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

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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.

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