High Throughput Screen and High Information Follow-Up Tests for Genotoxicants

Project SummaryCurrent batteries of genetic toxicology assays exhibit several critical deficiencies. First, the throughput
capacity of in vitro genotoxicity tests is low, and does not meet current needs, especially for early, high volume
screening environments that need to prioritize chemicals for further testing and/or development. Second,
conventional assays provide simplistic binary calls, genotoxic or non-genotoxic. In this scheme there is little or
no information provided about genotoxic mode of action. This is severely limiting, as it does not generate key
information necessary for prioritizing chemicals for further testing, guiding subsequent assays’
endpoints/experimental designs, or conducting risk assessments. Finally, most current assays do not place
requisite emphasis on dose response relationships, and therefore do not contextualize the results in terms of
potency. These deficiencies prevent genotoxicity data from optimally contributing to modern risk assessments,
where all of these capabilities and high information content are essential. We will solve these issues by
developing, optimizing, and validating a two-tiered testing strategy based on multiplexed DNA damage
responsive biomarkers and high-speed flow cytometric analysis. The first-tier focuses on throughput and is
used to prioritize likely genotoxicants for more comprehensive analysis in second tier testing. Specifically, it
involves a collection of several multiplexed biomarkers that will be used to identify likely genotoxic agents and
provide a preliminary assessment of genotoxic mode of action. The gH2AX biomarker detects DNA double
strand breaks, phospho-histone H3 identifies mitotic cells, nuclear p53 content reports on p53 activation in
response to DNA damage, the frequency of 8n+ cells measure polyploidization, and the ratio of nuclei to
microsphere counts provides information about treatment-related cytotoxicity. The second tier focuses on
information content and considers many more concentrations as well as additional biomarkers, including
micronucleus formation. Collectively, the tier two results provide definitive predictions about test chemicals’
genotoxic potential, mode of action, and potency. Over the course of this project we will study more than 3,000
diverse chemicals in order to understand the performance characteristics and generalizability of the two-tiered
testing strategy. An interlaboratory trial will be conducted with prototype assay kits to assess the transferability
of the methods, with the ultimate goal of providing the Nation with commercially available kits and testing
services.