Mutation Research, 116 (1983) 281-287 281 Elsevier Biomedical Press

Mutagenicity of cyanate, a decomposition product of MNU

M.S. Melzer, R.T. Christian *, J.F. Dooley, B. Schumann, H.L. Su and S. Samuels

Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267 (U.S.A.)

(Received 3 February 1982) (Revision received 28 June 1982)

(Accepted 18 August 1982)

Summary

Knox reported that the short-term effects of the carcinogen methylnitrosourea (MNU) were due to the formation of its decomposition product, the cyanate ion. He showed that cell survival and DNA synthesis decreased as the concentration of MNU and the cyanate ion ( N C O - ) increased in the medium. Further, the product of MNU decomposition comigrated with N C O - when added to his chromato- graphic test system. However, Knox did not study the mutagenicity of MNU or its breakdown products. We compared the mutagenicity of MNU and potassium cyanate (KNCO) in mammalian cells. Our results demonstrate that, although it is toxic to cells, KNCO does not induce ouabain-resistant mutants in cultured Chinese hamster cells (V79).

Knox (1976) reported that the cytotoxic effects of methylnitrosourea (MNU) and ethylnitrosourea (ENU), both potent carcinogens in rodents and in cultured cells, were due to the formation of their decomposition product, the cyanate ion, rather than to the alkylating property of these agents. He presented evidence that the cyanate ion is responsible for short-term effects and cytotoxicity. Knox demon- strated that cell survival and DNA synthesis decreased as the concentration of the cyanate ion ( N C O - ) and MNU increased in the culture medium. He also presented evidence, based on a paper chromatography study, that N C O - along with methanol and gaseous nitrogen are the breakdown products of MNU. Further, the product of MNU decomposition comigrated with N C O - when added to his chromatographic test system. However, Knox did not study the mutagenicity of MNU or its

* To whom inquiries and requests for reprints should be addressed.

0165-1218/83/0000-0000/$03.00 © Elsevier Biomedical Press

282

breakdown products. Roberts and Sturrock (1973) have reported that MNU induces an increase in the frequency of azaguanine-resistant mutants of V79-379A cells. Similarly, Jenssen and Ramel (1980) have demonstrated that MNU produces a non-linear dose-responsive increase in the recovery of 6-thioguanine-resistant mutants of V79 cells. However, there is little information in the literature concerning the mutagenicity of N C O - .

Wei et al. (1980) have recently reported that MNU both carbamoylates and methylates DNA. Carbamoylation is a minor reaction compared to methylation. In further experiments using an in vitro DNA directed system for fl-galactosidase (f iG) they determined the effect of MNU and ENU on the function of the DNA template. They found that the degree of inhibition of B-galactosidase synthesis was directly related to the extent of total alkylation with either ENU or MNU. Potassium cyanate (KNCO) had no effect on DNA template function for f iG synthesis. They suggested, however, that KNCO did interfere with f i g synthesis by carbamoylation of non-DNA components of the protein synthetic machinery.

Panasci et al. (1977) reported that MNU and KNCO have equivalent carbamoy- lating activity in vitro. It has also been reported that proteins and lipids are extensively carbamoylated by MNU (Kukushkina et al., 1979). These findings suggest that MNU and possibly KNCO may interact with many critical targets in V79 cells resulting in cell damage at many sites. We have carried out an analysis to determine if an increase in mutation frequency is one of the results of these complex reactions.

Materials and methods

We have determined the ouabain-resistant (OUA R) mutation frequency for both MN U and KNCO in Chinese hamster cells (V79) selected at the N a + / K ÷ -ATPase locus. The methods used in this report are a modification of those which we have previously reported (Lankas et al., 1980). A 48-h expression period was chosen for these experiments because of reports from several laboratories on the optimal expression of OUA R mutants. Chang et al. (1978) reported that 48 h was the expression period for maximum recovery of OUA R mutants. Newbold et al. (1977) reported that the maximum recovery of OUA R mutants occurred at approximately 4 generations (48-62 h) post treatment. Thacker et al. (1978) showed that the recovery of EMS-induced OUA R mutants remained stable between 2 and 8 days of expres- sion. Lankas et al. (1980) showed that there was little change in OUA R mutation frequency between 1 and 6 weeks of expression. These studies, all using V79 cells, demonstrate that a phenotypic expression period of 48 h yields a relatively stable, maximum recovery of OUA R mutants.

