ESR evidence for the generation of reactive oxygen species from the copper-mediated oxidation of the benzene metabolite, hydroquinone: role in DNA damage

Yunbo Li, Periannan Kuppusamy, Jay L. Zweier, Michael A. Trush

Research output: Contribution to journalArticle

Abstract

In previous studies, we observed that Cu(II) strongly induces the oxidation of hydroquinone (HQ), producing benzoquinone and H2O2 through a Cu(II) Cu(I) redox cycle mechanism. The oxidation of HQ by Cu(II) also results in plasmid DNA cleavage. In this study, using ESR spectroscopy we have investigated whether this chemical-metal redox system can generate reactive oxygen species which induce DNA damage. In order to set the stage for the ESR experiments and the inhibitors to be used in these experiments, some preliminary O2 consumption and plasmid DNA cleavage experiments were performed. Mixing 100 μM HQ with 10 μM Cu(II) in phosphate-buffered saline (PBS) resulted in a marked consumption of O2 and the concomitant generation of H2O2, and extensive DNA degradation in φX-174 RF I DNA. The presence of Superoxide dismutase (SOD) or mannitol did not affect either the O2 consumption, H2O2 generation or DNA damage. In contrast, the Cu(I) chelators, bathocuproinedisulfonic acid (BCS) and glutathione (GSH), extensively inhibited the HQ/Cu(II)-mediated O2 consumption and DNA damage. The presence of catalase also prevented the DNA damage. Although the HQ/Cu(II)-mediated O2 consumption increased in the presence of azide, azide markedly inhibited the HQ/Cu(II)-induced DNA degradation, resulting in primarily open circles. Using ESR spectroscopy, it was observed that Cu(II) strongly mediated the formation of semiquinone anion radicals from HQ in PBS, which could be blocked by BCS. α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)-spin trapping experiments showed that the interaction of HQ with Cu(II) produced 4-POBN-CH3 and 4-POBN-CH(OH)CH3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting that hydroxyl radical or an equivalent reactive intermediate is generated from the HQ/Cu(II) system. The presence of catalase, BCS or GSH but not SOD completely prevented the formation of 4-POBN-CH3 adduct from the HQ/Cu(II) plus 4-POBN/DMSO system. This indicates that both H2O2 and Cu(I) are critical for the formation of reactive oxygen from the HQ/Cu(II) system. Anaerobic conditions induced an ∼ 85% decrease in the formation of 4-POBN-CH3 adduct. Reactive oxygen scavenger experiments showed that the formation of the 4-POBN-CH3 adduct was significantly inhibited by azide but not by mannitol. Overall, the above results indicate that through a copper-redox cycling mechanism the copper-mediated oxidation of HQ generates reactive oxygen species which may participate in DNA damage.

Original languageEnglish (US)
Pages (from-to)101-120
Number of pages20
JournalChemico-Biological Interactions
Volume94
Issue number2
DOIs
StatePublished - 1995

Fingerprint

Metabolites
Benzene
DNA Damage
Paramagnetic resonance
Copper
Reactive Oxygen Species
Oxidation
DNA
Oxides
Azides
Oxidation-Reduction
DNA Cleavage
Mannitol
Dimethyl Sulfoxide
Experiments
Catalase
Superoxide Dismutase
Acids
hydroquinone
Spectrum Analysis

Keywords

  • Copper
  • DNA
  • Electron spin resonance
  • Hydroquinone
  • Reactive oxygen
  • Spin trapping

ASJC Scopus subject areas

  • Toxicology

Cite this

ESR evidence for the generation of reactive oxygen species from the copper-mediated oxidation of the benzene metabolite, hydroquinone : role in DNA damage. / Li, Yunbo; Kuppusamy, Periannan; Zweier, Jay L.; Trush, Michael A.

In: Chemico-Biological Interactions, Vol. 94, No. 2, 1995, p. 101-120.

