Transgenic nmice with a A shuttle vector containing a lacI target gene were generated for use as a shortterm, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl (3-D-galactopyranoside (X-Gal). Phage with mutations in the lacl gene form blue plaques, whereas phage with a nonmutated lacl form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10-S to 1.7 x i0-0, depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the miceor cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the laI gene, when replicated in Escherichia cohi, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the WaI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance ofmutations (81 %) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.The lacd gene has been used extensively as a target for the identification and analysis of spontaneous and induced mutations, due in part to the ease of using a calorimetric assay to rapidly screen for mutations. Genetic and sequence analysis of spontaneous and induced mutations detected in several systems utilizing lacd (1-5) has resulted in an extensive accumulation of data regarding the sequence specificity of spontaneous and induced mutations in lacd, which can be of considerable comparative value through the use of the lacd target gene in whole animal assay systems.To combine the cost-saving aspects of short-term assays with the predictive power of whole animal assays, we previously described the development of a system that depends upon efficient recovery of a A phage shuttle vector from transgenic mouse genomic DNA thro...

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.

Human osteosarcoma (HOS) clonal cells transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were characterized, and compared to non-producer HOS cells transformed by Kirsten murine sarcoma virus (Ki-MSV). The MNNG- and virus-transformed cells grew in the aggregate form above an agar base, grew in soft agar, and had a high fibrinolytic activity. When inoculated into nude mice, all the chemically or virally altered cells produced tumors or tumor nodules. When transplanted into ATS-treated hamsters, the cells transformed by MNNG (0.01 mug/ml) and Ki-MSV produced tumors but MNNG (0.1 mug/ml) transformed cells did not produce tumors. The control HOS cells did not grow in the aggregate form but formed colonies in soft agar, and had low fibrinolytic activity and no capacity to form tumors in nude mice and ATS-treated hamsters. However, one of the control clonal lines had a high level of fibrinolytic activity. Cellular aggregation properties of human transformed cells did appear to correlate with tumorigenicity in nude mice.

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Commercial hexane is a solvent mixture of six-carbon isomers, consisting principally of n-hexane, 3-methylpentane, methylcyclopentane and 2-methylpentane. The potential of commercial hexane to produce chromosome aberrations was evaluated in both an in vitro assay using Chinese hamster ovary (CHO) cells and an in vivo cytogenetics assay using Sprague-Dawley rats. The CHO cells were exposed to media containing commercial hexane at concentrations of 0.014-0.42 microliters ml-1 in the presence and absence of an S-9 activation mixture. Cellular toxicity was observed at the higher dose levels, but no increase in chromosome aberrations was observed in either the non-activated or S-9-activated systems. For the in vivo cytogenetics assay, rats were exposed nose-only for 6 h per day for 5 consecutive days to commercial hexane vapor at target concentrations of 900, 3000 and 9000 ppm. Bone marrow cells were collected at 6 and 24 h after the midpoint of the last exposure. Metaphase cells were examined microscopically for chromosome aberrations. No statistically significant increases in aberrant cells were observed in the commercial hexane-exposed animals of any dose group at either of the bone marrow harvest times. In conclusion, commercial hexane did not produce chromosomal mutations under the conditions of these studies.

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