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
AU - Vettorazzi, Ariane
AU - Azqueta, Amaya
PY - 2024/1
Y1 - 2024/1
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 MMS (200mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO , 0-4mM, 3h) 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 methyl methanesulfonate (MMS, 100-800µM, 1h) and hydrogen peroxide (H O 7.7-122.5µM, 5minutes) 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 MMS (200mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO , 0-4mM, 3h) 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 methyl methanesulfonate (MMS, 100-800µM, 1h) and hydrogen peroxide (H O 7.7-122.5µM, 5minutes) 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 - CometChip
KW - comet assay
KW - enzyme
KW - high-throughput
KW - in vitro
KW - in vivo
U2 - 10.1016/j.tox.2023.153690
DO - 10.1016/j.tox.2023.153690
M3 - Article
SN - 0300-483X
VL - 501
JO - Toxicology
JF - Toxicology
M1 - 153690
ER -