hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII

Abstract

The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to detect oxidative DNA damage. In the present study, FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for the ability to modify the sensitivity of the comet assay. Mouse lymphoma L5178Y cells were treated with dimethyl sulphoxide (DMSO) as a standard solvent or reference agents known to induce oxidative damage (gamma irradiation and potassium bromate) or alkylation (methyl methanesulfonate, MMS; ethylnitrosurea, ENU). Using DMSO even up to toxic concentrations, no increase in breaks was seen with FPG, ENDOIII or hOGG1. With gamma irradiation (1-10 Gy), dose-related increases in breaks were seen with all three enzymes. FPG and hOGG1 gave similar increases in breaks after potassium bromate treatment between 0.25 and 2.5 mmol/l, but ENDOIII showed an increase only at the highest concentration, 2.5 mmol/l. Following MMS treatment (5-23 micromol/l), FPG induced a dramatic increase in breaks compared with control levels and ENDOIII also showed a significant but smaller increase; in marked contrast, hOGG1 gave no increase. With ENU (0.5-2.0 mmol/l), increases in breaks were seen with FPG and ENDOIII at 1 and 2 mmol/l but, again, no increase was observed with hOGG1. These data indicate that all three endonucleases recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage. Therefore, caution should be taken when using FPG and ENDOIII in the comet assay with an agent that has an unknown mode of action since any additional strand breaks induced by either enzyme cannot necessarily be ascribed to oxidative damage. The use of hOGG1 in the modified comet assay offers a useful alternative to FPG and is apparently more specific for 8-oxoguanine and methyl-fapy-guanine.

Transcription factor NRF2 protects mice against dietary iron-induced liver injury by preventing hepatocytic cell death

“…DNA damage was measured using the alkaline comet assay as human 8-hydroxyguanine DNA glycosylase 1 (hOGG1)-sensitive sites. hOGG1 is considered to detect 8-OHdG in cell culture experiments with great specificity [14]. Fig.…”

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

“…For this modified assay, treatment with the enzymes precedes the unwinding step. The addition of lesion-specific endonucleases increases the sensitivity of the comet assay as well 13 .…”

Antineoplastic Drugs as a Potential Risk Factor in Occupational Settings: Mechanisms of Action at the Cell Level, Genotoxic Effects, and Their Detection Using Different Biomarkers

“…FPG je specifi čna za otkrivanje oksidiranih purina: 8-okso-7,8-dihidrogvanin, 2,6-diamino-4-hidroksi-5-formamidopirimidin i 4,6-diamino-5-formamidopirimidin te drugih purina koji imaju otvorene prstenove. ENDOIII prepoznaje oksidirane pirimidine kao što su timin glikol i uracil glikol (183,184). Nedostatak FPG-modifi ciranog komet-testa je što osim oksidiranih purina otkriva i AP-mjesta, različite purine s otvorenim prstenovima te adukte u molekuli DNA.…”

“…Enzim hOGG1 može popraviti oksidirane baze samo kad su sparene s citozinom pa je puno specifi čniji od FPGenzima koji prepoznaje 8-oksogvanin kad je sparen s citozinom, gvaninom ili timidinom. hOGG1 je strukturno srodan s ENDOIII, a funkcionalno srodan s FPG; dakle posjeduje AP-litičku aktivnost, iako slabiju od FPG, zajedno s aktivnosti glikozilaze (184). Usporedbe i prednosti navedenih modifi kacija opisane su na tablici 5.…”