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Whitehead Researchers Develop In VitroMouse Assay to Test for Genotoxicity

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Researchers at the Whitehead Institute of MIT and Harvard have developed an in vitro assay for assessing a compound’s toxicity that may prove to be much cheaper than the erythroid in vivo micronucleus assay and the Ames test.
 
The assay, which the researchers believe may eventually be transitioned to human use, could enable researchers to determine toxicity much sooner in the drug-discovery process, which could make the process much more efficient.  
 
Unlike the current MN assays, the Whitehead assay is a cell-culture system that could allow hundreds or thousands of tests to be performed from the bone marrow of a single mouse, said Joe Shuga, the doctoral candidate at MIT who developed the assay.
 
According to Shuga, the investigators showed that the lineage-marker negative population of adult mouse bone marrow underwent erythropoietic differentiation according to established in vivo patterns.
 
The research, which appeared online this week in the Proceedings of the National Academy of Science, also showed that erythrocytes generated in vitro were able to form MN when exposed to direct-acting genotoxic agents, he said.     
 
Greater Throughput, Cheaper Cost
 
According to Mark Collins, marketing manager in the cellular imaging and analysis division at Thermo Fisher Scientific, a “hot topic” has been the desire to improve throughput and productivity in genotoxicity as more compounds need to be tested for genotoxicity both for pharmaceutical and environmental reasons.
 
Also driving the desire to automate genotoxicity testing has been a requirement in Europe to move genotox testing for nonpharmaceutical uses out of animals and into cell-based systems by 2009.
 
The in vivo MN assay is generally conducted later in the drug-discovery process, Shuga told CBA News this week, because a large group of animals is necessary to encompass the multiple doses and time points that are required for a single compound.
 
Using this technique, researchers can dose a single mouse with a single candidate compound at a single dose level and typically assay the bone marrow one to two days later, said Shuga. If the bone marrow could be used to run hundreds to thousands of assays — as the researchers believe is possible based on their gross estimates — then the assay could be run earlier in the drug-development process, he said.
 
With a higher-throughput, in vitro version of this screen, researchers may decide to test a wide panel of drug candidates early in drug discovery to get some idea if problems would be encountered down the road with the in vivo MN assay, Shuga said. It could be applied earlier as a prescreen, he said, which could be a way for pharmaceutical companies to reduce R&D costs.
 
“What you’d want is a multiplexed culture system such as we’ve described, and have that feed into a high-throughput analysis system,” Shuga said. “I imagine within a year of industrial development, that could be up and running very smoothly.”
 

“Who would have thought that there would be interest in the in vitro rodent model, because it allows access to aspects of mechanisms that are difficult to get at in vivo?”

Right now, pharmaceutical companies prefer rodent models for studying genotoxicity. But using an in vitro technology to tease out where the events are occurring may be easier than using an in vivo system, which inherently is more complex, according to Linda Griffith, S.E.T.I. professor of biological and mechanical engineering and director of the Biotechnology Process Engineering Center at MIT.
 
She said drugmakers have suggested they would be interested in using an in vitro model. “We were surprised to find that there was interest from the pharmaceutical industry in having this particular assay done in vitro using rodent cells,” said Griffith. “Who would have thought that there would be interest in the in vitro rodent model, because it allows access to aspects of mechanisms that are difficult to get at in vivo?”
 
Human Evolution
 
Shuga said this work may eventually be transitioned to work with human tissue. “In the paper, we did not actually use any human tissue, but it is known that you can also obtain these erythroid progenitors from peripheral blood, bone marrow, and cord blood,” Shuga said. “So you may be able to derive these erythroid progenitors from human stock, and conduct the assay in vitro from there. That’s more of an area for future research, though.”
 
The team is currently evaluating a framework for doing this work with human tissue, said Griffith, who added that such research would copy what was done with the murine system.

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