Methods and kits for eukaryotic gene profiling

Details Methods and kits for eukaryotic gene profiling Methods and kits for eukaryotic gene profiling

1995-07-21

1998-09-22

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 29, 435 32, 435870, 435 912, 536 231, 536 243, 536 2433, 536 241, 424600, 424 941, 4242781, 4241781, C12Q 768, C07H 2102, C07H 2104, A01N 5900

Patent

active

058112316

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD OF INVENTION

This invention provides methods and diagnostic kits for identifying and characterizing toxic compounds. These methods and diagnostic kits measure transcription or translation levels from genes linked to native eukaryotic stress promoters, especially those of mammals. The kits and methods of this invention utilize at least one stress promoter from each of the following groups: redox stress, DNA stress, protein stress and energy/ionic stress. The invention also provides methods and diagnostic kits for identifying and characterizing compounds that are toxic to specific organs, such as skin and the eye, as well as for each of the individual stresses indicated above. The methods and diagnostic kits 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. A two year 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, much as animal diet, may impair the H. Abelson, "Diet and Cancer in Humans and Rodents", Science, 255, p. 141 (1992)!. And prior determinations on dioxin toxicity, based on guinea pig Orders New Look At Dioxin", Nature, 352, p. 753 (1991); L. Roberts, "More Pieces in the Dioxin Puzzle", Research News, October 1991, p. 377!. It is therefore apparent that there is an urgent need for a quick, inexpensive and reliable alternative to toxicity testing in animals.
Several short-term alternative tests are available. For example, the Ames Assay detects carcinogens which cause genetic reversion of mutant strains of Salmonella typhimurium. However, the Ames Assay cannot detect either non-mutagenic carcinogens or non-carcinogenic toxins. The yeast carcinogen assay system d

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