Copper redox-dependent activation of 2-tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

Yunbo Li, Andrew Seacat, Periannan Kuppusamy, Jay L. Zweier, James D. Yager, Michael A. Trush

Research output: Contribution to journalArticlepeer-review


The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2-tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O2 and the concomitant generation of H2O2. Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O2 and the generation of H2O2 and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide-N-tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH3 and POBN-CH(OH)CH3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH3 adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H2O2 with Cu(I). Incubation of supercoiled φX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.

Original languageEnglish (US)
Pages (from-to)123-133
Number of pages11
JournalMutation Research - Genetic Toxicology and Environmental Mutagenesis
Issue number2
StatePublished - Jul 25 2002
Externally publishedYes


  • 2-tert-Butyl(1,4)hydroquinone
  • 8-Hydroxy-2′-deoxyguanosine
  • Copper
  • DNA strand breaks
  • Hepatocytes
  • Reactive oxygen species

ASJC Scopus subject areas

  • Genetics
  • Health, Toxicology and Mutagenesis


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