The number of DNA oxidation-induced lesions was expressed as the difference between the enzyme- and the buffer- treated gels.įor the aim of evaluating the degree of damage, comet images were scored visually using an optical microscope. For each gel treated with hOGG1 there was a gel incubated in parallel, but with buffer alone.
hOGG1 is an endonuclease that recognizes lesions 8-oxo-dGuo and creates single strand breaks at those sites. In order to examine basal levels of oxidation-induced damage in DNA, nucleoids embedded in agarose were incubated with 50 µL hOGG1 in buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, 100 µg/mL bovine serum albumin) at 0.08 U per gel, for 45 min at 37 ✬. The efficacy of DNA repair was taken as the relative difference between DNA damage immediately after challenge and after 90 min of repair. This last was done by resuspending the washed cells in RPMI 1640 medium containing 20 % fetal calf serum, and incubating the cells at 37 ✬ for 90 min, which were further placed on ice to stop DNA repair, and embedded in agarose and the comet assay run. Some of the challenged cells were washed and then embedded in agarose and run through the comet assay as described above, to measure its resistance to challenge, while some of the challenged cells were used to assess DNA repair. This was induced by exposure to 200 µmol/L hydrogen peroxide (H 2O 2, made up in PBS), for 5 min at 4 ✬. Slides were: a) fixed for 10 min in a solution containing 15 % trichloroacetic acid, 5 % zinc sulphate heptahydrate, and 5 % glycerol b) washed three times with deionized water c) placed back-to-back in a horizontal staining jar d) stained for 35 min in dark conditions with shaker using 75 mL of freshly prepared stain solution composed by 34 mL of vigorously mixed stock solution B (0.1 % ammonium nitrate, 0.1 % silver nitrate, 2.5 % tungstosilicic acid, 0.15 % formaldehyde, v/v) and 66 mL of stock solution A (5 % sodium carbonate) e) washed three times with deionized water f) immersed 5 min in a stop solution (acetic acid 1 %) and g) slides were air-dried.Ī modification of the basic alkaline comet assay was introduced to test the cells’ response and their capacity to repair after a controlled in vitro oxidative challenge. Finally, the slides were washed three times in neutralizing buffer (0.4 M Tris, pH 7.5) to remove alkali and detergents, and were stained using a silver staining protocol. After the unwinding, DNA was electrophoresed at 0.8 V/cm and 300 mA for 25 min all these steps were carried out in subdued light. No significant differences in DNA damage were observed between controls and patients (p > 0.05), but DNA repair capacity was significantly slower in NF1 patients (p 0.05), pero la capacidad de reparación del ADN fue significativamente más lenta en los pacientes con NF1 (p 13.0) at 4 ☌. In this study, the comet assay was used to evaluate levels of basal single strand breaks, H 2O 2 oxidation-induced DNA damage, and repair capacity in lymphocytes of NF1 patients compared to healthy control subjects. NF1 is characterized particularly by café-au-lait spots and fibromatous tumors of the skin.
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder which displays considerable inter- and intra-familial variability in phenotypic expression. Playa, CP 10600, La Habana, Cuba.Ģ Centro Provincial de Genética, Holguín, Cuba. Reinaldo Gutierrez 1, Judith Pupo 1, Gretel Riverón 1, Ana M González 2, Anamarys Pandolfi 1, Aimara de Armas 1, Mildrey Cásido 1, Iris Rojas 1ġ Centro Nacional de Genética Médica, CNGM. DNA damage and repair capacity in patients with neurofibromatosis type 1ĪDN dañado y capacidad de reparación en pacientes con neurofibromatosis tipo 1