Abstract: Environmental pollution with carcinogenic substances seems to be one of the main factors promoting neoplasmic cell degeneration in humans and animals. Although it is obvious as history, the presence of carcinogens in some regions of many countries have to be evidenced. Biomonitoring in such cases requires accurate, sensitive, fast and specific biological methods to assess pollution with carcinogens. Recent validations of the most widely used test of Ames showed that about 50% of the known carcinogens remain undetectable by the test. This provoked the development of other short-term tests, such as alkaline elution, nick-translation, single-cell gel electrophoresis, restriction mutation assay, micronucleous expression, DEL assay. Although some of them were evaluated as promising methods, these assays can not specifically detect carcinogens and are not applicable for large-scale environmental studies.
We have constructed a principally new short-term assay for detection of carcinogens, based on the induction by carcinogens of the transposition of an engeneered oncogene like element in Saccaromyces cerevisiae, the Ty1 retrotransposon. The natural inertness of yeast cells to genotoxins was eliminated by introduction into the tester cells of a mutation which enhances cellular permeability and increases, in this way, the sensitivity of the test. Each transposition of the engeneered oncogene gives rise to a colony of selective medium making the Ty1 assay a quantitative test. The principle novelty of the Ty1 test consists in using a genetic element with structure, cell-cycle and expression control which resemble oncoviruses except that there is no extracellular phase in replication. The test is easy to perform, requires no special equipment, it is fast and cheep.
The characterization of the Ty1 test was done with pairs of laboratory genotoxins that are very similar in chemical structure - one being a carcinogen (for instance benzo (a) pyrene, the second having mutagenig activity without being carcinogen (benzo (e) pyrene). We studied three groups of genotoxins - carcinogen and mutagens, carcinogenic and not carcinogenic heavy metals, free (=carcinogenic) and conjugated (=not carcinogenic) bile acids. Results obtained in concentration-dependent and time-dependent experiments evidenced a positive answer of Ty1 test with all studied carcinogens and negative results with noncarcinonenic mutagens. The Ty1 test has a wider detection spectrum and is positive to carcinogens undetectable with other short-term tests. The specific positive response of Ty1 test to carcinogens was proved in environmental studies. Samples of soil, water and air for which the presence of carcinogens has been evidenced by chemical analyzes were positive in Ty1 test, while negative results were obtained with samples polluted with not carcinogenic mutagens. The results obtained with laboratory and environmental genotoxins evidenced that the Ty1 assay is a short-term test that specifically detects carcinogens.
Several explanations can be proposed for the specific response of Ty1 test to carcinogens. First, the similarity in structure, cell-cycle and function between Ty1 retrotransposons and retroviral oncoviruses may be considered as a precondition for similar responses to carcinogens. Second, the transposition of Ty1 to new locations of the genomic DNA creates genome instability and appearance of different DNA damages as it was found in tumors, induced by carcinogens. While the other short-term tests were developed to detect one genetic damage, the Ty1 test responds positively to all DNA lesions, induced by carcinogens, which increases the detection spectrum. Third, a survey of the literature shows that carcinogens, that are positive in Ty1 test, are also strong generators of reactive oxygen species (ROS). We favor this possibility and studied in details the role of elevated ROS levels for the positive answer of Ty1 test to carcinogens. Data will be presented for proving that the specificity of Ty1 test to carcinogens is due to the induction by Ty1-positives of a burst of ROS, while the Ty1-negative mutagens without carcinogenic potential have little effect of ROS production in yeast cells.