Sequence analysis of tka(-)-1 and tkb(+)-1 alleles in L5178Y tk+/- mouse-lymphoma cells and spontaneous tk-/- mutants.

The mouse-lymphoma mutagenesis assay detects forward mutations at the heterozygous thymidine kinase (tk-1) locus in L5178Y tk+/- 3.7.2C cells. This assay of genotoxicity is widely used to quantitate the mutagenic potential of a variety of chemical and physical agents. A NcoI heteromorphism between the tka- and tkb+ alleles allows the use of Southern blotting to broadly detect two major categories of mutations. These consist of deletions of the functional allele, characterized by absence of a 6.3-kb tk-hybridizing band, and apparent point mutations, indistinguishable from wild-type on blots. Rarely, Southern blots reveal a partial deletion of tkb. The variety of lesions recorded at the heterozygous tk-1 locus may be representative of events important in mammalian carcinogenesis and may include a greater range of mutagenic events than can be observed at hemizygous test loci. To further assess the ability of the mouse-lymphoma assay to detect a variety of mutations and to allow identification of point mutations, we have sequenced the entire tk-1 coding region from both tka- and tkb+ alleles of L5178Y 3.7.2C mouse-lymphoma cells. Sequences were obtained using PCR amplified double-stranded DNA templates prepared from cytoplasmic RNA from the heterozygous cell line. The two alleles were found to differ by a single TA to GC transversion, altering one amino acid in the deduced amino acid sequence. 4 spontaneous mutants were also sequenced and demonstrated a variety of mutations, including a 6-base pair in-frame deletion, a CG to GC transversion upstream of the start codon, a mutant apparently lacking expression of the tkb allele, and a mutant with apparent wild-type coding sequence for both tka- and tkb+ alleles. The diverse nature of the mutants isolated from L5178Y cells suggests that the mouse-lymphoma mutagenesis assay is capable of detecting a number of mutation types, enhancing the utility of the assay in studying the range of genetic lesions important in human disease. The lesions produced are readily analyzed using a combination of Southern blotting and sequence analysis.

Evidence for chromosomal replicons as units of sister chromatid exchanges.

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 micron with a peak value around 0.3 to 0.6 micron regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: II. Results with 20 chemicals.

Twenty chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary cells (CHO). These chemicals were tested with and without an added metabolic activation system (rat liver S9 fraction). Four chemicals were negative in both assays, 1 induced ABs only, and 15 were positive for SCEs; 6 of these 15 also induced ABs. The effect of cell harvest time on the ability to detect the induction of chromosomal aberrations was examined for six chemicals. Five of these had caused at least one of the following: cell cycle delay, aberrations observed in first division metaphase cells in the SCE assay, or a weak response in the standard AB assay (10-12-hr growth period). Three chemicals, chlorinated trisodium phosphate, 1,2-dibromo-3-chloropropane, and tetrakis(hydroxymethyl)phosphonium chloride, were positive using both the standard and extended harvest times. N-Nitrosodimethylamine and diphenhydramine HCl were only positive using an extended harvest time, and malonaldehyde was negative using both standard and extended harvest times.

Toxicity and mutagenicity of radiation from fluorescent lamps and a sunlamp in L5178y mouse lymphoma cells.

Unfiltered broad spectrum radiation emitted by black light, cool white, and black light blue fluorescent lamps and a sunlamp, is both toxic and mutagenic to L5178Y mouse lymphoma cells when the cells are irradiated in phosphate-buffered saline. The increase in mutant frequency seen after exposure of the cells is linear throughout the range of exposures tested. The linear increase in mutagenesis is observed even at exposure levels which do not cause significant toxicity. To facilitate comparison of the differing rates of mutagenesis derived from exposure-response curves obtained for each light source, we have defined a parameter, joule-equivalent mutagenesis (jem), equal to mutants per 10(5) survivors per joule per square meter. Jem values are calculated using the integrated irradiance of each lamp. Based on jem values, the relative mutagenicity of the various lamps tested (compared with a germicidal ultraviolet lamp) is 3 x 10(-3) for the sunlamp, 1 x 10(-4) for the black light and cool white lamps, and 3 x 10(-5) for the black light blue lamp. The toxic and mutagenic effects of the lamps are in reasonable agreement with their relative spectral output from 290 to 330 nm.