Synergistic Induction of DNA Strand Breakage Caused by Nitric Oxide

by Nitric Oxide Together with Catecholamine: Implications for Neurodegenerative Disease. Yumiko Yoshie† and Hiroshi Ohshima*. Unit of Endogenous Can...
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Chem. Res. Toxicol. 1997, 10, 1015-1022

1015

Synergistic Induction of DNA Strand Breakage Caused by Nitric Oxide Together with Catecholamine: Implications for Neurodegenerative Disease Yumiko Yoshie† and Hiroshi Ohshima* Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, Cedex 08, France Received February 19, 1997X

Oxidative damage in neuronal cells and DNA has been implicated in the pathogenesis of various neurodegenerative diseases. We have demonstrated that DNA strand breakage is induced synergistically when plasmid DNA is incubated in the presence of both an NO-releasing compound (diethylamine NONOate, spermine NONOate, sodium nitroprusside) and a catecholamine (e.g., L-DOPA, dopamine, etc.). Either an NO-releasing compound or a catecholamine alone induced much fewer strand breaks. Tyrosine and tyramine as well as O-methylated derivatives of DOPA and dopamines did not exert this synergistic effect in the presence of NO. The DNA strand breakage induced by NO plus dopamine was inhibited by carboxy-PTIO (a trapping agent of NO and possibly other radicals), superoxide dismutase, and antioxidants such as N-acetylcysteine and ascorbate but not by HO• scavengers such as dimethyl sulfoxide, ethanol, and D-mannitol. These results suggest that the free HO• is not involved; rather a new oxidant(s) formed by the reaction between NO and catecholamine could be responsible for causing the DNA strand breakage. We propose that one of the responsible compounds is peroxynitrite (ONOO-), which is a strong oxidant and nitrating agent formed by the reaction between NO and O2•-. NO has been shown to oxidize catecholamines to form quinone derivatives, which lead to the generation of O2•- by the quinone/hydroquinone redox system. O2•- then reacts rapidly with NO to form peroxynitrite. However, it is also possible that other compounds such as NOx generated from catecholamines and NO may cause DNA damage. Our results implicate a synergistic interaction of catecholamines formed in dopaminergic neurons and NO formed by microglia or astrocytes or the two compounds produced within the same neuronal cells to produce a potent oxidant(s) which could cause damage in cells and DNA, thus playing an important role in the pathogenesis of various neurodegenerative diseases.

Introduction Oxidative damage in neuronal cells and DNA has been implicated in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and prion diseases (bovine spongiform encephalopathy, scrapies, and CreuzfeldtJakob disease), as well as in the etiology of brain cancer. There are several indications for this increased oxidative stress in neurodegenerative diseases, including increased iron levels, increased lipid peroxidation, increased nitrite [a marker for nitric oxide (NO) production] concentrations, decreased peroxidase and catalase levels, increased superoxide dismutase (SOD)1 levels, and decreased glutathione levels (1-4). Oxidative damage/stress may induce in neuronal cells apoptosis and/or necrosis, which have been proposed to play a crucial role in neurodegenerative disease (5, 6). * To whom correspondence should be addressed. Tel: (33) 4 72 73 84 85. Fax: (33) 4 72 73 85 75. E-mail: [email protected]. † Visiting scientist from Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108, Japan. X Abstract published in Advance ACS Abstracts, August 15, 1997. 1 Abbreviations: carboxy-PTIO, 2-(4-carboxyphenyl)-4,5-dihydro4,4,5,5-tetramethyl-1H-imidazol-1-yloxy, 3-oxide, potassium salt; DEANO, diethylamine NONOate; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; nNOS, neuronal NO synthase; SNP, sodium nitroprusside; SOD, superoxide dismutase; SPER-NO, spermine NONOate; SSB, single-strand breaks.

