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Interaction of UV and N-methyl-N′-nitro-N-nitrosoguanidine: Cytotoxicity and mutagenecity in V79 cells
Mutation Research, 152 (1985) 77-83 Elsevier
77
MTR 04099
Interaction of UV and
N-methyl-N'-nitro-N-nitrosoguanidine:Cytotoxicity and mutagenicity in V79 cells
Nitaipada Bhattacharyya and Sukhendu B. Bhattacharjee Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Calcutta-700 064 (India) (Received 27 December 1984) (Revision received 23 April 1985) (Accepted 7 May 1985)
Summary Killing and mutation by UV in the MNNG-exposed population of V79 cells, as well as by MNNG in the UV-irradiated population of these cells have been studied. It was observed that pretreatment with MNNG increased the killing and mutation by UV, whereas, pretreatment with UV had no effect upon killing and mutation by MNNG. The increase in sensitivity to UV due to pretreatment with MNNG was lost if UV exposure was delayed for 24 h after MNNG treatment.
The interaction between two different DNAdamaging agents has been used in a number of experiments to study whether there is any common pathway for the repair of the damage (Ahmed and Setlow, 1977a,b; Gruenert and Cleaver, 1981; Park et al., 1981; Park and Cleaver, 1982). Gruenert and Cleaver (1981) showed that in normal human cells and in XP (xeroderma pigmentosum) variant cells, 4 mM MMS (methyl methanesulphonate) treatment reduced the repair replication induced after UV exposures. For both types of cells, the reduction was almost the same whether the cells were exposed to MMS first and then to UV or to UV first then to MMS. It was also seen that when normal human cells were treated with N-methylN'-nitro-N-nitrosoguanidine (MNNG) and then with graded doses of UV, there was a reduction in the repair replication. But with XP (group G) cells, similar treatment did not affect repair replication. Address for correspondence: Dr. S.B. Bhattacharjee, Professor and Head, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Sector" 1, Block 'AF', Bidhan Nagar, Calcutta-700 064 (India).
The magnitude of repair replication induced by MNNG in XP cells was the same as in the normal cells. It is thus clear that in XP cells, UV-irradiation did not inhibit repair of alkylation damage. Park et al. (1981) showed that in CHO and normal human cells, the unscheduled DNA synthesis (measured by autoradiograph) induced by UV and MMS were less than that expected from the sum of the unscheduled synthesis seen in cells exposed to UV or MMS alone. The synthesis was found to be independent of the order in which the cells were exposed. It was also shown that exposure to 1 mM MMS caused a slight reduction in the fraction of dimers excised, and to 2 mM, almost completely inhibited excision (Park et al., 1981). Unscheduled DNA synthesis and excision of pyrimidine dimers in UV-irradiated CHO cells were inhibited by prior exposure to the alkylating agent MNNG (Park and Cleaver, 1982). The excision repair ability of Chinese hamster V79 cells treated with N-acetoxy-2-acetylaminofluorene and UV light was measured by 3 techniques. It was seen that the amount of repair from combined treatment was less than additive and in
0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
78 some cases less than that due to either agent (Ahmed and Setlow, 1977b). But in human cells, the same combination of treatment resulted in repair which was additive (Ahmed and Setlow, 1977a). For V79 cells, additivity in repair was observed following UV and MMS exposure (Ahmed and Setlow, 1980). Measurement of inhibition of DNA synthesis from the incorporation of radioactive thymidine showed an additive effect when M N N G was applied along with UV radiation (Gichner and Veleminsky, 1982). However, it is interesting to note that the conclusions arrived at depended on the cell type and the nature of the agents and end-points although killing and mutation had not been utilized in these studies. It may be worthwhile to see if the observations made on the basis of these two end-points corroborate the earlier findings. With this in mind, we studied the effect of M N N G treatment on UV-induced killing and mutation and also the influence of pre-exposure to UV on MNNG-induced effects on V79 cells, in terms of killing and mutation. We have been able to show that in such interactions the order in which the exposures are carried out is important. Material and methods
MNNG, neomycin sulphate and 8-azaguanine (AG) were purchased from Sigma Chemical Co., penicillin and streptomycin sulphate from local sources. M N N G was dissolved in sterilized double distilled water at a final concentration of 300 /~g/ml, immediately before use and diluted to the desired concentrations. An A G stock solution was made in dimethyl sulphoxide (DMSO). Details about the culture conditions, media etc., survival and mutation studies have already been published (Bhattacharjee and Pal, 1982; Bhattacharjee et al., 1982). In brief, exponentially growing cells were washed with phosphate-buffered saline (PBS), trypsinised, diluted and plated in the required number and allowed to attach for about 4 h at 37°C in a 5% CO 2 humidified atmosphere. After attachment, the medium is withdrawn and the cells exposed either, to M N N G in growth medium for 1 h, or to UV in the absence of medium. Fresh medium was then added and incubated f o r colony formation. For interaction
