Eosinophil peroxidase catalyzes bromination of free nucleosides and double-stranded DNA

Chronic parasitic infections are a major risk factor for cancer development in many underdeveloped countries. Oxidative damage of DNA may provide a me chanism linking these processes. Eosinophil recruitment is a hallmark of pa rasitic infections and many forms of cancer, and eosinophil peroxidase (EPO ), a secreted hemoprotein, plays a central role in oxidant production by th ese cells. However, mechanisms through which EPO may facilitate DNA oxidati on have not been fully characterized. Here, we show that EPO effectively us es plasma levels of bromide as a cosubstrate to brominate bases in nucleoti des and double-stranded DNA, forming several stable novel brominated adduct s. Products were characterized by HPLC with on-line UV spectroscopy and ele ctrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Ring assignme nts for brominated purine bases as their 8-bromo adducts were identified by NMR spectroscopy. Using stable isotope dilution LC/ESI/MS/MS, we show that while guanine is the preferred purine targeted for bromination as a free n ucleobase, 8-bromoadenine is the major purine oxidation product generated f ollowing exposure of double-stranded DNA to either HOBr or the EPO/H2O2/Br( -)system. Bromination of nucleobases was inhibited by scavengers of hypohal ous acids such as the thioether methionine, but not by a large molar excess of primary amines. Subsequently, N-monobromoamines were demonstrated to be effective brominating agents for both free nucleobases and adenine within intact DNA. A rationale for selective modification of adenine, but not guan ine, in double-stranded DNA based upon stereochemical criteria is presented . Collectively, these results suggest that specific brominated DNA bases ma y serve as novel markers for monitoring oxidative damage of DNA and the nuc leotide pool by brominating oxidants.