Comparison of toxicity and mutagenicity of methylnitrosourea, methylnitronitrosoguanidine and ICR-191 among human lymphoblast lines

The toxic and mutagenic effects of the alkylating agents methylnitrosourea (MNU) and methylnitronitrosoguanidine (MNNG) and of the frameshift mutagen, ICR-191 were compared among 3 human diploid lymphoblast lines, MIT-2, WI-L2 and GM 130. The MIT-2 and WI-L2 lines were both sensitive to the toxic and mutagenic effects of all 3 agents tested. The WI-L2 line was more sensitive to the toxic effects of MNU and MNNG than the MIT-2 line, while it was somewhat less sensitive to the mutagenic effects of these alkylating agents. The GM 130 line was strikingly resistant to both the toxic and mutagenic effects of the alkylating agents. The order of sensitivity to the toxic effect of ICR-191 was MIT-2 > WI-L2 > GM 130, while the order of sensitivity to the mutagenic effects of this frameshift mutagen was GM 130 > MIT-2 > WI-L2. These results point to the importance of accounting possible variations in mutability among individuals when extrapolating from any single mutagenicity assay for human risk assessment.     

Enhancement by caffeine of N-methyl-N-nitrosourea-induced mutations and chromosome aberrations in Chinese hamster cells

N-Methyl-N-nitrosourea (MNU) increased the induction of mutations to 8-azaguanine resistance in Chinese hamster cells in a dose-dependent manner. Mutations were only observed with toxic concentrations of MNU. Since a plot of the fraction of cells surviving alkylation against the extent of methylation of DNA exhibited a shoulder it followed that there was a threshold level of DNA reaction which did not lead to mutations possibly due to efficient repair of DNA damage. Post-alkylation incubation in medium containing caffeine decreased cell survival while at the same time it increased the induced mutation frequency. Mutation frequency was increased whether caffeine was present for 48 h or for a further 12 days in the presence of the selective agent 8-azaguanine. MNU caused chromatid aberrations in Chinese hamster cells and these reached a value of 15% of the treated cells by 48 h after methylation. Post-alkylation incubation in caffeine increased the percentage of cells showing chromosomal damage to a maximum of 86% of treated cells by 40 h after alkylation. A large proportion of cells exhibited completely fragmented or shattered chromosomes. The proportion of cells showing the presence of micronuclei also dramatically increased following incubation of methylated cells in caffeine. These results are discussed in terms of the possibility that damage to DNA is responsible for the lethal, mutagenic and cytological effects of MNU in Chinese hamster cells, and that there is a caffeine sensitive step(s) in the repair of the DNA damage which is responsible for these effects.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

Experiments on the effect of the medium in mutation tests with the HGPRT system in cultured mammalian cells

Higher mutation frequencies were observed on 8AG than on 8AG medium when HGPRT-deficient mutants were being selected in V79 Chinese hamster cells.2 alternative explanations for the effect of the medium were considered, namely (1), that mechanisms are present that cause resistance to 8AG only, in addition to that (or those) causing resistance to both drugs, and (2), that mutants with low HGPRT content survive on 8AG but not on 6TG medium, owing to lower affinity of 8AG for the enzyme. The second explanation was favoured as a result of various experimental approaches, including kinetics of expression on the 2 media, cross-resistance at different expression times and serial selection on the 2 media.     

Enhanced survival and reduced mutation and aberration frequencies induced in 79 Chinese hamster cells pre-exposed to low levels of methylating agents

Exposure of V79 Chinese hamster cells to a single very low (sub-toxic and sub-clastogenic) dose of MNU or MNNG made these cells resistant to the toxic, mutagenic and clastogenic activities of the same agents given 6 h later. Cell survival was increased nearly 2-fold under optimal conditions when compared with the non-pretreated controls. Aberration frequencies were reduced to nearly half the control values (cells not pretreated). This was observed for a wide range of pretreatment concentrations and at different recovery times. The effect of mutagen pretreatment was most pronounced with respect to the induction of TG resistance, which became drastically reduced. The data indicate the existence of an adaptive repair pathway in V79 cells which may be induced by very low levels of methylating agents and which is error-free in handling lesions responsible, at least partially, for reproductive cell death, mutations and chromosomal aberrations.     

