William J. Caspary

Endogenous xenobiotic enzyme levels in mammalian cells

The response of mammalian cell lines to chemicals depends, in part, on the exogenous activation system used for the induction of a biological response. This could be attributed to differences in the expression of enzymes involved in xenobiotic metabolism. We have measured the activities of benzo[a]pyrene hydroxylase, dimethylaminoazobenzene N-demethylase, catalase, superoxide dismutase, peroxidase and glutathione-S-transferase in human lymphoblast TK6, mouse lymphoma L5178Y, Chinese hamster ovary (CHO) and lung (V79) and mouse C3H10T1/2 cell lines as well as in primary hepatocytes and S9 preparations of liver from male F344 rats. Nitroreductase was also measured in some of these preparations. Human lymphoblast TK6 and mouse C3H10T1/2 cells had the capacity to metabolize dimethylaminoazobenzene and the latter cell line also metabolized benzo[a]pyrene, indicating the presence of constitutive mono-oxygenase activity. Cytochrome P450 could not be detected spectrophotometrically in the cell lines. Western blot analysis indicated that P450 from the P450IIA family is expressed in C3H10T1/2 cells. Reactivity was also observed with an antibody to P450IA2; however, the identity of this protein remains uncertain. Superoxide dismutase, catalase and peroxidase, which protect cells against oxygen radical damage, were found in all the cell lines and in rat hepatocytes and S9. The human lymphoblast TK6 cell line, however, had the least of each of these three enzymes. Glutathione-S-transferase activity was detected at varying levels in all cell types. Nitroreductase activity was high in S9 and Chinese hamster ovary cells and lower in mouse lymphoma and Chinese hamster V79 cells.

Mutagenic activity of fluorides in mouse lymphoma cells.

The L5178Y mouse lymphoma cell forward-mutation assay was used to test for the mutagenic activity of sodium and potassium fluoride at the thymidine kinase locus. Mutants were detected by colony formation in soft agar in the presence of trifluorothymidine. Mutagenic and toxic responses were observed in the concentration range of 300-600 micrograms/ml with both sodium and potassium fluoride. Approximately 3-fold increases in mutant frequency were observed for concentrations in the 500-700 micrograms/ml range that reduced the relative total growth to approximately 10% in the absence or presence of a rat-liver S9 activation system. A sample of 30% sodium fluoride-70% sodium bifluoride (NaHF2) induced a similar mutagenic response but was more toxic with respect to the fluoride concentration. A specificity for fluoride ions in causing mutagenesis was indicated by the fast that much higher concentrations of sodium or potassium chloride were necessary to cause toxicity and increases in the mutant frequency. The possible involvement of chromosomal changes was signaled by the predominant increase in the small colony class of mutants.

Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures

Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size.

The mutagenic activity of selected compounds at the TK locus: Rodent vs. human cells

The mutagenic (TFT resistance) and toxic responses of mouse lymphoma (MOLY) L5178Y cells and human lymphoblast (HULY) TK6 cells were compared for 13 chemicals. The mutagenic activities of 8 of the 13 chemicals (62%) examined in the HULY and MOLY assays are in agreement - the results being judged positive in both assays. However, a dramatic difference is observed when the two conditions of metabolic activation are considered separately; the overall concordance of 8/13 has been achieved by combining a 13/13 (100%) agreement in the absence of S9 with a 1/6 (17%) agreement in the presence of S9. In the absence of S9, the concentration ranges, lowest significant doses, and shapes of the concentration-response curves for both toxicity and mutagenicity were similar in spite of the differences in exposure times (4 h for MOLY, 20 for HULY) and expression times (2 days for MOLY, 3 days for HULY). The general agreement observed in the absence of S9 contrasted with the differences manifested in its presence. 6 compounds which were negative in the absence of S9 were tested in both the MOLY and HULY assays in the presence of S9. Of the 6 chemicals, only 1 was positive in both MOLY and HULY under the latter condition; 4 others were positive in MOLY and negative in HULY whereas 1 was positive in HULY and negative in MOLY.

Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells

5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. Supravital UV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.

An in situ protocol for measuring the expression of chemically-induced mutations in mammalian cells

The generation of expression curves and the evaluation of mutagenic responses of mammalian cells using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio with time and the mutant fraction will not accurately represent the number of mutational events that occurred. The mutant fraction may also inaccurately assess the number of mutations if these mutations are expressed over a number of generations during the time before selection. We previously showed that recovery of L5178Y mouse cell mutants is not complete when mutations are allowed to express in suspension because slowly growing mutants and/or mutant progenitors are diluted out during this time (Rudd et al., 1990). In order to more accurately quantitate the mutagenic response of the cells, we developed an in situ procedure which segregates and immobilizes cells during expression. Because of this immobilization, slowly growing mutant progenitors and mutants expressed at different times will have an equal probability of being scored as mutants. Thus, one mutation leads to one mutant colony and the measurement of the mutagenic response of the cells to the chemical accurately reflects the mutational events that occurred. We plated L5178Y tk+/- mouse cells in semisolid medium immediately after treatment. As the cells grew and formed microcolonies, the selective agent TFT was added as an overlay at specified times, permitting only TFTr cells to survive. In this procedure, each mutation was captured as an individual colony; consequently, the measured mutation fraction accurately reflected the mutational events that occurred at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We previously described the in situ protocol for L5178Y cells and showed that the spontaneous mutation rate measured was 50-fold greater than when the cells expressed the phenotype in suspension (Rudd et al., 1990). From this we concluded that the slow growth phenotype was expressed before TFT resistance. In the present paper, we evaluate the effect of chemical treatment on the mutation fraction as a function of the time to TFT addition. Using the in situ protocol, we generated expression curves for three nucleotide analogs, 5-azacytidine, TFT and AraC. The numbers of TFTr colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Location of iron in the Fe2+-bleomycin complex as observed by 13C NMR spectroscopy

