Loss of P53 heterozygosity is not responsible for the small colony thymidine kinase mutant phenotype in L5178Y mouse lymphoma cells

The mouse lymphoma L5178Y Tk+/- 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk-/- mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/- cells or Tk-/- mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/- 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.     

Molecular analysis of ouabain-resistant mutants of the mouse lymphoma cell line L5178Y

We have carried out molecular and biochemical analyses on severalspontaneous and mutagen-induced ouabain-resistant mutants fromthe mouse lymphoma cell line L5178Y. Mutant cells were muchmore resistant than wild-type cells to the toxic effects ofouabain. The gross structures and copy numbers of two ouabain-resistantgenes were unaltered in the mutants, as was the expression ofthe Na, K-ATPase gene. Uptake of ⁸⁶Rb⁺ was more resistant toouabain in the mutants than in wild-type cells. There was noevidence for an inducible uptake mechanism. The Na, K-ATPaseactivity in extracts of the mutants was, in all cases examined,more resistant than wild-type cells to the inhibitory actionof 10³ M ouabain. At 10⁵M ouabain, the Na, K-ATPase activityin three out of eleven mutants was actually more sensitive toouabin than in wild-type cells. These data suggest that theouabain resistance of the mutants probably results from singlebasechanges in the alpha subunit of the Na, K-ATPase gene, ratherthan from amplification or overexpression of ouabain resistancegenes. The latter types of alterations have been described forouabain-resistant lines derived in other laboratories.     

Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

N-ethyl-N-nitrosourea (ENU) is a potent monofunctional ethylating agent that has been found to be mutagenic in a wide variety of organisms from viruses to mammalian germ cells. To elucidate the mutagenicity of ENU at the Tk(+/-) locus of mouse lymphoma cells and to confirm the ability of the mouse lymphoma assay (MLA) to detect both point mutations and large DNA alterations, Tk(+/-) L5178Y cells were exposed to different doses of ENU. Treatment of the cells with ENU resulted in a linear dose response with mutant frequencies of up to 16-fold over control. Evaluation of mutant clone size showed that 36% of the 100 microg/ml ENU-induced clones (66% in control) were small colony mutants and 64% (34% in control) were large colony mutants. DNA isolated from mutants in the control culture and the 100 microg/ml ENU treatment group was analyzed for loss of heterozygosity (LOH) using allele-specific PCR. The majority of the small colony mutants, both ENU-treated (97%) and spontaneous (91%), lost the Tk1b allele. The percentage of allele loss in ENU-induced large colony mutants was distinctly different from that of the control. Twenty-three percent of ENU-induced large colony mutants lost their Tk1b alleles, whereas 73% of the large colony mutants from the control culture lost the allele (P < 0.001). Overall, 50% of the Tk mutants from the 100 microg/ml ENU-treated cultures (86% in control) showed LOH. Our data indicate that ENU is a potent mutagen in mouse lymphoma cells and that 100 microg/ml ENU induces equal numbers of point mutations and chromosomal mutations. This study serves to verify that the MLA detects both point mutations and chromosomal mutations.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979). 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. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine.HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline.HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline.2 HCl, methyl viologen, nickel sulfate.6H2O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. Apart from 2-biphenylamine.HCl, 2-chloroethanol, disperse yellow 3, ethylene thiourea, FD and C yellow number 6, phenol, and 1,1,2-tetrachloroethane, rat liver S9 mix was not a requirement for these compounds. Chemicals not identified as mutagens were acid red, 11-aminoudecanoic acid, boric acid, 5-chloro-o-toluidine, coumaphos, cyclohexanone, decabromodiphenyl oxide, di(2-ethylhexyl)adipate, ferric chloride, fluometuron, melamine, monuron, phenesterin, phthalimide, reserpine, sodium dodecyl sulfate, 4,4-sulfonyldianiline, tetrachloroethylene, and zearalenone. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these were benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay. II: 18 coded chemicals

Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay by the use of procedures based upon those described by Clive and Spector [Mutat Res 44:269-278, 1975] and Clive et al [Mutat Res 59:61-108, 1979]. 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. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.     

