Double-stranded DNA breaks are serious lesions that contribute to the pathogenesis of cancer and chronic illnesses. Free radical injury is an important mechanism for the production of DNA modifications that eventually lead to chromosomal breaks. The generation of free radicals is increased in sickle cell disease (SCD) owing to the presence of the relatively unstable HbS and recurrent ischemia-reperfusion events. In the present study, a sensitive reticulocyte micronucleus assay was used to determine the frequency of chromosomal breaks in patients with SCD (

Offer et al. FASEB J. 2005;19:485
). A micronucleus is a piece of a chromosome left behind in a reticulocyte as a consequence of DNA double-stranded break after the nucleus is extruded. Blood samples were obtained from patients with SCD (n=33) and healthy volunteers (n=23). After immunomagnetic enrichment of CD71+ reticulocytes (Trf-Ret), RNA was removed with RNAse, and the cells were stained for miconuclei using the DNA dye 7-aminoactinomycin D (7-AAD) followed by flow cytometric evaluation. The appearance of reactive oxygen species in cytosol and membrane lipid oxidation in RBCs were measured by flow cytometry using the fluorescent markers 2, 7-dichlorofluorescin diacetate (DCF) and C11-BODIPY, respectively. The frequency of micronucleated Trf-Ret was significantly higher (p<.0001) in patients with SCD (mean 3.061/10,000 Trf-Ret, range 0.256 to 9.779) as compared with normal controls (mean 0.750/10,000 Trf-Ret, range 0.043 to 2.659). Mean DCF (125.1%) and C11-BODIPY (117.6%) fluorescence intensities were significantly greater than the mean control value (100%, p 0.0001). Our data indicate that patients with sickle cell disease have increased double stranded DNA-breaks compared with healthy controls, and suggest that elevated production of oxidants could contribute to the development of genetic damage in these patients.

Topics:
chromosomes, erythrocytes, micronucleus, sickle cell anemia, dna, free radicals, cancer, chronic disease, dyes, endoribonucleases

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2005, The American Society of Hematology
2005
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