Molecular analysis of 5-azacytidine-induced variants in mammalian cells.

5-azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC-->CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known.

Aflatoxin B1-induced mitotic recombination in L5178Y mouse lymphoma cells.

Aflatoxin B1 is a human hepatocarcinogen. It is also a known point mutagen in bacteria and mammalian cells. This mutagenic activity may be at least partly responsible for its carcinogenic activity. However, recent studies show that aflatoxin B1 induces mitotic recombination in the yeast Saccharomyces cerevisiae. Because numerous reports have implicated mitotic recombination in mechanisms leading to carcinogenesis and because no one has shown that aflatoxin B1 induces recombination in mammalian cells, we decided to examine the ability of aflatoxin B1 to induce recombination in a mammalian cell line. We used a combination of methods, analysis for loss of heterozygosity and whole chromosome in situ hybridization, to identify mechanisms of chromosome mutation, including mitotic recombination in the mammalian L5178Y mouse lymphoma cell system. Our experiments revealed that mitotic recombination caused approximately 60% or more of the aflatoxin B1-induced mutagenic lesions in this cell system. Thus, mitotic recombination plays an important role in aflatoxin B1-induced mutagenesis in mammalian cells and possibly in chemically induced mutagenesis and carcinogenesis. This work suggests that multiple genetic lesions may be involved in aflatoxin B1-induced pathology.

Analysis of large and small colony L5178Y tk-/- mouse lymphoma mutants by loss of heterozygosity (LOH) and by whole chromosome 11 painting: detection of recombination.

Analysis of 122 spontaneous large and small colony mutants derived from L5178Y tk +/- mouse lymphoma cells at 28 heteromorphic microsatellite loci on chromosome 11 showed that extensive loss of heterozygosity (LOH) is common in both large colony and small colony mutants, eliminating most chromosome 11 loci as candidates for a putative growth control locus. These results, in conjunction with historical cytogenetic data, suggest that a putative growth control locus lies distal to the thymidine kinase (Tk1) gene, near the telomere. Thirty seven mutants were hybridized with a chromosome 11-specific whole chromosome painting probe for analysis of rearrangements. Generally, painting confirmed earlier observations that large colony mutants are karyotypically normal, whereas small colony mutants frequently have detectable rearrangements. A point probe distal to Tk1 revealed no evidence of chromosome breakage in small colony mutants that appeared normal on whole 11 painting and had no LOH. Therefore, the molecular difference between large and small colony mutants remains unknown. Models to explain large and small colony mutants consistent with our findings are presented, including loss of a putative growth control gene, differential mechanisms of chromosome breakage/repair and second site mutations as explanations for small colony mutants. Painting revealed translocations and aneuploidy and showed that non-disjunction was not a common explanation for complete LOH. The most common finding was that large regions of LOH do not result from deletions, demonstrating that these cells can detect recombination events as well as previously observed chromosomal rearrangements, deletions and point mutations.

Micronuclei containing whole chromosomes harbouring the selectable gene do not lead to mutagenesis.

Loss of heterozygosity is one genetic change observed in many tumours. We do not know whether the loss of chromosomal material through micronucleus formation is a viable mechanism associated with, and possibly leading to, genetic disease. Previously, we treated L5178Y mouse lymphoma cells with four aneugens. Although these aneugens induced micronuclei containing predominantly whole chromosomes, they did not induce mutations at Tk1, the selectable gene, under the same non-toxic conditions in which they induced micronuclei. This suggested that the induction of micronuclei containing whole chromosomes was not an early event leading to phenotypically expressed mutations in these cells under the conditions used. However, it is possible that chromosome 11, on which Tk1 resides, may be under-represented in the micronucleus population. To find out the frequency of induction of micronuclei containing chromosome 11, we applied fluorescence in situ hybridization using a chromosome 11 paint to micronuclei induced by colcemid and vinblastine. We found that the numbers of micronuclei containing chromosome 11 are more than sufficient to be detectable as mutations if these micronuclei lead to viable mutants. We conclude that the formation of micronuclei containing whole chromosomes does not lead to viable, dividing mutants in this system.

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.

5-Azacytidine-induced 6-thioguanine resistance at the gpt locus in AS52 cells: cellular response.

Treatment of AS52 cells with 5-azacytidine resulted in an induction of 6-thioguanine-resistant [6TG] colonies, which reached a maximum by an expression time of 9 days. Dose responses for both cytotoxicity and mutation induction were determined following treatment with 5-azacytidine. At 20 microM treatment, 5-azacytidine exposure resulted in about 50% survival. Mutant frequency reached a maximum of 10 microM. At concentrations between 10 and 20 microM, 5-azacytidine was a potent mutagen but did not exhibit a dose response. Although many compounds both induce cell death and affect the growth rate of cells, 5-azacytidine specifically induced cell death and did not affect the doubling time of the surviving treated cell population.