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NO, produced from L-arginine by three distinct isoforms of NO synthase, plays various important roles in the central nervous system (7, 8). NO, a freely diffusible gas with free radical properties, functions as a neurotransmitter and participates in synaptic plasticity such as long-term potentiation in the hippocampus and longterm depression in the cerebellum (7, 8). Excess NO, however, could be also responsible for neurotoxicity associated with NMDA-receptor activation (9), ischemiareperfusion (10), and cold-induced brain edema (11), although the involvement of NO in neuronal injury and death remains controversial. 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson’s disease. Recent studies have shown that inhibition of neuronal NO synthase (nNOS) by a specific inhibitor (7-nitroindazole) significantly inhibited MPTPinduced neurotoxicity in mice (12, 13) and baboons (14). Mice lacking nNOS have also been shown to be significantly more resistant to MPTP-induced neurotoxicity than wild-type mice (13). The NO-mediated tissue injury may be mainly caused by the reaction with superoxide (O2•-) to form peroxynitrite (15, 16). Peroxynitrite is a strong oxidant and nitrating agent, which oxidizes sulfhydryl groups, induces lipid peroxidation and nitrates tyrosine residues in proteins to form 3-nitrotyrosine (15, 16). Peroxynitrite also induces DNA strand breaks (1719) and oxidative damage in isolated DNA in vitro (20). © 1997 American Chemical Society

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Peroxynitrite reacts rapidly with guanine and 2′-deoxyguanosine to form the adducts 8-nitroguanine (21, 22) and 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-deoxyguanosine (23), respectively. Cells treated with NO (24, 25) or peroxynitrite (26, 27) undergo apoptosis. Catecholamines, including the neurotransmitter dopamine and its precursor L-DOPA, have been implicated as sources of reactive oxygen species (28-30). Dopamine and L-DOPA can easily autoxidize, especially in the presence of transition metals such as iron and copper, to generate reactive oxygen species, including O2•-, hydrogen peroxide (H2O2), and hydroxyl radical (HO•) (31). Furthermore, quinone derivatives formed by oxidation of catecholamines are also considered to mediate toxicity by covalently binding to sulfhydryl or other nucleophilic groups of biological macromolecules in the cell (28-30). We have recently found that concurrent incubation of plasmid DNA with an NO-releasing compound and a polyhydroxyaromatic compound such as catechol or 1,4hydroquinone leads to a synergistic induction of DNA strand breaks (32). This DNA breakage is inhibited by SOD and NO-trapping agents such as carboxy-PTIO or oxyhemoglobin, suggesting that the simultaneous presence of both NO and O2•- is required to exert this synergistic effect on the DNA damage. As catechol is structurally similar to biologically important catecholamines such as L-DOPA, dopamine, and epinephrine, we have examined the effects of combinations of catecholamines and NO, both formed in the central nervous system, on the induction of DNA strand breakage. We report here that catecholamines and NO act synergistically to induce more deleterious effects on DNA.

Materials and Methods Chemicals and Biochemical Reagents. Diethylamine NONOate (DEA-NO), spermine NONOate (SPER-NO), and 2-(4carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1yloxy 3-oxide, potassium salt (carboxy-PTIO) were obtained from Cayman Chemical Co. (Ann Arbor, MI). Catecholamines such as L-DOPA, 6-hydroxy-DOPA, (-)-epinephrine, and 3-O-methylDOPA (3-methoxytyrosine) were purchased from Sigma Chemical Co. (St. Louis, MO). 6-Hydroxydopamine and DL-norepinephrine were purchased from Aldrich (Milwaukee, WI) and Fluka Chemie AG (Buchs, Switzerland), respectively. Superoxide dismutase (SOD), catalase, dimethyl sulfoxide (DMSO), and sodium nitroprusside (SNP) were obtained from Sigma. Xanthine oxidase from cow milk was purchased from Boehringer Mannheim (Mannheim, Germany). The plasmid pBR322 and desferrioxamine were from Pharmacia (Uppsala, Sweden) and Ciba-Geigy Laboratoire (Rueil-Malmaison, France), respectively. 6-Nitrodopamine was synthesized by bubbling NO gas (Alphagas, Lyon, France) slowly into 0.5 M sodium phosphate buffer, pH 7.5, containing dopamine, as described previously (33, 34). All other chemicals were commercially available. Reactions of Plasmid DNA with NO-Releasing Compounds and Catecholamines. The experiments were carried out by incubating plasmid pBR322 DNA (100 ng) in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM diethylenetriaminepentaacetic acid (DTPA) and either 0.1 mM NOreleasing compound (SNP, DEA-NO, SPER-NO) alone, 0.1 mM catecholamine [dopamine, L-DOPA, 3-O-methyl-DOPA (3-methoxytyrosine), 6-hydroxy-DOPA, etc.] alone, or these two compounds in combination at 37 °C for 1 h (final volume 10 µL). For comparison, the experiments were also performed by incubating plasmid pBR322 DNA in the presence of 5 mU/mL xanthine oxidase plus 0.5 mM hypoxanthine [which catalyzed under our conditions the formation of approximately 1.5 nmol/ min O2•-, determined spectrophotometrically by measuring cytochrome c reduction at 550 nm (35)] with or without 0.1 mM

