Genotoxicity and carcinogenicity are common properties of chemicals. The Carcinogenic Potency Database, which has data from long-term rodent cancer tests on 1485 chemicals, has positive toxicity results for over 60% of all chemicals tested. It is not surprising that genotoxicity is the cause of failure for many in vivo studies and clinical trials.
The Ames Test is required for FDA IND submission. While the Ames Test provides near-certain results that a compound is genotoxic, a negative result does not assure that the compound is not genotoxic. The Ames Test uses bacteria which lack many of the genotoxin targets found in mammalian cells which is the reason additional genotoxicity screens are required for regulatory purposes. Apredica's Ames Test for determination of mutagenicity of test agents determines genotoxicity by using the Ames MPF™ 98/100 Mutagenicity Assay.
Salmonella typhimurium strains TA100 and TA98 have point mutations that make them incapable of growing in medium unless histidine is supplied. Treatment with a genotoxic chemical causes a mutagenic event to occur, during which base substitutions or frameshifts within the His gene may cause a reversion to histidine prototrophy, and the mutated organisms become able to grow in histidine-deficient medium.
Approximately 107 TA100 or TA98 organisms are exposed to 6 serial dilutions of test agent at 37°C for 90 minutes in medium containing sufficient histidine to support approximately two cell divisions. The cultures are diluted in pH indicator medium lacking histidine and aliquoted into 48 wells of a 384-well plate. The plates are incubated for 48 hours at 37°C, then wells containing cells that have undergone the reversion to histidine prototrophy and have grown into colonies are counted for each dose and compared to a zero-dose (solvent) control. Each dose is done in triplicate to allow for statistical analysis of the data.
A two-fold increase in the number of revertant colonies upon exposure to test chemical relative to the zero-dose controls indicates that the chemical is mutagenic in the Ames MPF™ 98/100 assay.
A pre-screen determination of dose range can be performed to measure solubility and cytotoxicity of test agents.
The genotoxicity of metabolites of the test agents can be determined by performing the assay in the presence of liver S9 fractions.
Figure 1. Mutagenicity of Doxorubicin in Ames Assay in Salmonella typhimurium strain TA98.
Figure 2. Mutagenicity of Doxorubicin in Ames Assay in Salmonella typhimurium strain TA100.
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The in vitro micronucleus test identifies genotoxic carcinogens, i.e., carcinogens that act by causing damage to DNA. During cell division, if a chromosome or a fragment of a chromosome fails to be incorporated into one of the daughter nuclei - typically as a consequence of genetic damage - the chromosome may form its own micronucleus. This micronucleus can be detected using High Content Screening (HCS) techniques.
Apredica's in vitro micronucleus test for identifying genotoxicity uses fluorescent cellular imaging and HCS, which has many advantages over the manual methods typically used for the in vitro micronucleus test. These advantages include:
Apredica's HCS-based in vitro micronucleus test simultaneously collects multiple endpoints including, relative survival (cell count), membrane integrity (cytotoxicity assessment), and cell cycle information (binucleated cell frequency and proliferation index (CBPI). These parameters determine cell health (cytostasis) and micronuclei validity. The assay can also detect genotoxins requiring metabolic transformation by utilizing Aroclor-treated rat liver S9.
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Gentronix has developed a superior genotoxicity screening solution with its GreenScreen HC™ (Human Cells). GreenScreen HC's accuracy derives from its unique ability to both correctly identify genotoxins and to correctly identify non-genotoxins. Apredica is a Gentronix-Authorized Service Provider.
Validation studies1 have shown that GreenScreen HC™ identifies all mechanistic classes of direct-acting genotoxins including aneugens, topoisomerase inhibitors and DNA synthesis inhibitors. It can significantly reduce the number of problem compounds in preclinical development, thus minimizing the need for expensive and time-consuming mechanistic studies.
It is also important to determine if a candidate compound is metabolized to a genotoxic agent. The GreenScreen HC™ assay can also be performed with a source of exogenous metabolizing enzymes (S9)2 commonly used in genotoxicity testing to provide information on the pro-genotoxic properties of compounds.
GADD45a mediates the adaptive response to genotoxic stress. The patented GFP fluorescence reporter includes complex regulatory elements. The assay gives positive results for direct-acting mutagens, clastogens, as well as aneugens, and topoisomerase and polymerase inhibitors. Importantly, the assay gives correct negative results for non-carcinogens, many of which give misleading positive results in other in vitro tests.
Nine, 2-fold dilutions of each compound together with positive controls are set out in the microplate and growing cells are added to each well. After incubation and measurement in a microplate reader, software provides automated decisions and a clear graphical output.
The Comet assay, otherwise known as the the single cell gel electrophoresis (SCGE) assay, detects DNA damage at the individual eukaryotic cell level. It is a rapid yet sensititve method and relatively straightforward to perform with low compound requirements. The method uses electrophoresis on cells which have been exposed to the drug and then embedded in agarose microgels. If DNA fragmentation has occured then this is detected by migration from the nucleus and a comet-like appearance is observed with the head consitsting of intact DNA and the tail consisting of damaged DNA. The Comet assay provides a mechanistic insight into genotoxicity and complements other genotoxicity assessments.
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1. Hastwell P.W. et al., (2006) Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay. Mutation Research 607: 160-175.
2. Jagger C. et al., (2008) Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay. Mutagenesis pp.