Research output: Contribution to journalArticle

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abstract = "In previous studies, we observed that Cu(II) strongly induces the oxidation of hydroquinone (HQ), producing benzoquinone and H2O2 through a Cu(II) Cu(I) redox cycle mechanism. The oxidation of HQ by Cu(II) also results in plasmid DNA cleavage. In this study, using ESR spectroscopy we have investigated whether this chemical-metal redox system can generate reactive oxygen species which induce DNA damage. In order to set the stage for the ESR experiments and the inhibitors to be used in these experiments, some preliminary O2 consumption and plasmid DNA cleavage experiments were performed. Mixing 100 μM HQ with 10 μM Cu(II) in phosphate-buffered saline (PBS) resulted in a marked consumption of O2 and the concomitant generation of H2O2, and extensive DNA degradation in φX-174 RF I DNA. The presence of Superoxide dismutase (SOD) or mannitol did not affect either the O2 consumption, H2O2 generation or DNA damage. In contrast, the Cu(I) chelators, bathocuproinedisulfonic acid (BCS) and glutathione (GSH), extensively inhibited the HQ/Cu(II)-mediated O2 consumption and DNA damage. The presence of catalase also prevented the DNA damage. Although the HQ/Cu(II)-mediated O2 consumption increased in the presence of azide, azide markedly inhibited the HQ/Cu(II)-induced DNA degradation, resulting in primarily open circles. Using ESR spectroscopy, it was observed that Cu(II) strongly mediated the formation of semiquinone anion radicals from HQ in PBS, which could be blocked by BCS. α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)-spin trapping experiments showed that the interaction of HQ with Cu(II) produced 4-POBN-CH3 and 4-POBN-CH(OH)CH3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting that hydroxyl radical or an equivalent reactive intermediate is generated from the HQ/Cu(II) system. The presence of catalase, BCS or GSH but not SOD completely prevented the formation of 4-POBN-CH3 adduct from the HQ/Cu(II) plus 4-POBN/DMSO system. This indicates that both H2O2 and Cu(I) are critical for the formation of reactive oxygen from the HQ/Cu(II) system. Anaerobic conditions induced an ∼ 85{\%} decrease in the formation of 4-POBN-CH3 adduct. Reactive oxygen scavenger experiments showed that the formation of the 4-POBN-CH3 adduct was significantly inhibited by azide but not by mannitol. Overall, the above results indicate that through a copper-redox cycling mechanism the copper-mediated oxidation of HQ generates reactive oxygen species which may participate in DNA damage.",
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N2 - In previous studies, we observed that Cu(II) strongly induces the oxidation of hydroquinone (HQ), producing benzoquinone and H2O2 through a Cu(II) Cu(I) redox cycle mechanism. The oxidation of HQ by Cu(II) also results in plasmid DNA cleavage. In this study, using ESR spectroscopy we have investigated whether this chemical-metal redox system can generate reactive oxygen species which induce DNA damage. In order to set the stage for the ESR experiments and the inhibitors to be used in these experiments, some preliminary O2 consumption and plasmid DNA cleavage experiments were performed. Mixing 100 μM HQ with 10 μM Cu(II) in phosphate-buffered saline (PBS) resulted in a marked consumption of O2 and the concomitant generation of H2O2, and extensive DNA degradation in φX-174 RF I DNA. The presence of Superoxide dismutase (SOD) or mannitol did not affect either the O2 consumption, H2O2 generation or DNA damage. In contrast, the Cu(I) chelators, bathocuproinedisulfonic acid (BCS) and glutathione (GSH), extensively inhibited the HQ/Cu(II)-mediated O2 consumption and DNA damage. The presence of catalase also prevented the DNA damage. Although the HQ/Cu(II)-mediated O2 consumption increased in the presence of azide, azide markedly inhibited the HQ/Cu(II)-induced DNA degradation, resulting in primarily open circles. Using ESR spectroscopy, it was observed that Cu(II) strongly mediated the formation of semiquinone anion radicals from HQ in PBS, which could be blocked by BCS. α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)-spin trapping experiments showed that the interaction of HQ with Cu(II) produced 4-POBN-CH3 and 4-POBN-CH(OH)CH3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting that hydroxyl radical or an equivalent reactive intermediate is generated from the HQ/Cu(II) system. The presence of catalase, BCS or GSH but not SOD completely prevented the formation of 4-POBN-CH3 adduct from the HQ/Cu(II) plus 4-POBN/DMSO system. This indicates that both H2O2 and Cu(I) are critical for the formation of reactive oxygen from the HQ/Cu(II) system. Anaerobic conditions induced an ∼ 85% decrease in the formation of 4-POBN-CH3 adduct. Reactive oxygen scavenger experiments showed that the formation of the 4-POBN-CH3 adduct was significantly inhibited by azide but not by mannitol. Overall, the above results indicate that through a copper-redox cycling mechanism the copper-mediated oxidation of HQ generates reactive oxygen species which may participate in DNA damage.

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KW - Copper

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KW - Electron spin resonance

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KW - Reactive oxygen

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