buffer a t pH 7.9 containing 5 pM DTPA and 50 pM per base of sonicated calf thymus DNA was incubated with 1 mM phenylhydrazine (A) or 0.5 mM phenelzine (B, C) in the presence of 20 pM CuClz at 37 “C for the indicated durations. After the piperidine treatment, DNA fragments were electrophoresed on an 8% polyacrylamide, 8 M urea gel (18 cm X 50 cm) using a DNA-sequencing system, and the autoradiogram was obtained by exposing X-ray film to the gel. The relative amounts of oligonucleotides produced were measured by scanning the autoradiogram with a laser densitometer. Horizontal axis: The nucleotide number of human c-Haras-1 protooncogene starting with the BamHI site (27). (A) The 32P-5’-end-labeled%-basepair fragment (AuaI* 2247-PstI 2344) was used; phenylhydrazine + CuC12, 5 min. (B) The 32P-5’-endlabeled 98-base-pair fragment was used; phenelzine CuCl,, 10 min. (C) The 32P-5’-end-labeled341-base-pair fragment (XbaI 1906-AuaI* 2246) was used; phenelzine + CuClZ,10 min.

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spectrum of phenyl radical adduct (uN = 15.8 G, U H = 24.6 G) observed when DMPO was added to a buffer solution containing phenylhydrazine plus Cu(I1). The phenyl radical adduct was previously reported by Sinha (30).The formation of the adduct was not inhibited by catalase (spectrum A-2). Figure 8B-1 shows an ESR spectrum of a carbon-centered radical adduct observed when POBN was added to a buffer solution containing phenylhydrazine p l u ~Cu(I1). The signals (uN = 15.5 G , U H = 3.3 G) can be assigned to the phenyl radical adduct of POBN. The formation of the adduct was not inhibited by catalase (spectrum B-2). Signals of greater intensity were observed with phenylhydrazine plus Fe1*1(CN)63(spectrum B-3). Carbon-centered radical adducts were also observed with

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Nucleotide Number Figure 6. Comparison of site specificity of DNA cleavage induced by phenylhydrazine plus Cu(II), HzOzplus Cu(I), and HzOzplus Cu(I1). The 32P-5’-end-labeled337-base-pair fragment (PstI 2345-AuaI* 2681) in 200 p L of 20 mM sodium phosphate buffer at pH 7.9 containing 5 pM DTPA and 50 pM per base of sonicated calf thymus DNA was incubated a t 37 “C with 1 mM phenylhydrazine plus 20 pM CuClzfor 5 min (A), 0.5 mM HzOzplus 0.5 mM CuCl for 1min (B), or 1 mM HzOzplus 0.5 mM CuCl, for 10 min (C). After the piperidine treatment, DNA fragments were analyzed as described in the Figure 5 legend.

phenelzine. Figure 8C-1 shows an ESR spectrum of the carbon-centered radical adduct observed when POBN was added to a buffer solution containing phenelzine plus Cu(I1). The signals (uN = 15.7 G , U H = 2.8 G) can be assigned to the 2-phenylethyl radical adduct of POBN as reported by Ortiz de Montellano et al. (13).The formation of the adduct was not inhibited by catalase (spectrum C-2). Signals of much greater intensity were observed with phenelzine plus Fe11*(CN)63(spectrum C-3) as reported by August0 (31). Production of ‘OH during the Oxidation of Phenylhydrazine in the Presence of Hemin, Fe(II1)EDTA, or Peroxidase/H20z.Figure 9A shows *OHadduct (aN = U H = 14.8 G) and phenyl radical adduct ( U N = 15.8 G, U H = 24.6 G ) observed when DMPO was added to a buffer solution containing phenylhydrazine plus hemin. Addition of ethanol inhibited the formation of D M P M H (spectrum B). The spectrum may represent the overlap of the phenyl radical adduct and a-hydroxyethyl radical adduct (uN = 16.3 G , U H = 23.3 G ) which is thought to be produced during the reaction of ‘OH and ethanol. Man-

444 Chem. Res. Toxicol., Vol. 5, No. 3, 1992

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Nucleotide Number Figure 7. Site specificity of DNA cleavage induced by phenylhydrazine in the presence of hemin or Fe(II1)-EDTA. The 32P-5'-end-labeled 337-base-pair fragment (PstI 2345-AuaI* 2681) in 200 pL of 10 mM sodium phosphate buffer at pH 7.9 containing 5 pM DTPA and 50 pM per base of sonicated calf thymus DNA was incubated at 37 "C for 30 min with 1 mM phenylhydrazine plus 20 pM hemin (A) or 1 mM phenylhydrazine plus 20 pM Fe(II1)-EDTA (B). After the piperidine treatment, DNA fragments were analyzed as described in the Figure 5 legend.