Yoshie and Ohshima NO-releasing compound. The effects of various concentrations of dopamine and DEA-NO or SPER-NO on strand breakage were studied in 10 µL of 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA, either DEA-NO or SPER-NO (0-1 mM), and dopamine (0-1 mM). Similarly, the effects of pH on strand breakage induced by 0.1 mM dopamine and 0.1 mM DEA-NO were studied using 100 mM sodium phosphate buffers (final pH 4.5, 6.0, 7.4, 9.0, and 10.5) containing 0.1 mM DTPA. The acidic (pH 4.5) and alkaline (pH 9.0 and 10.5) buffers were prepared by adjusting pH with phosphoric acid or NaOH. Effects of SOD, Catalase, Scavengers of NO and HO• Radicals, Antioxidants, and a Chelating Agent on NO plus Dopamine-Induced DNA Strand Breakage. The effects of SOD and catalase (500 or 5000 U/mL) on DNA strand breakage were studied by incubating plasmid pBR322 DNA (100 ng) in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA and 0.1 mM DEA-NO plus 1 mM dopamine at 37 °C for 1 h. Similarly the NO-trapping agents (carboxyl-PTIO), HO• scavengers (DMSO, ethanol, D-mannitol), antioxidants (ascorbic acid, N-acetylcysteine), and other compounds (sodium azide, desferrioxamine) were examined at 1 and 10 mM, except for uric acid (0.2 mM). Analysis of DNA Single-Strand Breaks. The conversion of the covalently closed circular double-stranded supercoiled DNA (form I) to a relaxed open circle form (form II) and a linear form (form III) was used to investigate DNA strand breakage induced by NO plus catecholamine, according to the method of Yermilov et al. (36). Percentages of supercoiled (form I), relaxed (form II), and linear (form III) forms were calculated by an Imaging densitometer Model GS-670 (BIO-RAD, Hercules, CA). From these values, the average number of single-strand breaks (SSB) per pBR322 DNA molecule was calculated according to the method of Epe et al. (37, 38), taking into account that the relaxed form (form II) when stained with ethidium bromide gives fluorescence intensity 1.4-fold higher than the supercoiled form (form I) and that a relaxation is caused by 1 SSB/DNA molecule. Results (mean ( SD) are expressed as numbers of SSB per 104 bp (pBR322 consists of 4363 bp) after correcting for the numbers of SSB in untreated pBR322 plasmid, which contained 5-15% form II (corresponding to 0.08-0.27 sites/104) but no detectable form III. All experiments were carried out in triplicate, and statistical significance was calculated using Student’s t-test. Analyses of Dopamine NO Reaction Products by UVVis Absorption Spectroscopy and High-Performance Liquid Chromatography (HPLC). The absorption spectrum of dopamine was recorded using a Uvikon 922 spectrophotometer (Kontron Instrument, Saint Quentin Yvelines, France) between 500 and 230 nm. Dopamine (0.1 mM) was incubated in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA and 0.05-1 mM DEA-NO at room temperature (∼26 °C) for up to 60 min, and the absorption spectra were recorded at 15 min intervals. Concentrations of DEA-NO and SPER-NO were determined by a UV spectrophotometer as reported previously (39). For comparison, dopamine was incubated at room temperature in 100 mM sodium phosphate buffer (pH 7.4) containing 0.05 mM DTPA and 0.1 mM Cu2SO4 or in 100 mM sodium phosphate containing 0.1 mM DTPA and NaOH (pH 10.6). The reaction products were analyzed by HPLC as described previously (33), using a Spectraphysics HPLC (Model SP 8800) equipped with a Nucleosil C18 column (250 × 4.6-mm, 10 µm; Interchrom, France) under isocratic conditions with 50 mM citric acid buffer (pH 3.0) at a flow rate of 1 mL/min. Dopamine and its oxidized/nitrated products were detected by UV spectrophotometry (Spectra Series UV 100, thermoseparation) at 280 and 360 nm. Under these conditions, retention times of dopamine, dopaquinone, and 6-nitrodopamine were 5.7, 15.1, and 17.8 min, respectively. Dopamine and dopaquinone were detected at 280 nm; 6-nitrodopamine was detected at both 280 and 360 nm. Similar experiments were also carried out with 0.1 mM epinephrine and 0.05-1 mM DEA-NO, and the reaction products were analyzed by UV-Vis spectrophotometry and HPLC.