For determination of OUA R mutagenesis, 3 × 105 V79 cells were seeded per 100 mm petri dish in 10 ml of complete Dulbecco's modified essential medium (DMEM) supplemented with 107o fetal calf serum (Gibco) and 50 #g gentamicin/ml (Sche- ring). For determination of plating efficiency (PE), 200 V79 cells were seeded per 60 mm petri dish in 5 ml of complete DMEM. After 3 h incubation at 37°C in a

283

humidified atmosphere of 5% CO2:95~ air, the cells were exposed to various concentrations of MNU (Cat No. NO132, Sigma Chemical Co., St. Louis, MO, U.S.A.), KNCO (Cat No. P-244, Fisher Scientific Co., Cincinnati, OH, U.S.A.) or left untreated. After 2 h of exposure, the mutagen and the control media were removed from the cultures. The cells were washed twice with Hanks' balanced salt solution, refed with complete DMEM, and reincubated at 37°C. Following a 48-h expression period, the medium was removed from the mutagenesis plates and replaced with complete DMEM containing 1 mM ouabain (Sigma Chemical Co., St. Louis, MO, U.S.A.). After a selection period of 14-18 days, with an ouabain medium change on day 9, the mutagenesis plates were fixed,stained and scored for OUA R colonies. PE plates were fixed, stained and scored for surviving colonies after 7-9 days in complete medium.

The experimental design for the data analysis was a randomized block factorial design with experiments as a blocking factor. The model was Yijk = M + E k + C i + Dj + CDij + Rijkl, where E k stands for experiment, Cj for chemical, Dj for dose level, CDij for chemical-dose interaction, and Rijkl for replication. Wey (1980) studied the distribution of mutation frequencies at the OUA R locus of V79 cells. He concluded that such frequencies were best analyzed after a square root transformation. Such a transformation produced homogeneity of variance and symmetry of residuals about the transformed means. Therefore Yijkl was taken to be the square root of the observed mutation frequency. Standard analysis of variance techniques were applied, with the Scheff~ method (Snedecor and Cochran, 1967) used for post-hoc multiple comparisons.

Results

We have determined the MNU- and KNCO-induced mutation frequency of V79 cells (Table l) for comparison with untreated V79 cells (2.50UA R mutants per l05 survivors). The results of the analysis of variance demonstrated a statistically significant difference (P ~< 0.0001) in both the main effect of chemical and of dose. The difference between the MNU- and KNCO-induced OUA R mutation frequency was highly significant at equimolar concentrations of 0.3 mM (P ~< 0.001) and 1.0 mM (P ~ 0.0001). At equimolar concentrations of 0.01, 0.03 and 0.1 mM MNU and KNCO, no significant differences between the chemicals was found in the induced mutation frequencies. Further tests for simple main effect indicated a dose effect in MNU, but no dose effect in KNCO.

While there was no increase in the KNCO-induced OUA R mutation frequency, there was also no decrease in cell survival, in contrast to that reported by Knox (1976). In order to compare the mutation frequencies for MNU and KNCO at approximately equal cytotoxicities, we performed a second series of experiments at slightly higher concentrations of the chemicals. The results of the analysis of variance for the second series of experiments demonstrated a statistically significant different (P ~< 0.0001) between the MNU- and KNCO-induced mutation frequen- cies, although no dose effect (1.0 vs. 1.6 raM) was detected for MNU or KNCO. The

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Fig. l. OUA R mutagenicity (I, e) and cytotoxicity (13, O) of MNU and KNCO in V79 cells. The values represent the mean of a minimum of 2 and a maximum of 5 separate Expts. from both series of experiments. The control OUA R mutation frequencies did not exceed 30UA R mutants per 105 survivors and maintained a plating efficiency (percentage of cell survival) of 70-75%.

results f rom both series of experiments were used to graphically illustrate the dose-responsive increase in the MNU- induced O U A R mutat ion frequency and the dose-responsive increase in the M N U - and KNCO- induced cytotoxicity (Fig. 1). These results demonstrate that K N C O did not increase the O U A R mutat ion frequen- cies even at concentrat ions which severely depressed cell survival. Furthermore, a plot of muta t ion efficiency (Newbold et al., 1980; Ehrenberg, 1971) (not shown) demonst ra ted an increase in the O U A R mutat ion frequency with increasing cell death for M N U , but not for K N C O . Our results demonstra te that K N C O is not the active, mutagenic breakdown product of M N U in these experiments.

