Methodology for Evaluating Oxidative DNA Damage and Metabolic Genotypes in Human Trabecular Meshwork

Abstract

In vitro studies have suggested the possible role of oxidative DNA damage in degenerative eye diseases such as glaucoma. We propose a method aimed at evaluating the oxidative molecular damage directly in the human trabecular meshwork (HTM) collected during surgery from patients affected by glaucoma. In the same DNA samples, we evaluated two genes involved in the cellular defense against oxidative stress, the glutathione S -transferase-encoding genes GSTM1 and GSTT1. DNA was extracted, using a high-performance phenol/chloroform procedure, from the HTM collected during surgery from nine glaucoma patients and five controls. Oxidative DNA damage was evaluated by determining the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by means of 32 P postlabeling, thin-layer chromatography, and electronic autoradiography. GSTM1 and GSTT1 polymorphisms were determined by polymerase chain reaction (PCR) and agarose electrophoresis. Sufficient DNA amounts were obtained from all examined specimens. 8-OHdG was detected in all samples, with a level of 4.0 ± 6.5 (mean ± SD) 8-OHdG molecules/10 5 normal nucleotides in the glaucoma patients and a level of 2.6 ± 2.2 in the controls. These results were obtained by using DNA amounts as low as 0.11 μg. The genotype status of GSTM1 and GSTT1 was successfully determined in all patients by analyzing an aliquot of the same DNA used for the 8-OHdG evaluation. This method allows for the use of samples collected from living subjects during ocular surgery in order to study the role of oxidative DNA damage in the pathogenesis of degenerative eye diseases such as glaucoma.

Keywords: :

• Glaucoma
• Glutathione
• Human Trabecular Meshwork
• 8-Hydroxy-2′-deoxyguanosine