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Figure 9. ESR spectra of the radical adducts of DMPO produced during hemin-catalyzed autoxidation of phenylhydrazine. The sample (100 pL) contained 0.5 mM phenylhydrazine, 20 pM hemin, and scavenger in 20 mM sodium phosphate buffer (pH 7.9) containing 5 pM DTPA. After the addition of 14.6 mM DMPO and incubation for 10 min at 37 OC, ESR spectra were measured as described under Experimental Procedures with a receiver gain of 1 X 1ooO. Spectrum A, no scavenger [the spectrum is assigned and 'OH to a mixture of phenyl radical adduct of DMPO (0) adduct of DMPO (a)];spectrum B, 800 mM ethanol; spectrum C, 1500 units/mL catalase.

Figure 8. ESR spectra of the radical adducts of POBN and DMPO produced by phenylhydrazine in the presence of Cu(I1) or ferricyanide. The sample (100 pL) contained phenylhydrazine (A, B) or phenelzine (C) in 20 mM sodium phosphate buffer (pH 7.9) containing 5 pM DTPA. Where indicated, CuCl,, K3Fem(cN),, and/or catalase were added. After the addition of 14.6 mM DMPO (A) or 10 mM POBN (B, C), aliquots of the solution were immediatelytaken in a calibrated capillary, and ESR spectra were measured at room temperature as described under Experimental Procedures with a receiver gain of 1 X 1000 (A), 4 X 10 (B), or 4 x 100 (C). Spectrum A-1, 0.5 mM phenylhydrazine + 20 pM CuCI,; spectrum A-2,0.5 mM phenylhydrazine + 20 pM CuCl, + 1500 units/mL catalase; spectrum B-1, 0.5 mM phenylhydrazine + 20 pM CuCl,; spectrum B-2, 0.5 mM phenylhydrazine + 20 pM CuC1, + 1500 units/mL catalase; spectrum spectrum B-3,lO mM phenylhydrazine + 30 mM K3Fe111(CN)6; C-1, 0.5 mM phenelzine + 20 pM CuC1,; spectrum C-2,0.5mM phenelzine + 20 pM CuCl, + 1500 units/mL catalase; spectrum C-3, 10 mM phenelzine + 30 mM K3Fe111(CN)6.

nitol also inhibited the formation of DMPO-OH (data not shown). Catalase inhibited the formation of DMPO-OH (spectrum C), while heat-denatured catalase did not (data not shown). Because phenylhydrazine plus hemin-induced DNA damage was inhibited by ethanol, mannitol, and catalase, the main active species causing DNA damage may

be 'OH rather than phenyl radical. The adducts of *OH and phenyl radical were also observed in the case of phenylhydrazine plus Fe(II1)-EDTA. When phenylhydrazine was incubated with peroxidase/H202 in the presence of DMPO, phenyl radical adduct (aN = 15.8 G, uH = 24.6 G) was detected (data not shown) as reported by Sinha (30). The adduct was not observed in the absence of H202.

Discussion The present results suggest that phenyl radical produced during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide causes moderate DNA damage. The small effect of piperidine treatment suggests that the breakage of the deoxyribose phosphate backbone occurs more than base alteration and/or liberation. In the case of phenelzine plus ferricyanide, slight DNA damage was observed. The sp2-hybridized phenyl radical is more active in hydrogen abstraction than sp3-hybridized 2phenylethyl radical (14). Therefore, it is considered that the abstraction of a hydrogen atom of a sugar moiety of DNA causes the backbone breakage. There were reports concerning the DNA alterations by 2-phenylethyl radical (23) and the formation of 8-methylguanine as a result of DNA alkylation by methyl radicals (24). However, the