Mutagenicity and clastogenicity of acrylamide in L5178Y mouse lymphoma cells

Acrylamide was tested without exogenous activation in L5178Y/TK+/- -3.7.2C cells for mutation at the thymidine kinase locus and for clastogenicity. Acrylamide gave a positive induced mutagenic response (approximately 70 mutants/10(6) survivors) when tested at 600-650 micrograms/ml. The highest dose tested (850 micrograms/ml) resulted in an induced mutant frequency of approximately 380 mutants/10(6) survivors (survival = 13%). Acrylamide induced almost exclusively small-colony mutants, indicating that it might be acting by a clastogenic mechanism. As predicted, acrylamide was clastogenic, inducing both chromatid and chromosome breaks and rearrangements. A clearly positive clastogenic response was observed at both the 750 micrograms/ml and 850 micrograms/ml doses, which showed 16 and 64 aberrations per 100 cells, respectively (background = 3 aberrations per 100 cells). These studies indicate that the L5178Y/TK+/- mouse lymphoma assay can detect some chromosomal mutagens (clastogens) that show little activity in other single gene mutation assays, the CHO/HPRT and Salmonella.     

Molecular aspects of chemical mutagenesis in L5178Y/tk +/- mouse lymphoma cells

Southern blot analyses were performed on DNA from at least 10 large and 10 small colony thymidine kinase-deficient (tk -/-) mutants induced by each of 10 mutagens [2-amino-N6-hydroxyadenine (AHA), ethyl methanesulfonate (EMS), methyl methanesulfonate, 2-acetylaminofluorene, methotrexate, caffeine, methapyrilene, 4-(9-acridinylamino)-methanesulfo-m-anisidide, hycanthone methanesulfonate and procarbazine]. Two molecular mutant genotypes were recognized upon digestion with NcoI and subsequent probing with a 1.1 kb cDNA insert from plasmid pMtk 4: (i) no detectable alteration, and (ii) the absence of the functional tkb allele as indicated by the absence of the 6.3 kb fragment. In combination with the previously established chromosomal nature of most small colony tk -/- mutants, this permitted the classification of these 10 mutagens according to the relative proportions of each of four classes of genetic damage they induced. AHA and EMS gave mutational spectra consistent with their point mutational effects in other systems. The other eight mutagens induced mostly small colony mutants, most of which had lost the entire original tkb allele. Methotrexate induced high frequencies of large colony mutants at the tk locus, most of which lacked the tkb allele, although it is weakly or non-mutagenic at the hemizygous hprt locus in these same cells. At least three of these mutagens-methotrexate, caffeine, methapyrilene (and possibly procarbazine)--lack structural alerts for DNA reactivity, implying a major class of non-DNA primary targets for mutagenicity in mammalian cells that interact secondarily with the chromosome. These results are discussed in relation to the known differences in sensitivity among various short-term tests for genotoxicity.     

Mutagenesis of L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells by the clastogen ellipticine

Ellipticine is a potent clastogen in CHO cells (Bhuyan et al: Cancer Res 32:2538-2544, 1972). The reported mutant frequencies produced by ellipticine at the hprt locus in CHO cells are less than or equal to 50/10(6) survivors (background approximately 2/10(6); survival = 10%) (DeMarini et al: Cancer Res 43:3544-3552, 1983; Singh and Gupta: Cancer Res 43:577-584, 1983; Environ Mutagen 5:871-880, 1983). In the present study, the mutagenic and clastogenic activities of ellipticine were evaluated in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. Unlike the results at the hprt locus, ellipticine is a potent mutagen at the tk locus, with as little as 50 ng/ml producing an induced mutant frequency of 142/10(6) survivors (background = 56/10(6); survival = 61%) and 198/10(6) survivors (background = 72/10(6); survival = 50%) in two separate experiments. This same dose of ellipticine induced 44 aberrations per 100 metaphases (background = 5/100 cells). At 400 ng/ml, ellipticine induced over 1,000 mutants/10(6) survivors at approximately 10% survival and produced 242 aberrations/100 cells. Under the test conditions, most of the aberrations were chromosome rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, almost all of the TK-deficient mutants were small colonies. Thus, ellipticine is a potent clastogen in both Chinese hamster cells and in mouse lymphoma cells; however, it is a potent mutagen at only the tk locus and not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing the loss of multiple loci. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the more efficient detection of mutagens that act primarily by a clastogenic mechanism.     

