TY - JOUR

T1 - Further development of CometChip technology to measure DNA damage in vitro and in vivo

T2 - Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay

AU - Collia, Miguel

AU - Møller, Peter

AU - Langie, Sabine A S

AU - Vettorazzi, Ariane

AU - Azqueta, Amaya

N1 - Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2024

Y1 - 2024

N2 - DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0-4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100-800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7-122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

AB - DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0-4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100-800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7-122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

KW - Male

KW - Animals

KW - Rats

KW - Comet Assay/methods

KW - Rats, Wistar

KW - DNA Damage

KW - DNA Repair Enzymes

KW - Gels

U2 - 10.1016/j.tox.2023.153690

DO - 10.1016/j.tox.2023.153690

M3 - Journal article

C2 - 38040084

VL - 501

JO - Toxicology

JF - Toxicology

SN - 0300-483X

M1 - 153690

ER -