Efficient mutation induction by 125I and 131I decays in DNA of human cells

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.     

Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells

Three classes of TFTr variants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells can be identified--large colony (lambda), small colony (sigma), and tiny colony (tau). The sigma and lambda mutants are detectable in the routine mutagenesis assay using soft agar cloning. The tau mutants are extremely slow growing and are quantitated only in suspension cloning in microwells. Variants of all three classes have been analyzed in the process of evaluating the usefulness of the thymidine kinase locus in L5178Y/TK+/- mouse lymphoma cells for detecting induced mutational damage. 150 of 152 variants from mutagen treated cultures and 163 of 168 spontaneous mutants were TFTr when rechallenged approximately 1 week after isolation (3 weeks after induction). All of the 41 mutants assayed for enzyme activity were TK-deficient. The sigma and tau phenotypes were found to correlate with slow cellular growth rates (doubling time greater than 12 h), rather than from effects of the TFT selection or mutagen toxicity. Cytogenetic analysis of sigma mutants approximately 3 weeks after induction shows an association between the sigma phenotype and readily observable (at the 230-300 band level) chromosomal abnormalities (primarily translocations involving that chromosome 11 carrying the functional TK gene) in 30 of 51 induced mutants studied. Using an early clonal analysis of mutants (approximately 2 weeks after induction) 28 of 30 sigma mutants showed chromosome 11 rearrangements. All lambda mutants studied (17 of 17 evaluated 3 weeks after induction and 8 of 8 evaluated 2 weeks after induction) showed normal karyotypes (at the 230-300 band resolution level), including the chromosome 11s. These observations support the hypothesis that sigma (and likely tau) mutants represent chromosomal mutations and lambda mutants represent less extensive mutations affecting the TK locus. The inclusion of sigma mutants in the total induced mutant frequency, as well as distinguishing them as a separate subpopulation of TK-deficient mutants, is, therefore, essential in obtaining maximum utility of the information provided by the L5178Y/TK+/- mouse lymphoma assay.     

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Mutagenicity of actinomycin D in mammalian cells due to clastogenic effects

Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism.     

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.     

Do trifluorothymidine-resistant mutants of L5178Y mouse lymphoma cells re-express thymidine kinase activity following 5-azacytidine treatment?

TFT is an effective selective agent for TK-deficient mutants of L5178Y TK+/- -3.7.2C mouse lymphoma cells. Mutants can be classified by colony size into small colonies (many of which show readily observable chromosome abnormalities associated with chromosome 11--the location of the TK gene) and large colonies (which may represent events affecting only the expression of the TK gene). The precise nature of the induced damage causing the loss of the TK-enzyme activity for both mutant type is not known and is currently under investigation. The hypomethylating agent 5-azacytidine can be utilized to investigate the possibility that mutants might be the result of a suppressed rather than an altered TK gene. Mutant cell lines are treated with 5-azacytidine and then evaluated for re-expression of the TK enzyme as measured by resistance to THMG. In these studies, 11 mutants have been evaluated. None of the 11, including 10 small-colony mutants (6 with chromosome 11 translocations) and 1 large-colony mutant, show a high conversion to TK competency following 5-azacytidine treatment.     

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants

Mouse lymphoma cells of the L5178Y TK+/- -3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK+/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.     

The activation of procarcinogens to mutagens by cultured rat hepatocytes in the L5178Y/TK mutation assay

Whole cell preparations derived from collagenase-treated rat liver were cocultivated overnight with stationary (non-shaking) cultures of L5178Y/TK+/- cells in the presence of 8 different chemicals selected as representative aromatic amine, polycyclic hydrocarbon, or nitrosamine procarcinogens. When tested in the presence of hepatocytes, 2-aminoanthracene, 2-aminofluorene, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, 3-methylcholanthrene, and benzo[a]pyrene all produced substantial dose-dependent increases in trifluorothymidine-resistant variants compared to solvent controls after 20 h total exposure time. Only N-nitrosodipropylamine (DPrN) and N-nitrosodiethylamine (DEN) produced any dose-related mutagenic activity in similar experiments where hepatocytes were omitted; however, the response for the DPrN was quite variable at high doses in the absence of hepatocytes and the mutagenic response for the DEN was consistently enhanced at all dose levels by the presence of hepatocytes. Benzanthracene was not active in the presence of whole hepatocytes, even when tested with cells from a rat pretreated 24 h earlier with 20 mg/kg benzanthracene. Excepting benzanthracene, these data suggest that rat hepatocytes can be used to active 3 types of procarcinogens to mutagens in the L5178Y/TK gene mutation assay.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

The mutagenic potency of 4 agents at the thymidine kinase locus in mouse lymphoma L5178Y cells in vitro: effects of exposure time