studies, cells were exposed to M N N G and then to UV, or first to UV and then to M N N G immediately after the first exposure, or with a desired intervening growth period between the two treatments. For mutation studies, approximately 106 cells were treated with M N N G or exposed to UV. Some of the MNNG-exposed cells were treated with UV immediately or after an intervening period growth and some UV-irradiated cells were treated with M N N G immediately or after subsequent growth for the desired time. Thereafter, the cells were grown for 7-9 days for expression, with 3-4 subcultures. After expression, cells were replated at 5 x 105 cells per 100 mm in Coming petri dishes [this cell number was determined by reconstruction experiments (data not shown)] in selection medium which contained AG (3 /~g/ml) in complete growth medium (final concentration of DMSO 0.1%). A further increase in the concentration of AG up to 6/~g/ml did not affect the yield of mutants (data not shown). About 93% of the AG-resistant clones were sensitive to HAT medium (complete medium containing hypoxanthine 10 _4 M, aminopterin 3 x 10 - 7 M and thymidine 10 _5 M). Each data point represents the average of 2-5 independent experiments, the standard deviations being denoted by bars. Results
Fig. 1 shows the UV survival of cells pretreated with MNNG. Treatment with MNNG, immediately before the UV exposures reduces the shoulder width in a dose-dependent manner and increases the slope of the survival curve significantly, although the rate of increase was the same within the M N N G concentration range 0.1 /~g/ml to 1 /~g/ml. Fig. 2 shows the effect of previous UV exposures on the sensitivity of the cells to the graded doses of MNNG. It can be seen that previous exposure to UV did not influence the M N N G induced killing. The effect of separating the exposures to two agents by intervening periods of incubation in growth medium is" illustrated in Fig. 3. When 15 J / m 2 UV-exposed cells were treated immediately,
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Fig. 1. Survival of V79 cells exposed to different UV fluences. Cells were pretreated with MNNG: 0.0/tg/ml ((3); 0.1/tg/ml (z,); 0.5/~g/ml ([3); 1.0/~g/ml (@).
with 1 /~g/ml M N N G for 1 h the survival level was 0.02, but when the M N N G treatment was delayed for 2 - 6 h, there was a sharp increase in survival. On further delay up to 24 h, there was no further increase in survival. But when cells were first treated with 1 ~ g / m l M N N G and then grown io°
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Fig. 3. Survival of V79 cells exposed to 15 J / m 2 UV, followed by 1 ~g/ml of M N N G treatment (@) or to 1/~g/ml of M N N G followed by 15 J / m E of UV-irradiation (~,).
for several hours, before the UV exposure, there was an increase in survival in 4 - 1 2 h, whereafter it remained constant up to 24 h. But in all cases, the survival level was lower than would have been obtained by exposure to the second agent alone. 10'
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CONCENTRATION OF MNNGy.g/ml Fig. 2. Sensitivity of UV-irradiated V79 cells to MNNG: UV fluences: 0 J / m 2 (Q), 8 J / m 2 (0), 12 J / m 2 (~.), and 20 J / m 2
(A).
Fig. 4. Sensitivity of M N N G
exposed cells held for 24 h to
subsequent UV exposures (1~), compared to the cells which were not held after M N N G treatment (~.). The symbol ((3) represents the UV survival of control untreated cells.
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Fig. 5. Survival of V79 ceils on treatment with M N N G . Cells having no pretreatment (Q), pretreated with 20 J / m 2 UV, either immediately before (r~) or 24 h before (~) M N N G treatments.
Fig. 7. Influence of preirradiation with UV on M N N G - i n d u c e d mutation. UV fluences 0 J / m 2 (Q)~ lO J / m 2 (.~.), 20 J / m 2 ([]), and 30 J / m 2 (A).
Fig. 4 represents the survival curves in which treatment with graded doses of UV was carried out, 24 h after the first M N N G treatment. It can be seen that the increase in the slope of the UVsurvival curve, due to M N N G pretreatment (as observed in Fig. 1) was lost due to a 24-h delay. The effect of holding UV-irradiated (20 J / m 2) cells in growth medium for 24 h on their subse-
quent sensitivity to M N N G is shown in Fig. 5. In terms of survival the cells behave as if they had not been exposed to UV before. Fig. 6 shows the yield of 8-azaguanine-resistant mutants by UV-light irradiation of cells previously treated with MNNG. It can be seen that due to pretreatment with MNNG, the rate of mutation induction by UV is increased, although the in-
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Fig. 6. Influence of pretreatment with M N N G on UV-induced yield of mutants. M N N G concentrations: 0 /~g/m] (Q), 0.I # g / m ] (,*,),'0.5 # g / m l (El), ].0 ~ g / m l (11).