Relationship between DNA alkylation and specific-locus mutation induction by N-methyl- and N-ethyl-N-nitrosourea in cultured Chinese hamster ovary cells (CHO/HGPRT system)

Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43--13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per mumol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7--26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per mumol nucleotide per mM. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringently-defined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01--2.0 mM); mean values were 2800 and 840 mutants per 10(6) clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.     

The relative effectiveness of 6-thioguanine and 8-azaguanine in selecting resistant mutants from two V79 Chinese hamster cells in vitro

The frequency of surviving colonies in two V79 cell lines exposed to either 6-thioguanine or 8-azaguanine was dependent on initial plating density. Different degrees of metabolic-co-operation were found to occur in the two cell lines and the loss of both spontaneous and added mutants occurred at a lower cell density when 6TG was used for selection than when 8 AZ was used in both cell lines. Both analogues were degraded on incubation in medium plus serum in the absence of added cells. Variation in serum batch had little effect on the rate of degradation or on the frequency of colonies recovered after treatment of V79 cell lines with 8AZ. The reasons for preferring 8AZ to 6TG as a selective agent are discussed.     

A comparison of diameters of micronuclei induced by clastogens and by spindle poisons

To compare the size of micronuclei induced by clastogens and by spindle poisons, bone-marrow smears were prepared 24 h after a single intraperitoneal injection of triethylenemelamine (TEM) (0.3 mg/kg) or vincristine (VCR) (0.125 mg/kg) into male mice. 100 micronucleated erythrocytes (MNEs) were randomly selected from each group and photomicrographed. Diameters of the cytoplasm (D) and the micronucleus (d) of each MNE were measured on the photographs. Relatively large micronuclei (d?D/4) were frequent (74%) in the VCR-treated group, and infrequent (2%) in the TEM-treated group. The frequencies of MNEs resulting in d?D/4 were determined for several other mutagens. All clastogens tested could be classified as TEM type, and all spindle poisons as VCR type.These findings indicate that it is possible to analyze the action site of micronucleus-inducing agents on the basis of the relative sizes of micronuclei.     

Mutagenesis by simian virus 40. II. Changes in substrate affinities in mutant hypoxanthine-guanine phosphoribosyl transferase enzymes at different pH values.

A number of 8-azaguanine-resistant clones selected from Chinese hamster cells infected with SV 40, and supposed to originate by virus infection was investigated to demonstrate and analyze genetic alterations occurring in the cells after infection. All resistant clones tested showed reduced but detectable activity levels of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The extent of reduction in the activity was not identical for different substrates. In all the clones tested, spontaneous mutants included, the pH optimum for the enzymic reaction with guanine was shifted to lower values. The reduced enzymic activities of resistant clones correlated with their colony-forming ability in corresponding selective media. The results support the suggestion that SV 40 is able to induce gene mutations.     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

Cytotoxicity and mutagenicity of alkylating agents in cultured mammalian cells (CHO/HGPRT system): mutagen treatment in the presence or absence of serum

Cytotoxicity and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system) were measured for a range of concentrations of 6 alkylating agents [methyl and ethyl methanesulfonate (MMS, EMS), N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidine (MNNG, ENNG), and methyl- and ethyl-nitrosourea (MNU, ENU)] to determine the effect of the presence or absence of serum during the time of mutagen treatment. Cultures were treated with the mutagens for 5 h, a time period which results in no growth inhibition in the absence of serum, to estimate the potential decrease in effective mutagen dose to the cells which might result from reactivity with the serum proteins. With all 6 agents, identical results were found for cytotoxicity and for mutagenicity regardless of the presence or absence of serum during treatment. This finding demonstrates that the use of serum in cell-culture medium does not present any problems in apparent dosimetry studies, at least with these alkylating agents.     

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.     

Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells.