Mutagenic potency at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cells (expressed as induced trifluorothymidine (TFT)-resistant mutants/total dose) was assessed for 4 agents (ethyl methanesulphonate (EMS), benzidine, 1,8-dinitropyrene (1,8-DNP) and ICRF 159) using short (3-4 h) and long (21-24 h) exposure times. The mutagenic potency of EMS was found to be essentially independent of concentration and exposure time when tested over a cytotoxic range consistent with routine testing procedures. Similar results were obtained with benzidine but for both 1,8-DNP and ICRF 159 mutagenic potency was found to be highly dependent on the concentration and exposure time. 1,8-DNP failed to induce any significant increases in mutant frequency when tested at concentrations up to 5 micrograms/ml using short exposure times, whereas the compound was active at concentrations as low as 0.1 microgram/ml when the exposure period was extended to 21 h. Under the latter conditions, however, the molar potency of 1,8-DNP was found to be inversely related to concentration over a range extending from 0.1 to 5 micrograms/ml. ICRF 159 induced increases in the frequency of TFT-resistant mutants using short or long exposure times. When a short exposure time was used, however, the mutagenic potency of the antitumour agent decreased with increasing concentration between 1 and 500 micrograms/ml. Although possible explanations can be offered to account for these observations the results illustrate potential problems which may arise in this system when comparing mutagenic potency values for a range of compounds with a view to assessing relative risk.     

Genotoxicity of 6 oxime compounds in the salmonella/mammalian-microsome assay and mouse lymphoma TK +/- assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/- assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.     

A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).     

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.     

Large macromolecules can be introduced into cultured mammalian cells using erythrocyte membrane vesicles

Plasmid 6.4 kbp DNA, 14 kbp DNA, lambda phage particles, all of which contained herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, or IgM molecules, were mixed with erythrocyte membranes and treated with neutral detergent. The transparent mixture was diluted with phosphate-buffered saline (PBS), followed by centrifugation to collect membrane vesicles containing the large macromolecules. 10-15% of 6.4 kbp, 3% of 14 kbp, 4-7% of the lambda phage particles and 14.5% of IgM were trapped within erythrocyte membrane vesicles. The membrane vesicles containing these molecules were fused with L cells, or rat F2408#20 cells, both of which are deficient in thymidine kinase activity. In each case, transformants were obtained. 2 X 10(5) - 7 X 10(5) phage PFU or 1.5 X 10(6) - 8 X 10(7) DNA molecules were required to obtain one transformant from L cells, but 2-3 X 10(7) phage PFU or 2 X 10(9) - 1 X 10(10) DNA molecules were required for one transformant from rat cells. Number of colonies which transiently expressed TK genes in L cells was also determined by autoradiography. The ratio of stable transformants to colonies positive for transient expression in cells treated with low doses of DNA or lambda phage was 46-68%. The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells by HSV-TK DNA.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but 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 functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

Mutagenicity of methyl bromide in a series of short-term tests

Methyl bromide is commonly used as a soil fumigant in greenhouses. In the framework of a toxicological evaluation, it was tested for possible genotoxic properties in two bacterial test systems (the fluctuation test using Klebsiella pneumoniae and the plate test using Salmonella typhimurium TA100 and TA98), two systems using mammalian cells in vitro (forward mutations at the TK and HPRT loci in L5178Y mouse lymphoma cells and unscheduled DNA synthesis in primary rat-liver cells) and in the sex-linked recessive lethal test using Drosophila melanogaster. Methyl bromide was active in all tests except the DNA-repair assay. The results indicate a relatively low mutagenic efficiency of the compound, as expected from its alkylating properties.     

Comparative induction of gene mutations and chromosome damage by 1-methoxy-1,3,5-cycloheptatriene (MCHT), 1. Results from a battery of standard tests

The ability of 1-methoxy-1,3,5-cycloheptatriene (MCHT) to induce gene mutations and chromosome breaks has been examined in a battery of standard assays. MCHT was not mutagenic to 5 strains of Salmonella, with or without S9 fraction. In L5178Y TK+/- mouse lymphoma cells, MCHT induced TK-/- mutants in the presence but not in the absence of S9 fraction. In V79 Chinese hamster cells, MCHT induced azaguanine-resistant mutants in the presence and absence of S9 but the effect was considerably reduced in the absence of S9. MCHT resulted in no increases in chromosome aberrations in cultured human lymphocytes, with or without S9 fraction, neither was there any increase in micro-nucleated polychromatic erythrocytes in treated mice. MCHT thus appears on the basis of these results, to be possibly a specific gene mutagen (rather than clastogen) for mammalian cells. This uncommon mutagenicity profile has been investigated further in an accompanying paper (Cole et al., 1990) and has proved to be an oversimplification.