DNA Strand Breaks Induced by Catecholamine and NO

Figure 1. Typical agarose gel electrophoresis of pBR322 plasmid DNA incubated with either an NO-releasing compound (DEA-NO) alone, a catecholamine (L-DOPA, dopamine, 6-hydroxydopamine, 3-O-methyl-DOPA) alone, or the two compounds in combination. The experiments were carried out by incubating pBR322 plasmid DNA (100 ng) in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA and either 0.1 mM DEA-NO alone (lane 2), 0.1 mM catecholamine (lanes 3-6), or the two compounds in combination (lanes 7-10) at 37 °C for 1 h (final volume 10 µL).

Results Results obtained from a typical agarose gel electrophoresis of pBR322 plasmid DNA which was incubated with either a catecholamine (L-DOPA, dopamine, 6-hydroxydopamine, 3-O-methyl-DOPA) alone, an NO-releasing compound (DEA-NO) alone, or the NO-releasing compound and catecholamine in combination are shown in Figure 1. Incubation of the plasmid DNA with L-DOPA and dopamine resulted in a small increase in conversion of the covalently closed circular doublestranded supercoiled DNA (form I) to a relaxed open circle (form II) (lanes 3, 4), compared to that of the nontreated plasmid (lane 1). 6-Hydroxydopamine alone induced about 37% of form II, corresponding to ∼0.63 SSB/104 bp (lane 5). DEA-NO at 0.1 mM did not increase the formation of form II significantly (lane 2). However, when the plasmid was incubated in the presence of both DEA-NO and a catecholamine (L-DOPA, dopamine, 6-hydroxydopamine), significant conversion of form I to form

