P.R. Harbach

System issue: Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacl transgene in the Big Blue® Rat-2 embryo cell line

Big Blue™ Rat‐2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]‐induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 μg ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 × 10−5. Dose‐related increases in mutant frequency were observed following ENU treatment. Kinetics (time course, of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 μg ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C → A:T transitions (five at CpG sites), six (23%) G:C → T:A transversions, three (11.5%) G:C → C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion + insertion) mutation. Among 46 ENU‐induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C → A:T transitions (four at CpG sites), five (13.5%) A:T → G:C transitions, four (10.8%) G:C → T:A transversions, 11 (30%) A:T → T:A transversions, and two (5.4%) A:T → C:G transversions. Nearly 50% of the base substitutions in the ENU‐treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU‐induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 μg/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

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.

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.

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.

Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay.

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue® transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU‐treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4°C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4°C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4°C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37°C or 24°C showed highest titers on G1217 at 37°C. The fraction of the G1217, 37°C titer seen in the other strains and conditions varied widely with the cII mutation. Environ. Mol. Mutagen. 32: 325–330, 1998

Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [3H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the 2 most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylaminofluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2, 3 and 4 were similar, although shifted about 3-10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.

Comparative mutagenicity testing of ceftiofur sodium: I. Positive results in in vitro cytogenetics

Abstract Preclinical safety evaluation of new drugs is routine prior to the use in humans or animals and genetic toxicology assays are an accepted part of the evaluation along with other more traditional measures of toxicity. A widely used battery of genetic toxicology assays includes an Ames Salmonella microsome assay, a mammalian cell mutation assay, a rat bone marrow micronucleus test and an in vitro assay for induction of chromosomal aberrations. Ceftiofur (U-6427E, NAXCEL®, EXCENEL®), a new generation cephalosporin antibiotic, was subjected to this battery of assays. The result of the first three (Ames test, V79/HPRT mammalian cell mutation assay and the micronucleus test) were negative, the in vitro assay for induction of chromosome aberrations in CHO cells gave positive results. The nature of the observed aberrations was primarily chromatid and isochromatid breaks, gaps and fragments with little evidence of chromosomal rearrangements in the absence of S9 metabolic activation. The aberration increase was only seen following an extended (44 h) exposure to drug; no evidence of clastogenic activity was seen in the presence of S9 metabolic activation or at shorter treatment times. The severe treatment conditions required to produce the clastogenic effects also produced very high levels of mitotic inhibition and thus the observation of chromosomal aberrations is unlikely to be biologically meaningful.