An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.

Big Blue® mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor‐intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073–9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C → A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3′ flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G → A and G → T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G → A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations. Environ. Mol. Mutagen. 33:132–143, 1999 © 1999 Wiley‐Liss, Inc.

The study of hprt mutations in cynomolgus monkey T-lymphocytes is part of our effort to understand the mechanisms of mutagenesis in vivo. This primate model allows us to study mutations and their kinetics at the molecular level under well-controlled conditions using recently developed techniques for selection of mutant T-cells and polymerase chain reaction (PCR) amplification of hprt cDNA, which is directly sequenced. This is the first report of the sequence of the coding region of the cynomolgus monkey hprt gene and PCR/DNA sequence analysis of seven spontaneous mutant T-cell clones, as well as 23 mutant clones isolated 63 and 601 days after treatment with ethylnitrosourea (ENU, 77 mg/kg, intraperitoneal). cDNA was reverse transcribed from hprt mRNA directly from a lysate of about 2-4 x 10(3) cells, and a 700 bp fragment including the coding region was amplified by PCR and sequenced. Of the seven spontaneous mutants, only one point mutation (GC----AT transition) was detected, and the other six failed to amplify by PCR, possibly due to functional deletions. Of the 14 mutant clones isolated 63 days after ENU treatment, nine base substitutions were detected in ten clones: four transitions (three AT----GC and one GC----AT) and five transversions (four AT----TA and one AT----CG). Of the nine mutants isolated 601 days after ENU treatment, six single base substitutions were detected in six clones (five AT----TA and one AT----CG transversions), and one mutant had a large deletion or insertion. No changes were detected in three clones (one Day 63 and two Day 601 clones). In summary, only one of 15 single base substitutions isolated after ENU treatment was a GC----AT transition mutation and the rest were transitions and transversions at AT sites.

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