Fig. 8. Interaction of UV and M N N G on the induction of mutation. After 10 J / m 2 UV treatment cells were held for different periods before treament with 0.2 # g / m l M N N G was done (A). Alternatively, cells treated with 0 . 2 / ~ g / m l of M N N G were held for various periods before being exposed to 10 J / m 2 UV (®).
81 crease in rate was the same for concentrations of M N N G ranging between 0.1 ~g/ml and 1/~g/ml. Fig. 7 shows the variation in yield of 8azaguanine-resistant mutants induced by M N N G treatment of previously UV-irradiated cells. The induction of mutation was not in any way influenced by UV pretreatment. Fig. 8 represents the induction of 8-azaguanine-resistant mutants on separation of the UV and M N N G treatments by varying periods of intervening growth. The UV dose used was 10 J / m 2 and the MNNG dose 0.2 /~g/ml. The exposure conditions were that UV was used first, followed by M N N G treatment or M N N G was used first followed by UV exposure. It is seen that the mutant frequency was higher when the cells were exposed to M N N G before UV exposure than in the reverse case. If the UV treatment was carried out immediately after MNNG exposures or delayed for up to 4 h, the increase in mutation frequency was almost same. But if UV treatment was delayed for 18 h, there was a decrease in mutant frequency. The value was almost the same as that obtained with exposure to UV first and then to MNNG. In the latter case, the yield was equivalent to the summation of yield obtained by the two agents acting independently. This was also true for a growth period of 24 h between the two exposures. Discussion
It has been known for some time that the pathways for repair of UV damage and alkylation damage are different (Cleaver, 1971; AltamiranoDimas et al., 1979). Amongst all the cellular reaction products by an alkylating agent, alkylation of DNA is doubtless the most important for genetic toxicology (Brendet and Ruhland, 1984). It has been observed that N-7 alkylguanine and N-3 alkyladenine formed after treatment with alkylating agents could be removed quickly in CHO and mouse cells (Gincher and Veleminsky, 1982) and also in V79 cells (Warren et al., 1979). However 0-6 alkylguanine and methylated triesters formed after MNNG treatment could not be removed in the case of V79 cells (Warren et al., 1979). In XP cells, repair replication after treatment with monofunctional alkylating agents was normal (Cleaver,
1971; Stich et al., 1973), although on transformation, such cells could not properly eliminate 0-6 alkylguanine but could release N-7 alkylguanine normally (Goth-Goldstein, 1977). Increased killing and mutation by UV in cells pretreated with MNNG could be due to any of the following reasons or to a combination of them: (I) Direct interaction of the DNA lesions in close proximity; (II) Overlap of the two repair pathways; (III) MNNG might chemically alter DNA such that its sensitivity to UV is increased; (IV) Interaction via nucleotide pool and repair enzymes. In this connection it should be noted that pretreatment with UV did not in any way affect the MNNG induced killing or mutation. It was shown by Cleaver et al. (1979) that the number of dimers formed per 107 dalton of mammalian DNA was 2 per 10 J / m 2 of UV. If we extrapolate the data to our case, approximately 2-6 dimers are formed per 107 dalton on our UV treatments. We can also extrapolate the data presented by Roberts e t al. (1971) to find the approximate DNA alkylation product in our case is between 250 and 700 per 107 dalton. Thus the first possibility does not seem likely because of the small number of photoproducts. The second possibility, i.e. overlap of two repair pathways, also does not seem likely because had this been the case, one would have expected sensitization to UV by MNNG treatment as well as sensitization to MNNG by UV treatment. The major photoproduct of UV-irradiation, thymine dimers, is unlikely to have significant influence on the methylation of the purines; but methylation of the oxygen atoms of pyrimidines (Farmer et al., 1973; Singer, 1976) may influence the action of UV on pyrimidines, giving a likely explanation of the observation that MNNG treatment affected subsequent UV-induced effect, although UV exposures did not influence MNNGinduced effects. It has been claimed that UV repair involved a large complex of enzymes (Haynes, 1966), one or more alkylations per enzyme would thus be able to inhibit the repair of UV-induced damage, resulting in increased killing and mutation by UV in MNNG-exposed cells, compared with cells which were treated with UV alone. A similar proposition had been made for mammalian cells (Gruenert
82
and Cleaver, 1981; Park and Cleaver, 1982) and for bacteria (Correia and Tyrrel, 1979) to explain the inhibition of UV-induced repair by alkylating agents. Thus the increased killing and mutation induction by UV in MNNG-treated cells could also be due to the inhibition of repair enzymes. Another important point to note is that DNA bases in the nucleotide pool could also be alkylated (Topal and Baker, 1982). Such alkylation would give rise to an increase in the mutation yield induced by UV. On UV-irradiation, no such possibility exists. Clearly, this observation might offer an alternative explanation for the results obtained by interaction studies. Had this been the case, we would have expected sensitization to MNNG on previous UV-irradiation. In this connection, the existence of sublethal damage in UV-irradiated cells might play a significant role. It has been shown that repair of such damage can be inhibited by MNNG treatment, as evidenced from the loss of the shoulder width from UV-survival curves. For MNNG treatment, there is no shoulder in the survival curve and UV-irradiation does not influence the killing by MNNG. Repair of sublethal damage has been claimed to be error-proof (Jostes and Painter, 1980) and its inhibition would naturally increase the load upon the 'error-prone' systems, giving more mutations. This suggestion can offer an explanation for the results obtained in the interaction studies. Acknowledgements We are grateful to Ashis Kumar Datta and R.M. Tewary for excellent technical assistance.