The alkylating agent MMS was toxic to mouse lymphoma L5178Y cells and decreased their growth rate. A dose-dependent induction of thioguanine- and thymidine- but not ouabain-resistant variants was observed. The prolonged period for expression of thioguanine-resistant variants observed with other mutagens was also found in these studies. A comparison of MMS and EMS showed that MMS on a molar basis was approximately 10 times more toxic than EMS. With mutation, however, when evaluated at equal levels of cell killing MMS and EMS induced the same number of thymidine-resistant variants. For thioguanine-resistant variants MMS was approximately 10-fold less efficient than EMS, while for ouabain-resistance MMS, unlike EMBS, produced no variants at all. The ouabain results were further compared with positive results obtained using a modified Luria--Delbrück fluctuation test.     

Ultraviolet light-induced mutation of diploid human lymphoblasts

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited ³H-TdR uptake into DNA. A shoulder of about 6 J/m² and a D₀ of 6 J/m² was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m²; above 6 J/m², complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m² caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation.Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 j/m²), moderately toxic fluences (8.8 J/m²) and toxic fluences (17.6 J/m²). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show to shkow enchanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.     

Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5:10(-5) per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10(-6) in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with gamma-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than gamma-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.     

A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation.

A mammalian cellular system, utilizing Syrian hamster embryo cells, was developed for the concomitant study of neoplastic transformation and somatic mutation. Chemically induced somatic mutation of the cells was assayed at two genetic loci. Mutants deficient in hypoxanthine phosphoribosyl transferase (HPRT) were detected by the production of colonies resistant to 8-azaguanine (AG^r) or 6-thioguanine (TG^r) and mutants with an altered Na⁺/K⁺ ATPase were detected by the production of colonies resistant to ouabain (Oua^r). Colonies resistant to each of the three selective agents were isolated and characterized. AG^r and TG^r resistant cells maintained their resistance to the selective agent after isolation and growth in the absence of the drug, displayed a low reversion frequency, and possessed less than 1% of the HPRT activity of the wild-type cells. AG^r cells were also resistant to the cytotoxicity effects of 6TG. Oua^r cells also maintained their resistance to ouabain and were less sensitive to the inhibition of ⁸⁶Rb uptake by ouabain than the wild-type cells. The spontaneous frequency of all three types of resistant cells was <10^?6, but the mutation frequency was significantly increased following exposure of the cells to known mutagens in a dosage-dependent manner. These properties indicate that AG^r and TG^r cells posess a mutation in the structural or regulatory gene for HPRT, and that Oua^r cells have an altered Na⁺/K⁺ ATPase.The factors involved in the quantification of the mutation frequencies of hamster embryo cells following exposure to carcinogens were determined. Cytotoxicity was assayed by a reduction in the cloning efficiency of the treated cells. The recovery efficiencies of the resistant cells were measured by reconstitution experiments and the degree of cross feeding effects of HPRT^? cells was determined. The expression time of the mutations following exposure of the cells to carcinogens was also examined, and the mutation frequencie at the two loci of hamster embryo cells following exposure to MNNG or benzo(a)-pyrene (B(a)P) were determined. Employing this system, a quantitative comparison can be made between the frequencies of somatic mutation and morphological transformation.     

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Mutagenesis by N-acetoxy-2-acetyl aminofluorene of chinese hamster V79 cells is unaffected by caffeine

8-Azaguanine (AZG)- and 6-thioguanine (TG)-resistant cells (mutants) were induced in Chinese hamster V79-4 cells by 0.1--2.5 microgram/ml N-acetoxy-2-acetyle aminofluorine (AcAAF) treatments in the presence of 5% fetal bovine serum (FBS). The frequency of resistant colonies increased from 1 to 47 per 10(5) survivors. The effect of caffeine (50--200 microgram/ml) during the mutagenesis expression period was determined by adding caffeine 1--24 h after AcAAF. The medium was replaced after 48 h exposures so that caffeine was absent during subsequent selection with AZG or TG. No significant change in the AcAAF-induced mutant frequency occurred with any treatment combination although caffeine greatly enhanced the lethality associated with AcAAF treatments. Thus, caffeine interferes with postreplication repair in V79-4 cells without affecting the probability of error of the repair process. These results were obtained with a quantitative mutagenesis assay in which the cells were reseeded prior to selection to achieve maximum expression without interference from metabolic crossfeeding. In contrast, the commonly used in situ assay is subject to serious interference from crossfeeding and yields an artifactual enhancement of AcAAF mutagenesis by caffeine.     

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.