Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma.

Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.

Alternative methods in toxicology tests: In vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develope alternative methods to reduce the required number of animals. The newin vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by thein vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity within vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems forin vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mytochondrial activity, intracellular content and leakage of enzymes into the medium.

In vitro micronucleus test with kinetochore staining: evaluation of test performance.

In the framework of the coordinated programme 'Genomic Mutations' sponsored by the Commission of European Communities, eight known or suspected spindle poisons (cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine) were tested in a modified in vitro micronucleus test in Chinese hamster cells. Micronuclei (MN) with or without kinetochore were analyzed by staining of the kinetochore with an antikinetochore antibody (CREST staining). Mitotic index and ana-telophase:mitosis ratio were also recorded to evaluate cytotoxicity and c-mitotic effects of tested chemicals. CREST-positive MN were induced by diazepam, thiabendazole and vinblastine. Hydroquinone, pyrimethamine, econazole and cadmium chloride induced MN that contained both entire chromosomes and acentric fragments. Negative results were obtained with thimerosal. The results obtained indicate that the detection of CREST-positive MN is a powerful assay to identify spindle poisons. Furthermore, this method provides a useful tool to ascertain the origin(s) of induced MN.

Alternative methods in toxicology tests: in vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develop alternative methods to reduce the required number of animals. The new in vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by the in vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity with in vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems for in vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mitochondrial activity, intracellular content and leakage of enzymes into the medium.

Light-dependent activation of 7,12-dimethylbenz[a]anthracene to a potent genotoxicant.

7,12-Dimethylbenz[a]anthracene (DMBA), which is widely used in mutagenesis and experimental carcinogenesis, is activated to a mutagen by white fluorescent light. A 40 min exposure to white fluorescent light of Salmonella typhimurium TA98 plates treated with DMBA, in the absence of exogenous metabolism, resulted in an approximately 30-fold increase in the number of histidine revertants. This phenomenon also occurs, with lesser intensity, with other promutagens, such as benzo[a]pyrene or 2-acetylaminofluorene, and in other Salmonella tester strains. Moreover, white fluorescent light is able to activate DMBA to a toxicant for Chinese hamster V79 cells in culture, resulting in very low cell survival. Under these conditions, white fluorescent light-activated DMBA was shown to cause chromosomal aberrations, but not gene mutations, as determined by resistance to thioguanine. This white fluorescent light-dependent activation of DMBA seems to be related to the formation of reactive species, as the addition of vitamin E results in a reduction in the number of histidine revertants induced by white fluorescent light in S. typhimurium TA98.

Genotoxicity studies on ebrotidine.

Five genotoxicity studies on ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio] ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542), including at least four of the battery of tests recommended by toxicological regulatory guidelines for new drugs, were conducted. These tests were the Ames test for determination of bacterial gene mutations, sex-linked recessive lethal mutation test in Drosophila for gene mutations in eukaryotic systems, in vitro chromosome aberration test and micronucleus test for evaluation of structural and numerical aberrations, and sister chromatid exchange frequency test for assessment of non-specific damage to chromatin. Negative and positive controls were used in all the experiments. The effects were investigated in the absence or presence of metabolic activation by S-9 microsomal fraction from rat liver homogenate. A dose range toxicity study was also performed to determine the dosage levels or concentrations to be tested for the assessment of genotoxic effects. None of the tests showed a significant increase in the genotoxic parameters, both in vitro and in vivo in somatic or germ cells. It is, therefore, concluded that ebrotidine has not caused mutagenic or clastogenic effects in any of the experimental systems tested.

Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test.

Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70-80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.

Industrial Genotoxicology Group collaborative trial to investigate cell cycle parameters in human lymphocyte cytogenetics studies.

Human lymphocyte cultures have been used for many years for assessing the in vitro clastogenic potential of test substances. In these assays the harvest time should be based on the cell cycle time in order to ensure that cells are sampled at an appropriate time for the detection of clastogenic effects. The sources of variation in the cell cycle time in routine cytogenetic assays have not been well studied. Consequently 13 laboratories, all members of the Industrial Genotoxicology Group, participated in a collaborative study to measure the variation in cell cycle time in cultured human peripheral blood lymphocytes under various conditions. The study was performed in two phases, spaced 6 months apart. The average generation time (AGT) was measured by the incorporation of bromodeoxyuridine. Very similar AGTs were found in the presence and absence of S9 mix. The mean AGT (mean of four donors) in each laboratory varied from 11.2 to 17.1 h, indicating there is significant variability in cell cycle times of human peripheral blood lymphocytes between laboratories. There was greater variation between laboratories than within laboratories. A comparison of AGT values at 72 h performed in experiments at least 6 months apart indicated good reproducibility in most laboratories. The study indicates that a 24 h post-treatment harvest may result in the analysis of very few first division cells unless very significant cell cycle delay is induced by the test substance. It was also found that a post-harvest time equivalent to 1.5 cell cycles will result in an approximately equal mixture of first and second division cells and therefore should by suitable for assessing both the induction of chromosome aberrations and polyploidy.