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II was observed (lanes 7-9). On the other hand, 3-Omethyl-DOPA did not induce strand breakage in either the absence or presence of NO (lanes 6, 10). Similar synergistic effects on the induction of singlestrand breakage were observed with various catecholamines including L-DOPA, dopamine, epinephrine, and norepinephrine in combination with NO-releasing compounds such as DEA-NO, SPER-NO, and SNP (Table 1). On the other hand, no obvious DNA damage was induced by 3-O-methyl-DOPA, 3-O-methyldopamine, tyrosine, or tyramine in the presence of any NO-releasing compounds, in contrast to the above catecholamines (Table 1). Among various catecholamines tested, 6-hydroxy-DOPA and 6-hydroxydopamine in the presence of NO-releasing compounds induced SSB most strongly (2.0-4.4 SSB/104 bp). Combination of 6-hydroxy-DOPA and NO also induced formation of a linear form (form III) in addition to relaxed open circular (form II) DNA, indicating that these compounds caused double-strand breaks in DNA. For comparison, we examined DNA strand breakage induced by O2•- generated by xanthine oxidase and hypoxanthine in the absence or presence of NO-releasing compounds. Under the present conditions, it was found that about 1.5 nmol/min O2•- was generated by 5 mU/ mL xanthine oxidase and 0.5 mM hypoxanthine. A significant induction of SSB (0.72-2.72 SSB/104 bp) was observed only in the presence of NO-releasing compounds (Table 1). As shown in Figure 2A, dopamine and DEA-NO induced SSB very rapidly (150 times) than those of other catecholamines such as dopamine (28). It is therefore possible that 6-hydroxy-DOPA and 6-hydroxydopamine generate greater amounts of O2•-, which reacts with NO to generate increased levels of peroxynitrite, resulting in stronger induction of SSB than other catecholamines. On the other hand, although the DNA strand breakage caused by peroxynitrite is markedly inhibited by desferrioxamine and uric acid (32), these compounds were less effective against the strand breakage induced by NO and dopamine (Figure 5A). These results suggest that, in addition to peroxynitrite, the reaction between catecholamines and NO may also yield other types of compounds, which could cause DNA damage directly. One possible mechanism could be a reaction between semiquinone radical and NO resulting in the formation of semiquinone-NO adduct(s), which may induce strand breakage by releasing NOx. On the other hand, it has been reported that the reaction of various catecholamines with NO gas yields 6-nitro derivatives (33, 34), which may cause DNA damage. Our data, however, show that no detectable levels of 6-nitro derivatives are formed under our experimental conditions using NO-releasing compounds. Therefore it is unlikely that 6-nitrocatecholamines are involved in DNA damage. Catecholamines and NO have been separately postulated to play a role in the pathogenesis of various neurodegenerative diseases (see Introduction). However, it has never been considered that these two compounds act synergistically to cause cellular and/or DNA damage. Many recent studies have demonstrated that interaction between NO and catecholamines may occur in vivo. For example, NO has been implicated in regulation of the release of neurotransmitters such as norepinephrine (44, 45) and dopamine (46, 47) in vivo. More direct evidence is also accumulating, including the identification in the mammalian brain (48) of 6-nitroepinephrine, the reaction product between NO and epinephrine (33, 34). Increased release of both dopamine and NO has been also shown to occur during neuronal damage caused by cerebral ischemia-reperfusion (10, 49, 50). Under physiological and pathological conditions, NO is synthesized by three distinct isoforms of NO synthase, i.e., constitutive neuronal, constitutive endothelial, and inducible types of NO synthase. All of these isoforms play important roles in the central nervous system (7, 8). Heme-NO complexes in human substantia nigra (51) as well as increased concentrations of nitrite, an in vivo oxidized product of NO, in cerebrospinal fluids (4) have been reported in patients with Parkinson’s disease. Excess NO could be also responsible for neurotoxicity associated with NMDAreceptor activation (9) and cold-induced brain edema (11). Thus, the interaction between catecholamines and NO may occur under a variety of pathophysiological conditions and may result in the production of a new oxidant(s), including peroxynitrite, which may induce cellular and/or DNA damage. In conclusion, we have demonstrated that DNA strand breakage is induced synergistically by NO and catecholamines. Our results imply that catecholamines formed in dopaminergic neurons and NO formed by microglia or astrocytes or the two compounds produced within the

DNA Strand Breaks Induced by Catecholamine and NO

same neuronal cells may interact synergistically to produce a potent oxidant(s) which could cause damage in cells and DNA, thus playing an important role in the pathogenesis of various neurodegenerative diseases.

Acknowledgment. The authors thank Dr. J. Cheney for editing the manuscript, Mr. S. Auriol for technical assistance, and Mrs. P. Collard for secretarial work. Y.Y. is a recipient of a fellowship from the Japan Society for the Promotion of Science.

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