References Ahmed, F,E., and R.B. Setlow (1977a) Different rate limiting steps in excision repair of ultraviolet and N-acetoxy-2acetylaminofluorene damaged DNA in normal human fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 74, 1548-1552. Ahmed, F.E., and R.B. Setlow (1977b) DNA repair in V79 cells treated with combinations of ultraviolet radiation and Nacetoxy-2-acetylaminofluorene, Cancer Res. 37, 3414-3419. Ahmed, F.E., and R.B. Setlow (1980) DNA repair in V79 cells treated with combinations of physical and chemical carcinogens, Photochem. Photobiol., 32, 629-633. Ahmed, Z., and J. Laval (1984) Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methyl-
thymine-DNA metyltransferase and a O2-methylthymine DNA glycosylase, Biochem. Biophys. Res. Commun., 120, 1-8. Altamirano-Dimas, M., R. Skiar and B. Strauss (1979) Selectivity of the excision of alkylation products in a xeroderma pigmentosum, Mutation Res., 60, 197-206. Bhattacharjee, S.B., and B. Pal (1982) Tetracycline induced mutation in cultured Chinese hamster cells, Mutation Res., 101, 329-338. Bhattacharjee, S.B., S. Chatterjee and B. Pal (1982) Survival and mutation of Chinese hamster cells after ultraviolet irradiation and caffeine treatment, Mutation Res., 106, 137-146. Brendel, M., and A. Ruhland (1984) Relationship between functionality and genetic toxicology of selected DNA damaging agents, Mutation Res., 133, 51-85. Cleaver, J.E. (1971) Repair of alkylation damage in ultraviolet sensitive (xeroderma pigmentosum) human cells, Mutation Res., 12, 453-462. Cleaver, J.E., G.H. Thomas, J.E. Trosko and J.T. Lett (1972) Excision repair (dimer excision, strand breakage, repair replication) in primary cultures of eukaryotic (cattle) cells, Exp. Cell. Res., 74, 67-80. Cleaver, J.E., G.H. Thomas and S.D. Park (1979) Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light, Biochim. Biophys. Acta, 564, 122-131. Correia, I.S., and R.M. Tyrrell (1979) Lethal interaction between ultraviolet radiations and methyl methanesulphonate in repair-proficient and repair-defecient strains of Escherichia coli, Photochem. Photobiol., 29, 521-526. Farmer, P.B., A.B. Foster, M. Jarman and M.J. Tisdale (1973) The alkylation of 2'-deoxyguanosine and of thymidine with diazoalkanes, some observations on O-alkylation, Biochem. J., 135, 203-213. Gincher, T., and J. Veleminsk~, (1982) Genetic effects of Nmethyl-N'-nitro-N-nitrosoguanidine and its homologs, Mutation Res., 99, 129-242. Goth-Goldstein, R. (1977) Repair of DNA damaged by alkylating carcinogens is defective in xeroderma pigmentosum derived fibroblasts, Nature (London), 267, 81-82. Gruenert, D.C., and J.E. Cleaver (1981) Repair of ultraviolet damage in human cells and also exposed to agents that cause strand breaks, crosslinks, monoadducts and alkylations, Chem.-Biol. Interact., 33, 163-177. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) D N A repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Haynes, R.H. (1966) in General discussion, led by M. Delbruck, Radiation Res., 6 (Suppl.), 231-232. Jostes Jr., R.F. and R.B. Painter (1980) Evidence for repair of premutational damage from split X-ray doses, Radiat. Res., 84, 586-590. Lawley, P.D., D.J. Orr, S.A. Shah, P.B. Farmer and M. Jarman (1973) Reaction products from N-methyl-N-nitrosourea and DNA-containing thymidine residues, synthesis and identification of a new methylation product O4-methylthymidine, Biochem. J., 135, 193-201.
