Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-01-06
1996-12-31
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 29, 935 33, 935 38, 935 41, 935 43, C12Q 168, C12Q 102, C12N 1563
Patent
active
055893376
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF INVENTION
This invention provides methods and diagnostic kits for determining the toxicity of a compound. The methods and diagnostic kits of this invention employ a plurality of bacterial hosts, each of which harbors a DNA sequence encoding a different stress promoter fused to a gene which encodes an assayable product. Each of these stress promoters is induced by exposure to a different type of cellular stress. The stress promoters utilized in this invention, in toto, comprise those promoter which respond to redox stress, DNA stress, protein stress, energy stress and pH stress. The methods and diagnostic kits of this invention may be employed to characterize and quantify the toxicity of a compound, as well as to identify the cellular mechanism of its toxic action. Moreover, the methods of this invention yield information concerning the action of a compound on a subcellular level. This information may be utilized to design antitoxins to compounds found to be toxic and in active drug design.
BACKGROUND OF THE INVENTION
At least 55,000 chemicals are presently produced in the United States. Over 2,000 new chemicals are introduced into the market each year. Very few of these chemicals have been comprehensively tested for acute or chronic toxicity. For example, less than 1 percent of commercial chemicals have undergone complete health hazard assessment.
The Environmental Protection Agency ("EPA") has the authority to require toxicological testing of a chemical prior to commercial production, but that authority is rarely invoked. Less than 10 percent of new chemicals are subjected to detailed review by the EPA. In the interest of cost and speedy access to the market, the EPA often uses the toxicity of previously tested homologous compounds to gauge the toxicity of a new chemical.
The potential toxicity of new drugs is monitored by the Food and Drug Administration ("FDA"). For a New Drug Application (NDA), the FDA typically requires a large battery of toxicity, carcinogenicity, mutagenicity and reproduction/fertility tests in at least two species of live animals. These tests are required to last up to one year. The costs involved in completing these tests is enormous. For example, a typical 90-day exposure toxicity test in rats costs approximately $100,000. A two year toxicity test in rats costs approximately $800,000 [Casarett and Doull's Toxicology, 4th Edition, M. O. Amdur et al., eds. Pergamon Press, New York, New York, p. 37 (1991)].
Besides cost, animal testing also presents disadvantages in terms of time, animal suffering and accuracy. Typical toxicity tests are divided into three stages: acute, short term and long term. Acute tests, which determine the LD.sub.50 of a compound (the dose at which 50% of test animals are killed), require some 60-100 animals and a battery of tests for determining LD.sub.50, dose-response curves and for monitoring clinical end points, other than death. Short term tests usually involve at least 24 dogs and 90 rats and last from 90 days in rats to 6-24 months in dogs. Body weight, food consumption, blood, urine and tissue samples are frequently measured in the short-term tests. In addition, dead animals are subjected to post-mortem examinations. Long term tests are similar to short term tests, but last 2 years in rats and up to 7 years in dogs or monkeys.
Animal testing has come under criticism by animal rights activists and the general public because of the severe suffering inflicted on the animals. Moreover, recent evidence calls into question the accuracy of animal testing. For example, variables, such as animal diet, may impair the predictability of animal tests in determining carcinogenic properties [P. H. Abelson, "Diet and Cancer in Humans and Rodents", Science, 255, p. 141 (1992)]. And prior determinations on dioxin toxicity, based on guinea pig testing, are now being reevaluated [B. J. Culliton, "U.S. Government Orders New Look At Dioxin", Nature, 352, p. 753 (1991); L. Roberts, "More Pieces in the Dioxin Puzzle", Research News, October, 1991, p. 3
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Degen Nancy J.
Fleisher Mindy
Haley, Jr. James F.
Marks Andrew S.
The President and Fellows of Harvard College
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