In vivo accumulation of 8-hydroxy-2'-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families

Abstract

The present study was aimed at verifying the occurrence, if any, of in vivo oxidative DNA damage in FA homozygotes, their parents and siblings. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) was measured, by HPLC/EC, in DNA from circulating blood leucocytes from FA homozygotes and their relatives and compared with a group of paediatric and adult healthy subjects. The population studied consisted of: (i) 15 FA homozygotes; (ii) 24 FA heterozygotes; (iii) 11 siblings. The 8-OHdG level in FA homozygotes was significantly higher with respect to age-matched controls, with a mean level of 33.3 ± 6.8 (mean ± SE) and 3.9 ± 0.26 8-OHdG/10⁵ dG respectively. The FA parents (heterozygotes) also displayed higher 8-OHdG levels relative to controls. The release of hydroxyl ('OH) and 'OH-like radicals from leucocytes was determined by luminol-dependent chemiluminescence (LDCL) in a subgroup of FA homo- and heterozygotes, showing a very large in vivo formation of non-superoxide radicals. Chromosomal instability was also measured in the FA population. When relating either 8-OHdG or LDCL levels to spontaneous or diepoxybutane-induced chromosomal instability (S-CI and DEB-CI respectively), a significant correlation was observed between the 8-OHdG, LDCL and S-CI data. Within families a positive association was found between 8-OHdG levels in homozygotes and their related heterozygotes, suggesting segregation of the genetic defect(s) underlying the abnormal oxidative metabolism. The present study provides evidence for an in vivo pro-oxidant state in FA, in terms of excess formation of 'OH and 'OH-like radicals, and of DNA hydroxyl adducts. This finding appears to be shared by homozygotes and, to a lesser extent, by heterozygotes.