Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Application of alkaline unwinding assay for detection of mutagen-induced DNA strand breaks

The frequency of single-strand breaks in parental DNA and gaps in nascent DNA in various cells exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) was investigated by alkaline unwinding assay using two types of alkaline lysis conditions, 22°C lysis versus 0°C lysis. The DNA damage induced by MMS and MNU is considered to be characteristic of lesions produced in DNA by alkylating agents. The aim of our research project was to adjust this method to be able to detect the greatest number of DNA lesions induced by alkylating agents in parental DNA of different mammalian cells. In our experiments we used human cell lines EUE, GM637 and XP12, Chinese hamster V79 cells, and Syrian hamster embryo cells. The higher level of strand interruptions was detected under conditions of lysis of cells at 22°C. Probably the level of strand interruptions found after the lysis of cells at 22°C correlates with the increased number of disrupted alkali-labile sites of DNA. It is remarkable that the different lysis conditions did not influence the number of gaps detected in nascent DNA of alkylated cells. Comparing induction of breaks and gaps in radiolabelled strands of parental and daughter DNA under different lysis conditions, we succeeded in defining the optimum conditions for detection of alkali-labile sites of parental DNA.     

Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells

Studies were performed on the non-linear dose response for gene mutations induced by low doses of monofunctional methylating agents in V79 Chinese hamster cells. When treatment with methylnitrosourea was applied at the beginning of the S phase in synchronized cells, a linear dose-response curve was obtained, whereas application of the dose after gene replication resulted in a strong reduction of the number of induced mutations. Additional time for repair resulted in reduced dose response of MNU, indicating that an error-free repair process operates on methylated DNA in V79 Chinese hamster cells.     

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.     

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

Influence of anoxia and respiratory deficiency on the genotoxicity of some direct-acting alkylating agents in yeast

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.     

Chlorophyllin [CHLN] and the mutagenicity of monofunctional alkylating agents in Drosophila: the action of CHLN need not include an influence on metabolic activation

The effect of chlorophyllin (CHLN) on the mutagenicity of four monofunctional alkylating agents (MFAAs) was evaluated in the wing spot test in Drosophila. Three of the compounds are direct-acting (ethylnitrosamine (ENU), methylnitrosourea (MNU), and methylmethanesulfonate (MMS)) and one indirect-acting (diethylnitrosamine, DEN). Results indicate that the mutagenicity of all four compounds is strongly inhibited by CHLN. The findings are not in agreement with the conclusion of Romert et al. (1992) that CHLN has no effect on the mutagenicity of direct acting MFFAs inferred from their work with MNU and ethylmethanesulfonate (EMS) in the V79 and Salmonella in vitro test systems. The results suggest the possibility that the action of CHLN need not include an inhibiting effect on metabolic activation.     

Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes

We have established a specific correlation between the carcinogenic potency of a series of alkylating agents, with a mechanism of reaction ranging between Ingold's SN1-SN2 (ENU greater than MNU = MNNG greater than EMS greater than DMS = MMS) (Vogel et al., 1979; Bartsch et al., 1983) and specific target sites in the amino acids of nuclear proteins of cultured hepatocytes. More potent carcinogens, that react predominantly with an Ingold's SN1 mechanism, mainly alkylate the amino group of lysine and the guanido group of arginine. Weaker carcinogens, reacting with a mechanism closely resembling an Ingold's SN2, mainly alkylate the sulfhydryl group of the cysteine and the 3 position of the imidazolic ring of histidine. A compound with an intermediate type of reactivity alkylates, to a comparable extent, all 4 of the above-described positions. Although stable DNA damage brought about by alkylating carcinogens is considered to be the most likely cause of neoplastic transformation, epigenetic modifications may also play an important role in the process, especially because of their extreme stability. We have verified the existence of a linear correlation between the Swain-Scott substrate constant (S) of each compound and the amount of alkylation produced at the specific target sites. This type of correlation could be the basis of a 'short-term' genotoxicity assay in a battery of complementary tests.     

Influence of anoxia and respiratory deficiency on the genotoxicity of some direct-acting alkylating agents in yeast.

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory-deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.     

Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain

The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.     

Repair of methylated bases in mammalian cells during adaptive response to alkylating agents

Pretreatment of H4 (rat hepatoma) cells for 48 h with non toxic doses of alkylating agents methylmethane sulfonate, (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells more resistant to the toxic and mutagenic effects of these compounds. This adaptive response seems to reflect improved repair of methylated lesions in cellular DNA. Therefore, we measured the activity of the DNA-glycosylase for N-methylated purines (7-MeGua and 3-MeAd) and the activity of the O6-methylguanine-DNA methyltransferase in control and adapted cells. We show that the adaptive response does not significantly increase the DNA-glycosylase activity but involves the induction of methyltransferase molecules.     

