The effectiveness of three techniques to deliver a diazo dye suspension into the lungs of rats was compared. The intratracheal nebulization (ITN) technique delivered 10 l of the suspension per 5-ml puff of air in 10 puffs as an aerosol. The intratracheal fast instillation (ITFI) technique delivered 100 l of the suspension in a single 2-ml puff of air as droplets. The nose-only inhalation (NI) technique aerosolized the suspension at an analytical concentration that provided a calculated dose equivalent to 100 l of the suspension in a 2-h inhalation period. Immediately after dosing, all the rats were killed by exsanguination. The trachea was tied and the lung was inflated in situ with air. After fixation, 5m thick slices were prepared from each lobe of the lung at a plane perpendicular to the axis of the lobar bronchus at levels proximal, medial and distal to the hilus. The numbers of bronchi, bronchioli and alveolar ducts within four ranges of diameters and the proportion of each selected area of lung tissue with and without dye particles were quantified using electronic imaging analyzers. The results indicated that ITN and ITFI dispersed the particles evenly throughout most of the airways and in patches in the alveoli. The NI technique dispersed the particles homogeneously throughout the airways and the alveoli in the lungs. The mean number-percentage and the mean area-percentage data revealed that the doses delivered by ITN and NI were approximately 60% and 10%, respectively, of the ITFI dose. Thus, the ITFI technique appeared to be most suitable for pulmonary absorption and disposition studies where dosage precision is of primary concern. The ITN technique would need further improvement to meet the requirements for dose precision and particle distribution. For both ITFI and ITN, particle size was apparently not a critical determinant for deposition. The NI technique is suitable for inhalation toxicity studies where the pattern and uniformity of particle deposition is the primary concern.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

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.

We have investigated the use of cynomolgus monkeys (Macaca fascicularis) as a model of somatic cell mutagenesis in non-human primates. Using techniques described by Albertini (Mutation Research 150:411-422, 1985) for similar studies in humans, the frequency of TG-resistant T-lymphocytes in the peripheral blood was determined in animals that were either untreated or treated with ethylnitrosourea. The frequency of TG-resistant cells in untreated males was (mean +/- SD) 6.0 +/- 5.9 per 10(6) cells and for untreated females was 2.9 +/- 2.7 per 10(6) cells. The spontaneous frequency of TG-resistant cells for all animals was 4.2 +/- 4.44 per 10(6) cells. Maximum frequency of TG-resistant cells for two animals treated with a single I.P. dose of ENU was 45.1 and 77.9 per 10(6) cells. Substantial increases in frequencies of TG-resistant cells were not seen until at least 63 days after treatment. The TG-resistant phenotype of clones isolated in the assay was stable after growth for 2 weeks in the absence of selective agent. Many of the TG-resistant clones selected were frozen for future molecular analysis.

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