C.S. Aaron

Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozao: Linear relation of DNA alkylation per sperm cell (Dose) to sex-linked recessive lethals

The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.

The mouse bone marrow micronucleus test: evaluation of 21 drug candidates.

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.

Comparison of the AS52/XPRT and the CHO/HPRT assays: evaluation of 6 drug candidates

The purpose of this paper is to compare the result of testing a diverse group of chemicals in the CHO/HPRT and AS52/XPRT mutation assays. The AS52/XPRT system was as sensitive as the more widely used CHO/HPRT system in the case of the antitumor agents, and gave qualitatively similar results in all cases. On the basis of this and other experiments (Aaron et al., 1989) it appears that the AS52/XPRT system may be most useful in addressing mechanistic questions in mutagenesis. We recommend that the AS52/XPRT assay be used as the mammalian cell test system of choice in batteries used for identifying mutagens and genotoxic carcinogens.

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: Evaluation of 2-furoic acid and 7 drug candidates

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 8 compounds which were tested in the in-house genetic toxicology program. These compounds represent diverse chemical structure and most of them entered the screening program because they are biologically active in efficacy screens. All tests were carried out under Good Laboratory Practices Regulations of the U.S. Food and Drug Administration. None of the materials reported here produced an increase in UDS and therefore the UDS results with these compounds do not suggest potential for genotoxicity.

Comparative mutagenicity testing of ceftiofur sodium: III. Ceftiofur sodium is not an in vivo clastogen

Abstract Ceftiofur has been previously shown (Aaron et al., 1995a Mutation Res., this issue) to produce chromosome aberrations following extended treatment of Chinese hamster ovary cells in culture at high doses in the absence of S9 activation. The experiments described here address the relevance of this finding in vivo. Ceftiofur failed to induce aberrations in the bone marrow of CD-1 mice following intraperitoneal injection. Furthermore, ceftiofur did not induce micronucleated polychromatic erythrocytes in mouse bone marrow. In the experiments reported here no evidence of the induction of unscheduled DNA synthesis in the liver of rats treated in vivo was seen following oral exposure to high doses of the drug. However, the compound was foud to reach the bone marrow compartment in these experiments. Thus, the negative finding suggests that the in vitro finding of chromosome aberration induction by ceftiofur is not a cause of concern. This assertion is supported by mechanistic studies (Aaron et al., 1995b, Mutation Res., this issue) which show the cause of the in vitro findings to be most likely due cell cycle delay and by the observation that ceftiofur is rapidly metabolized in mammalian systems in vivo but not in the artificial conditions that occur in the in vitro experiments.

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 24 drug candidates.

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 24 drug candidates which were tested as coded compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of biological activity in a variety of preliminary drug efficacy screens. None of the compounds reported here produced an increase in UDS, and therefore, the UDS results with these materials do not suggest potential for mutagenesis or carcinogenesis.

The Salmonella mutagenicity test: Evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.

The CHO/HPRT assay: evaluation of 19 drug candidates.

The CHO/HPRT assay is used as part of basic mutagenicity screening batteries at the Upjohn Company. The results of this and other assays provides a way of identifying compounds likely to cause mutations in mammalian systems and for which additional testing may be required. This report provides results of testing 19 drugs and drug candidates for mutational properties in the CHO/HPRT assay. The results of these studies were uniformly negative. The diversity of structures and the fact that these compounds have potent (non-mutational) biological activity suggests that separation of mutagenic properties from other beneficial properties is feasible. These compounds have also been evaluated in several other assays (Aaron et al., 1989a-d) and were negative for genotoxicity. Thus, the results of this study and other available data fails to suggest any genotoxic hazard from these materials.

Comparative mutagenicity testing of ceftiofur sodium II. Cytogenitic damage induced in vitro by ceftiofur is reversible and is due to cell cycle delay

Abstract Ceftiofur, a new generation of cephalosporin antibiotic, used to combat bacterial respiratory disease in growing cattle and swine, has been tested in a battery of genetic toxicology assays (Aaron et al., 1995a) and been shown to produce chromosome aberrations in CHO cells following treatment for 44 h. No evidence of aberration induction was seen at shorter, i.e., 20 h, treatment time nor was any suggestion of clastogenicity seen in the presence of S9 metabolic activation. The experiments reported here were undertaken to determine significance of this observation and elucidate the reason for clastogenesis in the earlier experiments. Briefly, ceftiofur was found to not affect the pH or osmolality of the treatment solutions nor were enzymes generally associated with cell death released during the treatment period. The aberrations were found to be reversible, and thus, doubt was cast concerning the potential for direct DNA damage as a causative factor. The most profound effect of ceftiofur treatment at this level was the dramatic effect on cell cycle kinetics and therefore the clastogenic effects observed following exposure to cettiofur in vitro appear to be due to prolongation of the cell cycle.