About 700,000 new cases of cancer affect North Americans each year. It is estimated that from about 70 to about 90 percent of these new cancer cases are linked to environmental carcinogens. Epidemiologists estimate that at least about 70 percent of human cancer would be preventable if the main risk and antirisk factors could be identified. One epidemiological example of this phenomenon is colon and breast cancer. These are among the major types of cancer, but they are quite rare among Japanese living in Japan. However, Japanese living in the United States have a relatively high incidence of this disease.
There are in excess of about 80,000 chemicals in commercial production. Over 400,000 new organic compounds are synthesized every year, and at least 1,000 of them each year will eventually be introduced into economic use. There is a need to be able to determine which of these new compounds will cause cancer. However, it is difficult to predict without testing whether any particular chemical will cause cancer.
The most reliable means for determining whether a particular compound is carcinogenic is a long term assay, which generally is based on the experimental assessment of the potential of the substance to induce tumors in rodents. Long term assays usually take from 6 to 12 months to conduct, and they are relatively expensive. Because of the time and/or the expense involved, it is not feasible to conduct long term assays in many situations, especially where one is seeking a preliminary indication as to whether to proceed with the development of a particular substance.
The need for relatively fast and inexpensive means for preliminarily evaluating the cancer-causing potential of new chemicals has led to the development of many short term assays; some of these short term assays are described in column 4 (lines 13-44) of U.S. Pat. No. 4,701,406, the disclosure of which is hereby incorporated by reference into this specification. The most widely known of these short-term assays is the Ames Assay. This Assay is based upon the assumption that carcinogens will cause the genetic reversion of certain mutant strains of the bacteria Salmonella typhimurium. In other words, the mutant strains revert to their normal form in the presence of mutagens. A description of the Ames Assay may be found, e.g., in an article by Ames et al., "Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test," Mutation Research, vol. 31 (1975), pp. 347-364.
One disadvantage of the Ames Assay is that many classes of carcinogenic compounds consistently show poor responses in this assay. Thus, as is disclosed at column 4 of U.S. Pat. No. 4,701,406, the Ames Assay is not very useful for evaluating certain metals, steroid hormones, and chlorinated hydrocarbons which, although they are known to be carcinogens, give very poor responses.
One of the major problems with the Ames Assay is that, although it is useful for evaluating certain mutagenic compounds, it is not generally useful for evaluating carcinogenic compounds which are not mutagenic. See, for example, McCann et al., "Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals, Proc. Nat. Acad. Sci. USA, vol. 72, No. 129 (1975), pp. 5135-5139. Also see McCann et al., "Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals Discussion, "Proc. Nat. Acad. Sci. USA, vol. 73, No. 3 (1976), pp 950-954.
Short term tests involving mutation and recombination assays with the yeast Saccharomyces cerevisiae have been developed. However, these yeast assays are only able to detect about 74 percent of the known carcinogens as being positive. See, for example, an article by Zimmermann et al. appearing in Mutation Research, vol. 133 at pages 199-244 (1984).
The prior art teaches the use of both the Ames Assay and the aforementioned yeast assay in combination, but even the use of both of these assays fails to detect many nonmutagenic carcinogens. See, e.g., the aforementioned article by Zimmermann et al.
The prior art also teaches the use of other in vitro mammalian assays, see e.g. a book by Milman et al. entitled "Handbook of carcinogen testing", Noyes Publications, New Jersey, the publication of which is incorporated by reference into this specification. The mammalian assays also fail to detect many nonmutagenic carcinogens. The following examples are taken from the aforementioned reference. Thus for example the tests for sister chromatid exchange as well as for chromosome aberrations with mammalian cells are both negative for ethionene, ethylenethiourea and safrole. Thus, for example, the syrian hamster embryo clonal transformation procedure is negative with aniline. Thus, for example the rat liver foci assay is negative with safrole and thioacetamide.
Not only do the prior art short-term tests fail to show positive results with many known carcinogens, but they also usually fail to indicate whether a prospective carcinogen will cause genome rearrangement. There is a substantial body of literature indicating that compounds which cause genome rearrangement might cause cancer. Thus, it has been shown that the excision of retroviruses from genomes can cause cancer; see Bishop, Ann. Rev. Biochem. 52:301-354 (1983) and Bishop, Cell 42:23-38 (1985). Thus, it has been shown that amplification of specific human DNA sequences up to 120 times are associated with cancer, see Montgomery et al., Proc. Natl. Acad. Sci. USA 80:5724-5728 (1983) and Schwab et al., Proc. Natl. Acad. Sci. USA 81:4940-4944). Thus, it has been shown that immunoglobulin class switching in B lymphocyte differentiation is associated with cancer; see Brown et al., Proc. Natl. Acad. Sci. USA 82:556-560 (1985), Korsmeyer et al., Proc. Natl. Acad. Sci. USA 80:4522-4526 (1983), and Cleary et al., Proc. Natl. Acad. Sci. USA 81:593-597 (1984). Thus it has also been shown that rearrangements involving the T Cell receptor gene are associated with cancer; see Flug, Proc. Natl. Acad. Sci. USA 82:3460-3464 (1985) and Minden et al. Proc. Natl. Acad. Sci. USA 82:1224-1227 (1985). Thus, it has also been shown that amplification preceded by mutation of a gene is associated with cancer; see, e.g., Fujita, Proc. Natl. Acad. Sci. USA 82:3849-3853 (1985). Thus, it has also been shown that deletions in recessive oncogenes are associated with carcinogenesis such as retinoblastoma; see, e.g., Hansen and Cavanee, Cell 53:172-173 (1988), see also, Ponder, Nature 335:400-402 (1988). The role of genome rearrangement in carcinogenesis has also been discussed in more general terms in Klein, Nature 294:313-318 (1981), Pall, Proc. Natl. Acad. Sci. USA 78:2465-2468 (1981), Cairns, Nature 289:353-357 (1981), and Wintersberger, Naturwissenschaften 69:107-113 (1982).
It is an object of this invention to provide a short-term assay system which can be used to evaluate many bactericidal and antibiotic compounds.
It is another object of this invention to provide a short-term assay which can be used to evaluate many non-mutagenic compounds which are carcinogenic and which do not show positive results in the prior art Ames Assay, yeast assays and mammalian cell culture systems.
It is yet another object of this invention to provide a short-term assay system which can be used to evaluate many compounds or compositions which cause genome rearrangement.
USPTO Backgrounds
USPTO Backgrounds