83 Park, S.D., and J.E. Cleaver (1982) Inactivation of nucleotide excision repair in Chinese hamster ovary cells by exposure to an alkylating agent, Photoehem. Photobiol., 35, 419-421. Park, S.D., K.H. Choi, S.W. Hong and J.E. Cleaver (1981) Inhibition of excision repair of ultraviolet damage in human ceils by exposure to methyl methanesulphonate, Mutation Res., 82, 365-371. Roberts, J.J. (1978) The repair of DNA modified by cytotoxic, mutagenic and carcinogenic chemicals, Adv. Radiat. Biol., 7, 211-414. Roberts, J.J., J.M. Pascoe, J.E. Plant, J.E. Sturrok and A.R. Crathorn (1971) Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells, 1. The effect on Hela and Chinese hamster cell survival of alkylation of cellular macromolecules, Chem.-Biol. Interact., 3, 29-47. Sasaki, M.S. (1973) DNA repair capacity and susceptibility to chromosome breakage in xeroderma pigmentosum cells, Mutation Res., 20, 291-293.
Singer, B. (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents, Nature (London), 264, 333-339. Stich, H.F., R.H.C. San and Y. Kawazoe (1973) Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagen, Mutation Res., 17, 127-137. Topal, M.D., and M.S. Baker (1982) DNA precursor pool: A significant target for N-methyl-N-nitrosourea in C3H/101 clone 8 cells, Proc. Natl. Acad. Sci. (U.S.A.), 79, 2211-2215. Warren, W., A.R. Crathorn and KS. Shooter (1979) The stability of methylated purines and of methylated phosphotriesters in DNA of V79 cells after treatment with N-methyl-N-nitrosourea, Biochim. Biophys. Acta, 563, 82-88. Wolff, S., B. Rodin and J.E. Cleaver (1977) Sister-chromatid exchange induced by mutagenic carcinogens in normal and Xeroderma pigmentosum cells, Nature (London), 265, 347-349.
77
MTR 04099
Interaction of UV and
N-methyl-N'-nitro-N-nitrosoguanidine:Cytotoxicity and mutagenicity in V79 cells
Nitaipada Bhattacharyya and Sukhendu B. Bhattacharjee Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Calcutta-700 064 (India) (Received 27 December 1984) (Revision received 23 April 1985) (Accepted 7 May 1985)
Summary Killing and mutation by UV in the MNNG-exposed population of V79 cells, as well as by MNNG in the UV-irradiated population of these cells have been studied. It was observed that pretreatment with MNNG increased the killing and mutation by UV, whereas, pretreatment with UV had no effect upon killing and mutation by MNNG. The increase in sensitivity to UV due to pretreatment with MNNG was lost if UV exposure was delayed for 24 h after MNNG treatment.
The interaction between two different DNAdamaging agents has been used in a number of experiments to study whether there is any common pathway for the repair of the damage (Ahmed and Setlow, 1977a,b; Gruenert and Cleaver, 1981; Park et al., 1981; Park and Cleaver, 1982). Gruenert and Cleaver (1981) showed that in normal human cells and in XP (xeroderma pigmentosum) variant cells, 4 mM MMS (methyl methanesulphonate) treatment reduced the repair replication induced after UV exposures. For both types of cells, the reduction was almost the same whether the cells were exposed to MMS first and then to UV or to UV first then to MMS. It was also seen that when normal human cells were treated with N-methylN'-nitro-N-nitrosoguanidine (MNNG) and then with graded doses of UV, there was a reduction in the repair replication. But with XP (group G) cells, similar treatment did not affect repair replication. Address for correspondence: Dr. S.B. Bhattacharjee, Professor and Head, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Sector" 1, Block 'AF', Bidhan Nagar, Calcutta-700 064 (India).
The magnitude of repair replication induced by MNNG in XP cells was the same as in the normal cells. It is thus clear that in XP cells, UV-irradiation did not inhibit repair of alkylation damage. Park et al. (1981) showed that in CHO and normal human cells, the unscheduled DNA synthesis (measured by autoradiograph) induced by UV and MMS were less than that expected from the sum of the unscheduled synthesis seen in cells exposed to UV or MMS alone. The synthesis was found to be independent of the order in which the cells were exposed. It was also shown that exposure to 1 mM MMS caused a slight reduction in the fraction of dimers excised, and to 2 mM, almost completely inhibited excision (Park et al., 1981). Unscheduled DNA synthesis and excision of pyrimidine dimers in UV-irradiated CHO cells were inhibited by prior exposure to the alkylating agent MNNG (Park and Cleaver, 1982). The excision repair ability of Chinese hamster V79 cells treated with N-acetoxy-2-acetylaminofluorene and UV light was measured by 3 techniques. It was seen that the amount of repair from combined treatment was less than additive and in
0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
