Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens

Dan Xu; Chun-Hua Huang; Lin-Na Xie; Bo Shao; Li Mao; Jie Shao; Balaraman Kalyanaraman; Ben-Zhan Zhu

doi:10.1093/carcin/bgz021

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens

Carcinogenesis. 2019 Sep 18;40(9):1153-1163. doi: 10.1093/carcin/bgz021.

Authors

Dan Xu^{1

2}, Chun-Hua Huang^{1

2}, Lin-Na Xie^{1

2}, Bo Shao^{1

2}, Li Mao^{1

2}, Jie Shao^{1

2}, Balaraman Kalyanaraman³, Ben-Zhan Zhu^{1

2

4}

Affiliations

¹ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, The Chinese Academy of Sciences, Beijing, P.R. China.
² University of Chinese Academy of Sciences, Beijing, P. R. China.
³ Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA.
⁴ Linus Pauling Institute, Oregon State University, Corvallis, OR, USA.

PMID: 30870561
DOI: 10.1093/carcin/bgz021

Abstract

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.