Measurement of oxidative DNA damage by catechol estrogens and analogues invitro

The growth-promoting effects of estrogens in hormone-dependent tumor tissue s involve receptor-mediated pathways that are well-recognized; however, the role of estrogens in tumor initiation remains controversial. Estrogen meta bolites, primarily the catechol estrogens (CE's), have been implicated in t umor initiation via a redox cycling mechanism. We have developed metabolica lly stable CE analogues for the study of receptor versus redox cycling effe cts on DNA damage. Comparisons between hydroxy estradiols (HE2's), methoxy estradiols (ME2's), and hydroxymethyl estradiols (HME2) in potentiometric a nd DNA damaging studies were made. DNA damage was assessed in calf thymus D NA using 8-oxo-2'-deoxyguanosine (8-oxo-dG) as a genotoxic marker for oxida tive stress. Increases-in the number of 8-oxo-dG/10(5) dG were significant for each 2-HE2 and 4-HE2. Cu(II)SO4, a transition metal known to catalyze t he redox cycling of o-quinones, substantially increased the amount of DNA d amage caused by both GE's. However, DNA damage was only observed at concent rations of 10 mu M or higher, much greater than what is found under physiol ogic conditions. Furthermore, the presence of endogenous antioxidants such as glutathione, SOD,and catalase drastically reduced the amount of DNA dama ge induced by high concentrations of 2-HE2. There was no DNA damage observe d for the non-redox cycling HME2's, making these compounds useful probes in the study of receptor-mediated carcinogenesis. Thus, both 2-HE2 and 4-HE2 are capable of producing oxidative DNA damage at micromolar concentrations in vitro. However, since the amount of CE's has not been shown to surpass n anomolar levels in vivo, it is unlikely that free radical production via re dox cycling of CE's is a causative factor in human tumorigenesis.