78 some cases less than that due to either agent (Ahmed and Setlow, 1977b). But in human cells, the same combination of treatment resulted in repair which was additive (Ahmed and Setlow, 1977a). For V79 cells, additivity in repair was observed following UV and MMS exposure (Ahmed and Setlow, 1980). Measurement of inhibition of DNA synthesis from the incorporation of radioactive thymidine showed an additive effect when M N N G was applied along with UV radiation (Gichner and Veleminsky, 1982). However, it is interesting to note that the conclusions arrived at depended on the cell type and the nature of the agents and end-points although killing and mutation had not been utilized in these studies. It may be worthwhile to see if the observations made on the basis of these two end-points corroborate the earlier findings. With this in mind, we studied the effect of M N N G treatment on UV-induced killing and mutation and also the influence of pre-exposure to UV on MNNG-induced effects on V79 cells, in terms of killing and mutation. We have been able to show that in such interactions the order in which the exposures are carried out is important. Material and methods
MNNG, neomycin sulphate and 8-azaguanine (AG) were purchased from Sigma Chemical Co., penicillin and streptomycin sulphate from local sources. M N N G was dissolved in sterilized double distilled water at a final concentration of 300 /~g/ml, immediately before use and diluted to the desired concentrations. An A G stock solution was made in dimethyl sulphoxide (DMSO). Details about the culture conditions, media etc., survival and mutation studies have already been published (Bhattacharjee and Pal, 1982; Bhattacharjee et al., 1982). In brief, exponentially growing cells were washed with phosphate-buffered saline (PBS), trypsinised, diluted and plated in the required number and allowed to attach for about 4 h at 37°C in a 5% CO 2 humidified atmosphere. After attachment, the medium is withdrawn and the cells exposed either, to M N N G in growth medium for 1 h, or to UV in the absence of medium. Fresh medium was then added and incubated f o r colony formation. For interaction
studies, cells were exposed to M N N G and then to UV, or first to UV and then to M N N G immediately after the first exposure, or with a desired intervening growth period between the two treatments. For mutation studies, approximately 106 cells were treated with M N N G or exposed to UV. Some of the MNNG-exposed cells were treated with UV immediately or after an intervening period growth and some UV-irradiated cells were treated with M N N G immediately or after subsequent growth for the desired time. Thereafter, the cells were grown for 7-9 days for expression, with 3-4 subcultures. After expression, cells were replated at 5 x 105 cells per 100 mm in Coming petri dishes [this cell number was determined by reconstruction experiments (data not shown)] in selection medium which contained AG (3 /~g/ml) in complete growth medium (final concentration of DMSO 0.1%). A further increase in the concentration of AG up to 6/~g/ml did not affect the yield of mutants (data not shown). About 93% of the AG-resistant clones were sensitive to HAT medium (complete medium containing hypoxanthine 10 _4 M, aminopterin 3 x 10 - 7 M and thymidine 10 _5 M). Each data point represents the average of 2-5 independent experiments, the standard deviations being denoted by bars. Results
Fig. 1 shows the UV survival of cells pretreated with MNNG. Treatment with MNNG, immediately before the UV exposures reduces the shoulder width in a dose-dependent manner and increases the slope of the survival curve significantly, although the rate of increase was the same within the M N N G concentration range 0.1 /~g/ml to 1 /~g/ml. Fig. 2 shows the effect of previous UV exposures on the sensitivity of the cells to the graded doses of MNNG. It can be seen that previous exposure to UV did not influence the M N N G induced killing. The effect of separating the exposures to two agents by intervening periods of incubation in growth medium is" illustrated in Fig. 3. When 15 J / m 2 UV-exposed cells were treated immediately,
79 i0 °
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la.
tt~ D CO
I 5
I 10
I 15
I 20
25
30
UV FLUENCE J / M 2
1(3:
Fig. 1. Survival of V79 cells exposed to different UV fluences. Cells were pretreated with MNNG: 0.0/tg/ml ((3); 0.1/tg/ml (z,); 0.5/~g/ml ([3); 1.0/~g/ml (@).
with 1 /~g/ml M N N G for 1 h the survival level was 0.02, but when the M N N G treatment was delayed for 2 - 6 h, there was a sharp increase in survival. On further delay up to 24 h, there was no further increase in survival. But when cells were first treated with 1 ~ g / m l M N N G and then grown io°
i
i
i
i
i
L
4
~,
12
16
20
24
TIME BETWEEN THE DOSES (Hours)
Fig. 3. Survival of V79 cells exposed to 15 J / m 2 UV, followed by 1 ~g/ml of M N N G treatment (@) or to 1/~g/ml of M N N G followed by 15 J / m E of UV-irradiation (~,).
for several hours, before the UV exposure, there was an increase in survival in 4 - 1 2 h, whereafter it remained constant up to 24 h. But in all cases, the survival level was lower than would have been obtained by exposure to the second agent alone. 10'
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CONCENTRATION OF MNNGy.g/ml Fig. 2. Sensitivity of UV-irradiated V79 cells to MNNG: UV fluences: 0 J / m 2 (Q), 8 J / m 2 (0), 12 J / m 2 (~.), and 20 J / m 2
(A).
Fig. 4. Sensitivity of M N N G
exposed cells held for 24 h to
subsequent UV exposures (1~), compared to the cells which were not held after M N N G treatment (~.). The symbol ((3) represents the UV survival of control untreated cells.