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.     

In vitro DNA and nuclear proteins alkylation by 1,2-dimethylhydrazine

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.     

Repair of Alkylation Damage: Stability of Methyl Groups in Bacillus subtilis Treated with Methyl Methanesulfonate

Bacillus subtilis was not inactivated and was able to replicate even though approximately 3 x 10(4) methyl groups added by methyl methanesulfonate (MMS) were bound to the deoxyribonucleic acid (DNA) of each organism. No significant loss of methyl groups from the DNA occurred for several generations upon incubation of methylated wild-type or MMS-sensitive cells. Single-strand breaks were not observed in the DNA from cells treated at this low MMS dose. Higher doses of MMS resulted in significant killing of both wild-type and MMS-sensitive strains, and the DNA extracted from such treated cells sedimented more slowly than control DNA through alkaline sucrose gradients, indicating the presence of breaks or apurinic sites (or both). These breaks were repaired upon incubation of wild-type but not of MMS-sensitive strains. Repair of damage induced by alkylating agents is probably the repair of breaks which occur as a consequence of high levels of alkylation.     

Base alterations in yeast induced by alkylating agents with differing Swain-Scott substrate constants.

The base alterations induced by four alkylating agents, methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-methylurea (MNU), and N-nitroso-N-ethylurea (ENU), have been determined at the URA3 locus in the yeast Saccharomyces cerevisiae. The mutagen treatment was carried out on yeast cells in the logarithmic phase of growth. The mutants were selected by their resistance to 7.3 mM-5-fluoroorotic acid at pH 3.8. DNA sequence analysis was carried out by the dideoxy chain termination method. The alkylating agents were selected for their widely differing Swain-Scott substrate constants (s values), which are as follows: MMS, s = 0.83; EMS, s = 0.67; MNU, s = 0.42; ENU, s = 0.26. A higher s value is correlated with a higher ratio of 7-alkylguanine to O6-alkylguanine in native DNA in vitro. 125 forward mutations from URA3----ura3 were sequenced with marked differences in the mutational spectra being observed as the s value changed. Five hotspots were recorded for the four alkylating agents. They were all G.C----A.T transition mutations. There was one common hotspot for all of them; there were two additional ones for the two ethylating agents (ENU and EMS) and two different ones for MNU. Four of the five hotspots have the 5'-GG-3' sequence with the 3'-guanine mutated. It was seen that MMS, which has the highest Swain-Scott substrate constant, yielded the widest array of mutational types. As the substrate constants decreased, the types of mutations became more and more restricted to the G.C----A.T transitions and the A.T----T.A transversions. The transitions are consistent with the concept that mutations arise from O6-alkylation of guanine and alkylation of thymine. The transversions are consistent with the notion of N1-alkylation of adenosine or adenylic acid.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Factors modifying 3-aminobenzamide cytotoxicity in normal and repair-deficient human fibroblasts

3-Aminobenzamide (3-AB), an inhibitor of poly(ADP-ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3-AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3-AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3-AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3-AB for 36-48 hours following MMS treatment. During this same interval, 3-AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3-AB. Human mutant cells defective in DNA repair differed in their responses to 3-AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3-AB than control cells, while Huntington's disease cells had a near-normal response. Among UV-sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3-AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3-AB could be dependent on inability of the mutant cells to repair damage by other processes.     

Inhibitory effect of cadmium and mercury ions on induction of the adaptive response in Escherichia coli

Cadmium and mercury ions inhibited the promotion of ada and alkA gene expression in the adaptive process induced by methylating agents such as N-methyl-N-nitrosourea (MNU), methyl methanesulfonate (MMS) and methyl iodide in Escherichia coli. In fact, the induction of O6-methylguanine-DNA methyl-transferase (MGTase) by MNU was suppressed in E. coli in the presence of these metal ions. These ions potentiated mutagenesis induced by methylating agents such as MNU and MMS, but not that induced by ethylating agents, UV irradiation, or N4-aminocytidine. These comutagenic effects were observed in wild-type and umuC36 strains of E. coli but not in the ada-5 strain, which is unable to induce the adaptive response. These results suggest that the comutagenic effects of Cd2+ and Hg2+ are due to inhibition of ada and alkA gene expression promoted by methylated MGTase.