Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay.

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.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

2-Phenoxy-3-Trichloromethylquinoxalines Are Antiplasmodial Derivatives with Activity against the Apicoplast of Plasmodium falciparum.

The malaria parasite harbors a relict plastid called the apicoplast. Although not photosynthetic, the apicoplast retains unusual, non-mammalian metabolic pathways that are essential to the parasite, opening up a new perspective for the development of novel antimalarials which display a new mechanism of action. Based on the previous antiplasmodial hit-molecules identified in the 2-trichloromethylquinoxaline series, we report herein a structure-activity relationship (SAR) study at position two of the quinoxaline ring by synthesizing 20 new compounds. The biological evaluation highlighted a hit compound (3i) with a potent PfK1 EC50 value of 0.2 µM and a HepG2 CC50 value of 32 µM (Selectivity index = 160). Nitro-containing (3i) was not genotoxic, both in the Ames test and in vitro comet assay. Activity cliffs were observed when the 2-CCl3 group was replaced, showing that it played a key role in the antiplasmodial activity. Investigation of the mechanism of action showed that 3i presents a drug response by targeting the apicoplast and a quick-killing mechanism acting on another target site.