286

Discussion

Our results, demonstrating a non-linear dose-responsive increase in the MNU-in- duced OUA R mutation frequency, confirm the results of Jenssen and Ramel (1980). These authors demonstrated that low doses of 2 methylating agents (MNU and methyl methanesulfonate) induced non-linear dose-responsive increases in the re- covery of hypoxanthine-guanine phosphoribosyl-transferase-deficient (HGPRT) mutants of V79 cells. In contrast, they demonstrated that ethylating agents (ethyl- nitrosourea and ethyl methanesulfonate), UV radiation and X-rays induced linear dose-responsive increases in the recovery of H G P R T mutants. The threshold re- sponse (deviation from linearity) of mutation induction with methylating agents was postulated by these authors to be due to the V79 cell's capacity to excise O6-methyl guanine and incapacity to excise O6-ethyl guanine. Jenssen and Ramel (1980) further postulated that the repair of methylated DNA is error free and saturable up to a certain number of lesions, above which an error-prone repair process is operable. Warren et al. (1979) and Fox and Brennand (1980) have demonstrated that V79 cells have little or no capacity to excise O6-methyl guanine. Although explanation of our results in terms of the capacity of V79 cells to excise O6-methyl guanine is tenuous, we have demonstrated that 0.3 and 1.0 mM MNU induces a dose-responsive increase in the recovery of OUA R mutants.

Serebryanyi et al. (1970) showed that MNU induced both carbamoylation and methylation in animal DNA. Wei et al. (1980) demonstrated both reactions in phage DNA, although carbamoylation was a very minor reaction. Wei and his colleagues also showed that KNCO, a carbamoylating agent, had no effect on DNA template activity. However, KNCO did inhibit the complete fl-galactosidase synthesis system. These authors suggest that carbamoylation of the protein synthetic machinery is responsible for blocking the enzyme synthetic pathway. They further suggest that this is a potentially important toxic reaction. The results of the experiments reported here on the mutagenic and cytotoxic effects of MNU and KNCO support their results. MNU was both mutagenic and cytotoxic in these experiments. KNCO was not mutagenic at any concentration tested but was toxic at approximately the same molar concentration as MNU. The dose-resporise curves for the lethal events for the 2 agents were quite similar. There was no significant increase in the MNU-induced mutation frequency at concentrations that were not cytotoxic. Panasci et al. (1977) showed that MNU and KNCO have equal carbamoylating activity in vitro. The cytotoxicity found at equimolar concentrations supports the suggestion by Wei et al. (1980) that carbamoylation is responsible for the cytotoxicity of MNU. Both agents interact with many critical targets in the cell. These targets may include proteins and lipids as well as DNA. In light of these many interactions in V79 cells, the mutagenic event may be more complex than simple alkylation of DNA. However, our results are consistent with the hypothesis that methylation of D N A causes the OUA R mutants observed in these experiments.

287

A c k n o w l e d g e m e n t s

W e w o u l d l ike to t hank Dr . V i n c e n t Finel l i , Dr . S h a n e Q u e H e e a n d Dr . T e r e n c e

C o d y for the i r adv i ce a n d rev iew of the manusc r ip t .

Th i s w o r k was s u p p o r t e d by the N a t i o n a l In s t i t u t e o f E n v i r o n m e n t a l H e a l t h

Sc iences u n d e r C e n t e r G r a n t ES-00159.

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Fox, M., and J. Brennand (1980) Evidence for the involvement of lesions other than O6-alkylguanine in mammalian cell mutagenesis, Carcinogenesis, 1,795-798.

Jenssen, P., and C. Ramel (1980) Dose-response curves for the induction of 6-thioguanine-resistant mutants by low doses of monofunctional alkylating agents, X-rays and UV radiation in V79 Chinese hamster cells, Mutation Res., 73, 339-347.

Knox, P. (1976) Carcinogenic nitrosamides and cell cultures, Nature (London), 259, 671-673. Kukushkina, G.V., I.S. Sokglova and L.B. Gorbacheua (1979) Kinetics of carbamoylation of macromole-

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