Mutagenic responses of L5178Y mouse cells at the tk and hprt loci.

The expression of multiple recessive genes by aberrant mitotic lesions plays a major part in carcinogenesis. These lesions include intragenic mutations as well as chromosomal lesions. An in vitro model for studying carcinogenesis should respond to all these lesions. Mutagenesis studies that target hemizygous loci may not be useful in studying chromosomal mechanisms because lesions incorporating essential genes already missing on the inactive, homologous chromosome may be lethal to the cell. Cells heterozygous at the selectable gene may survive. Using L5178Y mouse cells, we compared the mutagenic responses at the heterozygous tk and hemizygous hprt loci to four chemicals-benzidine dihydrochloride, diglycidylresorcinol ether, nitrofen and p-benzoquinone dioxime. None of the compounds induced clear positive responses at the hprt locus. In contrast, all the compounds induced clear or marginal mutagenic responses at the tk locus. These data are consistent with the expectation that heterozygous loci can detect lesions that are refractory to hemizygous loci.

Mouse chromosome-specific painting probes generated from microdissected chromosomes.

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosome probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.

The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals.

Guidelines have been proposed to assess the potential of chemicals to affect human health. Written into these guidelines is the requirement that information be submitted on mutagenic activity. Although regulatory agencies accept mutagenicity data from both the hprt and tk loci in mammalian cells, many studies suggest that the L5178Y mouse lymphoma assay at the thymidine kinase locus is likely to detect a greater spectrum of mutagenic lesions. Thus, there is increasing emphasis being placed on this assay in many proposed and published guidelines. The L5178Y mouse lymphoma suspension protocol produces both small and large colonies which are the products of mutants growing at different rates. There is a reduction in the proportion of slowly growing mutants with respect to the total population of cells when expression is carried out in suspension. This potentially leads to quantitatively inaccurate assessments of the mutagenic activity of chemicals. Therefore an in situ procedure was developed that more accurately assesses the mutagenic activity of chemicals by maximizing the detection of small colonies. Many guidelines recommend tests that assess the clastogenic activity of chemicals. Some regulatory agencies accept data from the mouse lymphoma mutation assay to detect clastogens if the protocol is optimized for the detection of small colonies or if colony sizing data are submitted. The conventional suspension assay protocol is not sufficiently validated for this purpose. The in situ protocol has greater potential to meet these requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Micronuclei induced by modulators of methylation: analogs of 5-azacytidine.

Jones and coworkers demonstrated a qualitative correlation between 5-azacytidine and some of its analogs in inducing changes in cell morphology and their ability in preventing DNA methylation. Previously, we evaluated the same compounds to determine their ability to induce trifluorothymidine (TFT) resistance in L5178Y mouse cells and found that their mutagenic potency also correlated with their reported ability to induce morphological changes in C3H10T1/2 cells. Here, we examine four of the same analogs, 5-fluoro-2'-deoxycytidine, 5-azacytidine, 5,6-dihydro-5-azacytidine and 6-azacytidine, to find out if micronuclei induced by these compounds correlated with these effects. The most cytotoxic analog was 5-fluoro-2'-deoxycytidine, followed by 5-azacytidine. 5,6-Dihydro-5-azacytidine and 6-azacytidine were substantially less cytotoxic. All four compounds induced micronuclei. The lowest dose ranges at which responses were observed for micronucleus induction were -0.04 microM for 5-fluoro-2'-deoxycytidine, 0.2 microM for 5-azacytidine and 10-20 microM for 5,6-dihydro-5-azacytidine and 6-azacytidine. Lack of kinetochore staining in most of the micronuclei indicated that all four compounds were clastogenic. We note a general trend in the biological activity of these analogs: compounds that are specifically blocked at the 5 position such as 5-azacytidine and 5-fluoro-2'-deoxycytidine effect changes in cell morphology, cytotoxicity, TFT resistance and the induction of micronuclei at very low doses. 5-Azacytidine analogs that possess more chemically accessible 5 positions such as 5,6-dihydro-5-azacytidine and 6-azacytidine either require doses that are orders of magnitude greater to induce these effects or are unable to induce changes in cell morphology and TFT resistance at doses below which the compound is lethal to the cells.

In situ and suspension protocols for chemically-induced mutation at the tk locus in L5178Y MOLY cells: dose response and colony size distribution.