Multicolor spectral karyotyping of the L5178Y Tk+/- -3.7.2C mouse lymphoma cell line

The L5178Y/Tk+/- -3.7.2C mouse lymphoma cell line is characterized, at the cytogenetic level, by a karyotype involving both numerical and complex structural aberrations. While the karyotype is remarkably normal for a transformed cell line that has been in culture for almost half a century, there are a number of chromosomal alterations that because of their complexity cannot be fully characterized by routine or even high-resolution G-banding studies. Multicolor spectral karyotyping (SKY) was performed on the cell line in anticipation of identifying the previously unresolved chromosome aberrations and confirming interpretations previously identified by banding studies. New chromosome aberrations detected by SKY include numerical aberrations of chromosome 15, duplications of regions of chromosomes 4, 5, 12, and 18, and deletion of chromosome 14. Complex unbalanced translocations involved segments of chromosomes 6, 14, and 15. In total, the SKY technique was able to provide new refined designations on segments of eight different chromosome pairs (4, 5, 6, 9, 12, 14, 15, 18) and identified all three previously unidentified marker chromosomes. This analysis provides an updated standard reference for the karyotype of the L5178Y/Tk+/- -3.7.2C cell line used in the in vitro mouse lymphoma mutation assay.     

Comparison of chromosome aberration frequency and small-colony TK-deficient mutant frequency in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

The L5178Y/TK(+/-)-3.7.2C mouse lymphoma assay is used to quantitate the induction of thymidine kinase (TK)-deficient mutants. The mutants detected in the assay form colonies that can be distinguished as large or small. The induction of small-colony mutants has been associated with the induction of chromosome mutations. In the present paper, we compare the analysis of induced small-colony TK mutants with gross aberration analysis (the more classical approach to analyzing chromosomal damage). Data are presented for 34 mutagens. As expected, we find that while the induction of gross aberrations and the induction of small-colony TK mutants is correlated, there is no simple mathematical relationship between the two endpoints. The two markers evaluate different subpopulations of chromosome mutations. While either endpoint can be used to detect chromosomal mutations, it should be remembered that the small-colony TK mutants represent genetic events which are compatible with cell viability. Only those alterations compatible with cell viability are a significant risk for human carcinogenicity or mutagenicity.     

Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers (22). We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10^–2 after treatment with 1.5 g/ml ofN-methyl-N-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2–6 times more sensitive to UV than parental L51 T/t cells in terms of D₃₇ (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay to coded chemicals. I: Results for nine compounds

Nine substances were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al (Mutat Res 59:61-108, 1979). 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 coded chemicals were tested at least twice. Significant responses were obtained with calcium chromate, 3-(chloromethyl)pyridine, 1,2-epoxybutane, monochloroacetic acid, dicyclohexylthiourea, 2,4-diaminophenol hydrochloride, and pentachloroanisole. Apart from pentachloroanisole, rat liver S9 mix was not a requirement for the clearly mutagenic activity of any of these compounds. Compounds not identified as mutagens were 3-amino-1,2,4-triazole and sucrose.     

Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium and L5178Y mouse lymphoma cells. Except for the weak response obtained with ferrous fumarate, none of the compounds induced a mutagenic response in Salmonella. In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe+3) to ferrous (Fe+2). Responses with the elemental forms of Fe were divergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9. With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe+2 compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. The lowest effective doses (LED) for induction of mutagenicity were identified for these compounds and an LED ratio calculated. The LED ratio ranges from 1 for FeSO4 to 30 for carbonyl Fe, which are similar to oral LD50 values obtained in animal studies.     

Analysis of the genotoxicity of nine acrylate/methacrylate compounds in L5178Y mouse lymphoma cells

Nine acrylate/methacrylate esters were tested for the inductionof mutations, aberrations and micronuclei in cultured L5178Ymouse lymphoma cells without exogenous activation. With theexception of 2-ethylhexyl acrylate, and dicyclopentenyloxyethylmethacrylate which produced equivocal mutagenic responses, theother seven compounds (2-hydroxyethyl acrylate, dicyclopentenyloxyethylacrylate, tetraethylene glycol diacrylate, tetraethylene glycoldimethacrylate, trimethylolpropane triacrylate, trimethylolpropanetrimethacrylate, and pentaerythritol triacrylate) produced positivemutagenic responses with different potencies. For the mutagenicacrylates/methacrylates, primarily small-colony, trifluorothymidine(TFT)-resistant mutants were induced, suggesting a clastogenicmechanism that was supported by increased aberration and micronucleusfrequencies (except for trimethylolpropane trimethacrylate whichwas positive for aberration but not micronucleus induction).Generally, it was found that multifunctional compounds (esterswith > 1 functional vinyl group) required lower concentrationsthan monofunctional compounds to induce maximal cytotoxic, mutagenic,and clastogenic responses. In addition, acrylates were generallymore potent than their corresponding methacrylates. This informationand these comparative activities will provide some guidancefor setting priorities of concern for hazard consideration foracrylate/methacrylate ester compounds. ⁴To whom correspondence should be addressed     

Differential cell-cycle-dependent inhibition of DNA synthesis by hydroxyurea and cytosine arabinoside in cultured L5178Y mouse lymphoma cells

The effect of various concentrations of hydroxyurea and cytosine arabinoside on DNA synthesis, measured as 3H-thymidine incorporation, in exponentially growing and synchronized L5178Y cells in culture was investigated. The experiments revealed that each compound causes differential inhibition of DNA synthesis at various times of the S-phase. Maximum sensitivity to hydroxyurea occurred in cells in the late S, and maximum sensitivity to cytosine arabinoside in the early S-phase. When they were given simultaneously in low concentrations an additive effect was observed.     

Identification of potential biomarkers of genotoxicity and carcinogenicity in L5178Y mouse lymphoma cells by cDNA microarray analysis

In the present study, cDNA microarray analyses were performed with mouse cDNA chips in order to evaluate similarities and differences in the gene expression profiles for compounds differing in their genotoxic and carcinogenic potential. Eight test substances were evaluated, two each from four classes of compounds: genotoxic carcinogens (1,2-dibromoethane and glycidol), genotoxic noncarcinogens (8-hydroxyquinoline and emodin), nongenotoxic carcinogens (methyl carbamate and o-nitrotoluene), and nongenotoxic noncarcinogens (D-mannitol and 1,2-dichlorobenzene). Quadruplicate hybridization experiments were performed in order to identify a set of genes with significant expression changes for these four classes of substances. Twelve genes were consistently altered more than twofold by the genotoxic noncarcinogens while four genes were consistently regulated by the nongenotoxic carcinogens. One gene (Trp63) was identified whose expression was upregulated by all four genotoxic substances regardless of the presence or absence of carcinogenicity; this finding, however, was not confirmed by quantitative real-time RT-PCR. RT-PCR did confirm the change in expression of 9 of 15 genes (60%) identified by microarray analysis. Interestingly, the downregulated genes were least likely to be validated by real-time RT-PCR. Those genes showing more than a twofold change in expression level in response to at least one substance were further analyzed with hierarchical clustering after category assignment of each gene according to its main cellular function. Clustering revealed differences in the gene expression profiles between the genotoxic and nongenotoxic substances for genes involved in cell cycle control, the stress response, and the immune response. However, no clustering specific to all four carcinogenic substances was observed in any of the functional categories. Taken together, these results suggest that gene expression profiling in mouse lymphoma cells can provide valuable information for the evaluation of potential genotoxicity but may have limitations in predicting carcinogenicity.     

