GreenScreen HC™ genotoxicity assessment

Cyprotex have partnered with Gentronix, specialists in genotoxicity screening, to offer the GreenScreen HC™ assay.
The GreenScreen HC™ genotoxicity assay utilizes p53-competent human-derived TK6 cells to host the patented GADD45a-GFP reporter system. GADD45a has been implicated in the response to genome damage by genetic, biochemical and genomic approaches¹.
The assay uniquely delivers both highly specific and highly sensitive detection of genotoxic stress in a human cell line.
To detect ‘pro-genotoxins’ (i.e. chemicals which require metabolic activation to become genotoxic), the GreenScreen HC™ assay is performed both in the presence and absence of the post mitochondrial liver fraction, S9, typically prepared from chemically induced rat livers².
GreenScreen HC™ offers several advantages over existing in vitro mammalian genotoxicity assays including ease of use, speed, improved accuracy and reduced compound requirements.

The GADD45a-GFP (GreenScreen HC™) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens.

³Hastwell PW, Webster TW, Tate M, Billinton N, Lynch AM, Harvey JS, Rees RW and Walmsley RM (2009) Mutagenesis 24(5); 455-463

Protocol

GreenScreen HC™ Genotoxicity assay protocol

Test Article Concentration	9 serial dilutions, typically 2-fold; e.g. 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL
Incubation Time	GreenScreen HC™: 48 hour exposure time with results collected at 24 and 48 hour timepoints. GreenScreen HC S9™: 3 hour exposure in the presence of S9, followed by 45 hour recovery and response time. Measurement at 48 hour timepoint.
Quality Controls	1 % DMSO -Negative vehicle control GreenScreen HC™: Methyl methanesulfonate (High 50 μg/ml, Low 10 μg/mL) – Positive control GreenScreen HC S9™: Cyclophosphamide (High 25 μg/ml, Low 5 μg/mL) – Positive control Non-fluorescent control TK6 strain
Test Article Requirements	10 mg solid compound - Other compound preparations are acceptable
Metabolizing System	Typically, aroclor-1254 induced rat liver S9
Analysis Method	GreenScreen HC™ – Spectrophotometric reader GreenScreen HC S9™ – Flow cytometer
Data Delivery	Written report presenting overall results. Lowest effective concentration (LEC) for positive genotoxicity and cytotoxicity results. Excel worksheets with full graphical dose response data for genotoxicity and cytotoxicity.

Data

Data from the GreenScreen HC™ Genotoxicity assay

GreenScreen HC™ has been validated extensively against a wide range of different in vitro and in vivo genotoxicity methods. Results are published within the literature^1,3,4,5.

Figure 1
Schematic diagram illustrating the basic principles of the GreenScreen HC™ assay.

GreenScreen HC™ is a reporter assay which consists of a stably replicating plasmid including all the cis-acting regulatory elements of the human GADD45a gene, coupled to a gene encoding green fluorescent protein (GFP). GADD45a has a central role in genomic integrity, and genotoxic stress induces its transcription. The reporter system exploits p53-dependent, genotoxin-specific induction of human GADD45a expression and the up-regulation leads to production of GFP which is monitored by fluorescent detection (plate reader or flow cytometer). Cytotoxicity is also monitored by optical absorbance, proportional to cell proliferation, in the GreenScreen HC™ assay, and by uptake of propidium iodide dye, proportional to cell viability, in the GreenScreen HC S9™ assay.

Test	Positive (sensitivity)		Negative (specificity)		Concordance
Test	n	%	n	%	n	%
GreenScreen HC™	11/12	92	44/47	94	55/59	93
Bacterial mutation	4/12	33	41/45	91	45/57	79
In vitro cytogenetic	12/12	100	21/37	57	33/49	67
In vitro mammalian mutation	5/9	56	30/38	79	35/47	74
In vivo genotoxicity	12/12	100	40/45	89	52/57	91

Figure 2
Predictivity statistics for genotoxic carcinogenicity in a collection of 75 marketed pharmaceuticals³.

GreenScreen HC™ exhibits both high sensitivity and specificity for genotoxic carcinogens. If combining the figures for sensitivity and selectivity to generate concordance values, GreenScreen HC™ is found to be the best predictor of genotoxic carcinogens when compared with other methods for assessing in vitro and in vivo genotoxicity.³

References

¹ Birrell L et al. (2010) Mutation Research 695; 87-95
² Jagger C et al., (2009) Mutagenesis 24(1); 35-50
³ Hastwell PW et al., (2009) Mutagenesis 24(5); 455-463
⁴ Knight AW et al., (2009) Regul Toxicol Pharmacol 55; 188-199
⁵ Knight AW et al., (2009) J Biomol Screen 14; 16-30