We used EMS up to concentrations of 0.25 microliters/ml (292 micrograms/ml) to induce mutations at the tk locus in L5178Y MOLY cells, measured the cellular response by the in situ mutation assay protocol and compared these results to those obtained in a concomitant suspension assay. EMS induced mutagenic responses with both protocols. The mutant fraction for the solvent control was 89 mutants per million viable colonies for the suspension protocol and 426 mutations per million viable cells plated for the in situ protocol. These numbers increase to 447 and 2073 respectively, with 0.25 microliter/ml EMS treatment. Sizing curves indicated that the in situ protocol detected a greater proportion of smaller colonies than did the suspension protocol. Not only were the number of small colonies greater than large colonies in the in situ protocol, but their rate of increase was also slightly higher than that of the large colonies. The in situ protocol also reduces the time and cost of experimentally performing the assay compared to the suspension protocol. In this paper we compare the use of the suspension and in situ protocols to measure chemically-induced mutations and demonstrate that the latter method detects a larger fraction of induced mutations at the tk locus in L5178Y MOLY cells.

Is micronucleus induction by aneugens an early event leading to mutagenesis?

This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological consequence of micronuclei with mutation induction as measured by trifluorothymidine (TFT) resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vinblastine. All four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained whole chromosomes. However, these same compounds were unable to induce TFT resistance under three different treatment regimes. We concluded that these compounds, under conditions where they induce primarily kinetochore positive micronuclei, were not able to induce mutations. Thus, the induction of micronuclei containing whole chromosomes harboring a selectable gene is not an early event leading to mutations in these cells.

Loss of heterozygosity in spontaneous and chemically induced tumors of the B6C3F1 mouse.

The B6C3F1 mouse is used worldwide to gauge the carcinogenic hazard posed by chemicals to humans. An assessment of the ability of this rodent model to predict human neoplasia requires an evaluation of similarities and differences in the genetics of tumor formation between these two species. We examined 142 spontaneous and chemically-induced liver tumors isolated from the B6C3F1 mouse for losses of heterozygosity (LOH) at 78 polymorphic loci and compared these results to genetic changes known to occur in human hepatocellular carcinoma. Approximately a third of the 142 mouse tumors exhibited LOH, suggesting that tumor suppressor gene inactivation may be involved in the formation of mouse liver tumors. Most of the LOH observed was restricted to seven chromosome sites and most of the tumors that underwent LOH lost alleles from only one of those seven sites. The relatively few losses seen in these mouse tumors distinguished them from clinical stage human tumors in that, in the mouse tumors, interstitial deletions appeared more frequently than losses of whole chromosomes. Only four mouse tumors lost a whole chromosome. LOH occurred at loci of the mouse genome syntenic to areas of the human genome known to harbor the Wilms', retinoblastoma, APC, MCC and DCC tumor suppressor genes; these genes have never been associated with hepatocellular carcinomas. Losses observed on chromosomes 5 and 8 (syntenic to human chromosomes 4 and 16) suggest tumor suppressor genes that are common to hepatocellular carcinomas from both species, while losses on chromosome 9 suggest involvement of a previously unidentified tumor suppressor gene.

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)

An investigation of micronucleus and mutation induction by oxazepam in mammalian cells.

The benzodiazepines are a class of drugs that are widely used in the treatment of various psychiatric disorders. One member of this class, oxazepam, is also a common metabolite of several other benzodiazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is inconsistent, we investigated the oxazepam-induced formation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and L5178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all three cell lines. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were formed in the first mitosis after treatment. Kinetochore staining (CREST-antiserum) revealed the presence of kinetochores in approximately 50% of the micronuclei in all three cell types. This result was further confirmed by in situ hybridization in L5178Y cells and indicates the presence of whole chromosomes or centric fragments as well as acentric fragments in the oxazepam-induced micronuclei. The L5178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.

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.

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.

Responses of the L5178Y mouse lymphoma forward mutation assay: V. Gases and vapors.

A new protocol for testing vapors and gases in the L5178Y mouse lymphoma assay is presented. Four chemicals, propylene, 1,2-propylene oxide, 1,3-butadiene, and vinylidene chloride, were tested for their mutagenic potential. Cultures were exposed to the chemicals, which were delivered as vapors or gases, for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 microgram/ml. Each chemical was tested at least twice. Significant responses were obtained with 1,2-propylene oxide and vinylidene chloride, but neither cytotoxicity nor mutagenicity was induced by 1,3-butadiene; propylene could not be classified as either mutagenic or non-mutagenic in the assay. Rat liver S9 mix was not a requirement for the mutagenic activity of 1,2-propylene oxide, whereas the liver preparation markedly enhanced both the cytotoxicity and mutagenicity of vinylidene chloride.

Responses of the L5178Y mouse Lymphoma cell forward mutation assay. V: 27 coded chemicals.

Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay using procedures based upon those described by McGregor et al. (McGregor DB, Martin R, Cattanach P, Edwards I, McBride D, Caspary WJ (1987): Environ Mol Mutagen 9:143-160). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde, o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1-phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds. Chemical not identified as mutagens were N-4-acetylaminofluorene, chlorpheniramine maleate, chloropropamide, 1,4-dioxane, endrin, ethylene glycol, iron dextran, methapyrilene, sodium(2-ethylhexyl)alcohol