Supra-additive genotoxicity of a combination of gamma-irradiation and ethyl methanesulfonate in mouse lymphoma L5178Y cells

While testing for genotoxicity is usually performed on single chemicals, exposure of humans always comprises a number of genotoxic agents. The investigation of potentially synergistic effects of combinations therefore is an important issue in toxicology. Combinations of 511 keV gamma-radiation with the chemical alkylating agent ethyl methane-sulfonate were investigated in the in vitro micronucleus test in mouse lymphoma L5178Y cells. With combinations in the low dose linear effect range for the individual agents (0. 25-2 Gy and 0.8-3.2 mM, respectively), supra-additivity by 34-86% was seen. The synergism was more pronounced at the higher dose levels. Supra-additivity was confirmed in experiments using cytochalasin B and analyzing binucleate cells only, to control for putative effects on the cell cycle. Statistical significance was shown by a 2-factor analysis of variance with interaction. The results indicate that damage to DNA by gamma-radiation and alkylation could affect different rate limiting steps in the formation of micronuclei. Further investigations will have to show whether the observations are of general validity, in particular, whether other end-points of genotoxicity produce the same results and whether the degree of supra-additivity is always dose dependent. The latter would have a strong impact on risk assessment for mixtures at low doses.     

Development of an optimal S9 activation mixture for the L5178Y TK+/- mouse lymphoma mutation assay

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17-29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non-toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (less than or equal to 4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at greater than or equal to 50% or more of the maximum over the usual 4-hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3-methylcholanthrene (MCA), 2-acetylaminofluorene (AAF), benzo(a)pyrene (BAP), 9,10-dimethyl-1,2-benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)-possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.     

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.     

Reactivity of catecholamines and related substances in the mouse lymphoma L5178Y cell assay for mutagens

Using the L5178Y mouse lymphoma cell thymidine kinase locus and the Salmonella his locus assays, the mutagenic potentials of several catecholamines and related compounds were examined. No supplementary metabolic activation systems were used. In the mouse lymphoma assay, the dihydroxybenzenes catechol and hydroquinone had similar and appreciable mutagenic potentials, whereas resorcinol was less active. Derivatives of catechol, such as dopamine and epinephrine, were mutagenic, whereas the related monohydroxylated compounds tyramine and synephrine were inactive. The primary amine, arterenol, and the corresponding secondary amine, epinephrine, induced similar mutagenic responses. Carboxylation of the side chain of dopamine, giving L-dopa, reduced the maximum mutagenic response. The introduction of charged groups directly on to the aromatic ring also reduced mutagenic activity, while an intervening methylene reversed this effect. Thus, 3,4-dihydroxyhydrocinnamic acid was more active than 3,4-dihydroxybenzoic acid. The compound active at the lowest doses was 4-tert-butyl catechol. The activities of these compounds are highly dependent upon substituent groups. Experiments with superoxide dismutase gave circumstantial evidence for some of the mutagenic activity being due to superoxide anion. Active oxygen species might be responsible for some of the observed effects, but this cannot be concluded from the superoxide dismutase experiments. Mutagenic responses in Salmonella were very low but were significant for L-dopa in three strains and for epinephrine and arterenol in one strain. Limited DNA association studies of 14C-dopamine suggest interactions in L5178Y and Salmonella cells and in mouse liver. The mutagenicity of dopamine in L5178Y is reduced by high serum concentrations during the exposure period, while the apparent association with DNA is unaffected.