High-throughput approaches for genotoxicity testing in drug development: recent advances

Figures & data

Table 1 Current methods for screening compounds for genotoxicity

System	Test	Advantages	Disadvantages	Adaptations for high throughput	Regulatory approval
Prokaryotic systems	Ames test	Simple yet sensitive in detecting point mutations, frame shift, and base substitutions	Laborious, use of multiple strains, screening of very few compounds at a given point of time; prokaryotic system	Adapted to a 96- and 384-well plate format, using liquid media, referred to as fluctuation test	An integral part of standardized test to assess genotoxicity of compounds
	Ara D test	More sensitive than Ames in detecting point mutations	Laborious; time-consuming; prokaryotic system	Non-high-through put	Has not received regulatory approval
	SOS ChromoTest	Simple; rapid; high sensitivity; colorimetric end point; requirement of single strain; ability to evaluate highly toxic compounds	Prokaryotic system	Exhibits potential to be transformed into a high- throughput assay	Status unknown
Eukaryotic systems	Yeast DEL assay	Accurate and shows 92% similar results in comparison to Ames; eukaryotic system	Laborious; time-consuming	Adapted to a 96- and 384-well plate format	Status unknown
	Yeast mitotic gene-conversion assay	Detection of reverse- and repair- associated mechanism, thereby with increased sensitivity	Low potential to be transformed into high throughput	Adapted to a 96- and 384-well plate format	Included in the Code of Federal Regulations of USA
	Comet assay	Detection of DNA damage; allows for differentiation between genotoxicity- and cytotoxicity-induced DNA damage; in vitro and in vivo	Inability to detect short-lived DNA lesions; low reproducibility	Adapted to a 96-well format using suspension cultures and use of ATP levels to assess viability; microarray-based comet chip	Guidelines available for both in vitro and in vivo comet assay
	Micronucleus assay	Evaluation and quantification of chromosomal damage; high sensitivity when coupled with FISH; in vitro and in vivo	Laborious, time-consuming, variability in scoring cells	Adapted to using flow cytometry by Gentronix to ensure accurate scoring; automated by Cerep	In vivo micronucleus test is one of the standard battery of tests for assessing the toxicity of compounds
	Mouse lymphoma assay	Detects point and chromosomal mutation; eukaryotic system	High false-positive rate	Adapted for high-throughput screening in 96-well plate	Mandatory for regulatory compliance
Animal models	Transgenic mice	Close resemblance to humans	Time-, labor-, and resource-intensive; expensive	Not adaptable for the high-throughput format
	Zebrafish	Share 70% homology with humans; short generation time; easy to breed and low maintenance; transparent larvae and development of larvae outside body		Emerging in vivo model for toxicity studies	Limited regulatory approval
	Drosophila melanogaster	Complementary to yeast- and bacteria-based models; easy and low-cost maintenance; high number of testable offspring; DNA-repair systems similar to humans		Promising model for testing genotoxicity for in vivo studies	Recommended by ECVAM as an appropriate model for research and testing
Biomarkers as genotoxicity evaluators	3D-DIP-Chip* (Agilent Technologies) – change in acetylation levels of H3 histone Cell Ciphr (Cellumen Inc.) CellSensor (Life Technologies) Oligo GEArray (SA Biosciences) GreenScreen HC (Gentronix) – GADD45A BlueScreen HC (Gentronix) – GADD45A Anthem’s genotoxicity screen – p21, p53 GADD153	Assess genotoxicity at the molecular level, enhancing specificity and sensitivity	Each test has a different end point; therefore, drawing conclusions by comparison of data obtained may be difficult	All have been adapted for screening multiple compounds in short periods of time

Notes: Various in vitro and in vivo models used for detection of genotoxicity, their advantages and disadvantages, ability to be adapted for HTS, and their regulatory status as an HTS approach has been outlined. *DNA-damage detection by DNA immunoprecipitation microarray chip.

Abbreviations: FISH, fluorescence in situ hybridization; ECVAM, European Centre for the Validation of Alternative Methods; HTS, high-throughput screening.