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Fig. 5. Survival of V79 ceils on treatment with M N N G . Cells having no pretreatment (Q), pretreated with 20 J / m 2 UV, either immediately before (r~) or 24 h before (~) M N N G treatments.
Fig. 7. Influence of preirradiation with UV on M N N G - i n d u c e d mutation. UV fluences 0 J / m 2 (Q)~ lO J / m 2 (.~.), 20 J / m 2 ([]), and 30 J / m 2 (A).
Fig. 4 represents the survival curves in which treatment with graded doses of UV was carried out, 24 h after the first M N N G treatment. It can be seen that the increase in the slope of the UVsurvival curve, due to M N N G pretreatment (as observed in Fig. 1) was lost due to a 24-h delay. The effect of holding UV-irradiated (20 J / m 2) cells in growth medium for 24 h on their subse-
quent sensitivity to M N N G is shown in Fig. 5. In terms of survival the cells behave as if they had not been exposed to UV before. Fig. 6 shows the yield of 8-azaguanine-resistant mutants by UV-light irradiation of cells previously treated with MNNG. It can be seen that due to pretreatment with MNNG, the rate of mutation induction by UV is increased, although the in-
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TIME BETWEEN TWO EXPOSURES (HOURS)
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UV F'LUEHCE J / M 2
Fig. 6. Influence of pretreatment with M N N G on UV-induced yield of mutants. M N N G concentrations: 0 /~g/m] (Q), 0.I # g / m ] (,*,),'0.5 # g / m l (El), ].0 ~ g / m l (11).
Fig. 8. Interaction of UV and M N N G on the induction of mutation. After 10 J / m 2 UV treatment cells were held for different periods before treament with 0.2 # g / m l M N N G was done (A). Alternatively, cells treated with 0 . 2 / ~ g / m l of M N N G were held for various periods before being exposed to 10 J / m 2 UV (®).
81 crease in rate was the same for concentrations of M N N G ranging between 0.1 ~g/ml and 1/~g/ml. Fig. 7 shows the variation in yield of 8azaguanine-resistant mutants induced by M N N G treatment of previously UV-irradiated cells. The induction of mutation was not in any way influenced by UV pretreatment. Fig. 8 represents the induction of 8-azaguanine-resistant mutants on separation of the UV and M N N G treatments by varying periods of intervening growth. The UV dose used was 10 J / m 2 and the MNNG dose 0.2 /~g/ml. The exposure conditions were that UV was used first, followed by M N N G treatment or M N N G was used first followed by UV exposure. It is seen that the mutant frequency was higher when the cells were exposed to M N N G before UV exposure than in the reverse case. If the UV treatment was carried out immediately after MNNG exposures or delayed for up to 4 h, the increase in mutation frequency was almost same. But if UV treatment was delayed for 18 h, there was a decrease in mutant frequency. The value was almost the same as that obtained with exposure to UV first and then to MNNG. In the latter case, the yield was equivalent to the summation of yield obtained by the two agents acting independently. This was also true for a growth period of 24 h between the two exposures. Discussion
It has been known for some time that the pathways for repair of UV damage and alkylation damage are different (Cleaver, 1971; AltamiranoDimas et al., 1979). Amongst all the cellular reaction products by an alkylating agent, alkylation of DNA is doubtless the most important for genetic toxicology (Brendet and Ruhland, 1984). It has been observed that N-7 alkylguanine and N-3 alkyladenine formed after treatment with alkylating agents could be removed quickly in CHO and mouse cells (Gincher and Veleminsky, 1982) and also in V79 cells (Warren et al., 1979). However 0-6 alkylguanine and methylated triesters formed after MNNG treatment could not be removed in the case of V79 cells (Warren et al., 1979). In XP cells, repair replication after treatment with monofunctional alkylating agents was normal (Cleaver,
1971; Stich et al., 1973), although on transformation, such cells could not properly eliminate 0-6 alkylguanine but could release N-7 alkylguanine normally (Goth-Goldstein, 1977). Increased killing and mutation by UV in cells pretreated with MNNG could be due to any of the following reasons or to a combination of them: (I) Direct interaction of the DNA lesions in close proximity; (II) Overlap of the two repair pathways; (III) MNNG might chemically alter DNA such that its sensitivity to UV is increased; (IV) Interaction via nucleotide pool and repair enzymes. In this connection it should be noted that pretreatment with UV did not in any way affect the MNNG induced killing or mutation. It was shown by Cleaver et al. (1979) that the number of dimers formed per 107 dalton of mammalian DNA was 2 per 10 J / m 2 of UV. If we extrapolate the data to our case, approximately 2-6 dimers are formed per 107 dalton on our UV treatments. We can also extrapolate the data presented by Roberts e t al. (1971) to find the approximate DNA alkylation product in our case is between 250 and 700 per 107 dalton. Thus the first