Abstract The antineoplastic action of bleomycin is currently thought to arise from the degradation of cellular DNA by the iron-bleomycin complex. Bleomycin A2 has one iron binding site as revealed by the iron-titrations of bleomycin monitored optically. To probe the structure of the Fe2+-bleomycin complex, we studied the paramagnetic effects of its high spin ferrous iron on the nuclear relaxation rates ( 1 T 1 ) of the natural abundance carbon-13 atoms in the molecule. The presence of Fe2+ in bleomycin predominantly enhances the 1 T 1 of only four protonated carbon atoms in the molecule (C2, C3, C5, and C6). No other protonated carbon atoms are affected significantly. From the magnitudes of the paramagnetic effects of Fe2+ on the 13C relaxation rates, we obtain distances of 3.6, 4.1, 4.0, and 3.6 A from the metal to the C2, C3, C5, and C6 carbon atoms, respectively. These results are consistent with the metal ion-chelation of the α-amino group of the terminal diaminopropionic acid residue and the pyrimidine ring but do not implicate any other parts of the bleomycin molecule in binding to iron.

Mathematical model of L5178Y mouse lymphoma forward mutation assay

A mathematical model of the biological protocol for the Mouse Lymphoma L5178Y Forward Mutation Bioassay is presented. The model relates the mutant progenitor frequency (MPF), the number of cells per million surviving cells with DNA damage after exposure to the chemical, to the mutant frequency (MF), the number of TFT-resistant cells per million survivors. For a given expression time, the deterministic relationship is linear and the proportionality constant depends on the relative suspension growth factor (rg) and relative cloning efficiencies (rc) of mutants to those of wild type cells: MF = (rg X rc) X MPF. Experimental noise leads to variations in the values of rg and rc and lack of reproducibility in the system. If mutant progenitors and their progeny grow as well as wild-type cells and if all of the parental mutant progenitors express the mutant phenotype, then rg = 1/2 and rc = 1. Biological mechanisms, such as differential growth characteristics of mutant and wild-type cells or DNA repair, can make the mutant frequency an inaccurate estimate of the MPF. For the assay to be useful as a screen for the mutagenic activity of chemicals, rg X rc has to be reasonably constant from chemical to chemical.

Stable dicentric chromosomes induced by chemical mutagens in L5178Y mouse lymphoma cells

Stable, tandem dicentric chromosomes were discovered in two mutant cell colonies resulting from exposure of L5178Y mouse lymphoma cells to chemical mutagens. These unusual dicentrics were present in all metaphase cells examined from these colonies, even after approximately 65 cell generations in culture. Observation of cells in metaphase and anaphase suggests that the interstitial centromere in these dicentrics is non-functional, and that the terminal centromere is solely responsible for their orderly anaphase segregation.

Identification of a heteromorphic microsatellite within the thymidine kinase gene in L5178Y mouse lymphoma cells.

The objective of this work is to identify a heteromorphism within the thymidine kinase (Tk1) gene which can be used to assay for allele loss by means of PCR. Intron F of mouse Tk1 contains two (CA)n microsatellite sequences separated by 107 bp of non-repetitive sequence. We tested this region for heteromorphism in L5178Y mouse lymphoma cells. A PCR primer pair designated Agl1 yielded products of 396 and 194 bp from L5178Y tk+/- genomic DNA. The 194-bp product resulted from a secondary binding site between the two (CA)n repeats for the forward Ag11 primer and was not produced from tk-/- mutants that had lost the functional Tk1b allele. Agl2 primers produced two PCR products of 523 and approximately 440 bp and Agl3 primers produced products of 579 and approximately 500 bp. In both these cases, the difference in product size was approximately equal, indicating that Intron F is approximately 80 bp shorter in the non-functional Tk1a allele than in Tk1b. This heteromorphism forms the basis for an assay for allele loss by means of PCR. Agl1 and Agl3 primers yielded additional products of 91 and 274 bp, respectively, consistent with sizes expected from the mouse Tk1 pseudogenes (Tk1-ps). Our conclusions drawn from an analysis of 122 mutants for Tk1b loss using Agl2 primers agreed with previous analysis of the NcoI heteromorphism. Thus, a simple PCR-based analysis can identify Tk1b loss in the L5178Y mouse lymphoma cells.