DNA Damage by Phenylhydrazine

present results may indicate that the modification of guanine residue is a minor pathway in DNA damage induced by carbon-centered radicals, especially by phenyl radical, if the C-8 adduct of the guanine residue as well as the N-7 adduct is labile to the piperidine treatment. On the other hand, at low concentrations of phenylhydrazine or phenelzine (less than 0.5 mM), phenyl radical and 2phenylethyl radical do not seem to play an important role in the Cu(11)-,hemin-, or Fe(II1)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine, as supported by the following observations. First, catalase did not inhibit phenyl radical production by phenylhydrazine plus Cu(I1) whereas it inhibited DNA damage by phenylhydrazine plus Cu(I1). Second, phenyl radical production by phenylhydrazine in the presence of hemin or Fe(II1)EDTA was not inhibited by ethanol or mannitol which inhibited DNA damage by phenylhydrazine in the presence of hemin or Fe(II1)-EDTA. Phenylhydrazine in combination with hemin or Fe(111)-EDTA caused cleavage at every nucleotide without marked site specificity. This result suggests the involvement of 'OH, since previous study revealed that 'OH generated from Fe(II1)-NTA- or Fe(I1)-EDTA-catalyzed decomposition of H202causes DNA cleavage with such a site specificity (32,33). The DNA cleavage was inhibited completely by 'OH scavengers and partially by catalase. Consistently, in ESR spin-trapping experiments 'OH was trapped by DMPO upon the reaction of phenylhydrazine with hemin. Production of DMPO-OH was inhibited by 'OH scavengers and catalase. DMPO-OH were also observed with phenylhydrazine plus Fe(II1)-EDTA. Thus, it is considered that 'OH is generated from H202during the hemin- or Fe(II1)-EDTA-catalyzed autoxidation of phenylhydrazine and causes DNA damage. H20zmay be produced in reactions initiated by reduction of Fe(II1) by phenylhydrazine. We have reported that, in the presence of Mn(III), hydrazine and 1,2-dimethylhydrazine cause DNA damage The through an 'OH radical not formed via Hz02(25,N). present study showed that phenylhydrazine also caused Mn(II1)-mediated DNA damage. The mechanisms may be similar. In the case of phenelzine, Mn(II1)-mediated DNA damage was not observed. The obvious differences in structure-function activity noted for Mn(II1)-mediated DNA damage may be explained by in terms of oxidationreduction potentials of these hydrazines. Cu(I1) was shown to be more effective on phenylhydrazine-dependent DNA damage than Mn(111) and Fe(II1). Predominant piperidine-labile sites induced by phenylhydrazine plus Cu(I1) were found to be the thymine residue of 5'-GTC-3' sequence. A similar sequence-specific cleavage was also observed with phenelzine plus Cu(I1). In previous papers, we reported that hydrazine plus Cu(I1) (34) and methylhydrazines plus Cu(I1) (25) induce DNA cleavage at the thymine residue of 5'-GTC-3' sequence. Since 'OH scavengers have no or weak inhibitory effects on Cu(I1)-mediated DNA damage by phenelzine or phenylhydrazine, 'OH is not thought to be the main active species. The inhibitory effects of bathocuproine and catalase on phenylhydrazine plus Cu(I1)-induced DNA damage indicate that the copper-oxygen complex derived from the reaction of H202with Cu(1) may participate in the DNA damage. Cu(1) plus H202as well as Cu(I1) plus the hydrazines caused DNA cleavage at the thymine residue of 5'-GTC sequence, which was not observed with Cu(I1) plus H202 The different cleavage patterns suggest the existence of at least two different active coppel-oxygen complexes.

Chem. Res. Tonicol., Vol. 5, No. 3, 1992 445

The biological significance of copper has recently drawn much interest in connection with carcinogenicity and mutagenicity (35,36). Copper is an essential component of chromatin (37) and is known to accumulate preferentially in the heterochromatic regions (38). Abnormal copper accumulation in the liver of LEC rats developing spontaneous hepatoma has been recently demonstrated (39). Metabolites of carcinogenic benzene and o-phenylphenol, which have not been proved to be mutagenic in a bacterial test system, were shown to produce H202and cause DNA damage in the presence of Cu(I1) (40, 41). Tkeshelashvili et al. (42) reported that frequency of mutants produced by Cu(1) or Cu(I1) was equal to or greater than that produced by Fe(I1) and that the mechanism of copper-induced mutation was not simply the production of 'OH. Iron is another candidate for the activator of H202 in cellular DNA damage (43, 44). Although further research is necessary, the metal-mediated DNA damage through H20zseems to be relevant for the expression of the mutagenicity and carcinogenicity of phenylhydrazine and phenelzine.

Acknowledgment. We are grateful to Professor Masayuki Ikeda for his encouragement throughout this work. This work was supported by a research grant from the Fujiwara Foundation of Kyoto University, a Grand-in-Aid for Cancer Research from the Ministry of Health and Welfare, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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