possibility does not seem likely because of the small number of photoproducts. The second possibility, i.e. overlap of two repair pathways, also does not seem likely because had this been the case, one would have expected sensitization to UV by MNNG treatment as well as sensitization to MNNG by UV treatment. The major photoproduct of UV-irradiation, thymine dimers, is unlikely to have significant influence on the methylation of the purines; but methylation of the oxygen atoms of pyrimidines (Farmer et al., 1973; Singer, 1976) may influence the action of UV on pyrimidines, giving a likely explanation of the observation that MNNG treatment affected subsequent UV-induced effect, although UV exposures did not influence MNNGinduced effects. It has been claimed that UV repair involved a large complex of enzymes (Haynes, 1966), one or more alkylations per enzyme would thus be able to inhibit the repair of UV-induced damage, resulting in increased killing and mutation by UV in MNNG-exposed cells, compared with cells which were treated with UV alone. A similar proposition had been made for mammalian cells (Gruenert
82
and Cleaver, 1981; Park and Cleaver, 1982) and for bacteria (Correia and Tyrrel, 1979) to explain the inhibition of UV-induced repair by alkylating agents. Thus the increased killing and mutation induction by UV in MNNG-treated cells could also be due to the inhibition of repair enzymes. Another important point to note is that DNA bases in the nucleotide pool could also be alkylated (Topal and Baker, 1982). Such alkylation would give rise to an increase in the mutation yield induced by UV. On UV-irradiation, no such possibility exists. Clearly, this observation might offer an alternative explanation for the results obtained by interaction studies. Had this been the case, we would have expected sensitization to MNNG on previous UV-irradiation. In this connection, the existence of sublethal damage in UV-irradiated cells might play a significant role. It has been shown that repair of such damage can be inhibited by MNNG treatment, as evidenced from the loss of the shoulder width from UV-survival curves. For MNNG treatment, there is no shoulder in the survival curve and UV-irradiation does not influence the killing by MNNG. Repair of sublethal damage has been claimed to be error-proof (Jostes and Painter, 1980) and its inhibition would naturally increase the load upon the 'error-prone' systems, giving more mutations. This suggestion can offer an explanation for the results obtained in the interaction studies. Acknowledgements We are grateful to Ashis Kumar Datta and R.M. Tewary for excellent technical assistance.
References Ahmed, F,E., and R.B. Setlow (1977a) Different rate limiting steps in excision repair of ultraviolet and N-acetoxy-2acetylaminofluorene damaged DNA in normal human fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 74, 1548-1552. Ahmed, F.E., and R.B. Setlow (1977b) DNA repair in V79 cells treated with combinations of ultraviolet radiation and Nacetoxy-2-acetylaminofluorene, Cancer Res. 37, 3414-3419. Ahmed, F.E., and R.B. Setlow (1980) DNA repair in V79 cells treated with combinations of physical and chemical carcinogens, Photochem. Photobiol., 32, 629-633. Ahmed, Z., and J. Laval (1984) Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methyl-
thymine-DNA metyltransferase and a O2-methylthymine DNA glycosylase, Biochem. Biophys. Res. Commun., 120, 1-8. Altamirano-Dimas, M., R. Skiar and B. Strauss (1979) Selectivity of the excision of alkylation products in a xeroderma pigmentosum, Mutation Res., 60, 197-206. Bhattacharjee, S.B., and B. Pal (1982) Tetracycline induced mutation in cultured Chinese hamster cells, Mutation Res., 101, 329-338. Bhattacharjee, S.B., S. Chatterjee and B. Pal (1982) Survival and mutation of Chinese hamster cells after ultraviolet irradiation and caffeine treatment, Mutation Res., 106, 137-146. Brendel, M., and A. Ruhland (1984) Relationship between functionality and genetic toxicology of selected DNA damaging agents, Mutation Res., 133, 51-85. Cleaver, J.E. (1971) Repair of alkylation damage in ultraviolet sensitive (xeroderma pigmentosum) human cells, Mutation Res., 12, 453-462. Cleaver, J.E., G.H. Thomas, J.E. Trosko and J.T. Lett (1972) Excision repair (dimer excision, strand breakage, repair replication) in primary cultures of eukaryotic (cattle) cells, Exp. Cell. Res., 74, 67-80. Cleaver, J.E., G.H. Thomas and S.D. Park (1979) Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light, Biochim. Biophys. Acta, 564, 122-131. Correia, I.S., and R.M. Tyrrell (1979) Lethal interaction between ultraviolet radiations and methyl methanesulphonate in repair-proficient and repair-defecient strains of Escherichia coli, Photochem. Photobiol., 29, 521-526. Farmer, P.B., A.B. Foster, M. Jarman and M.J. Tisdale (1973) The alkylation of 2'-deoxyguanosine and of thymidine with diazoalkanes, some observations on O-alkylation, Biochem. J., 135, 203-213. Gincher, T., and J. Veleminsk~, (1982) Genetic effects of Nmethyl-N'-nitro-N-nitrosoguanidine and its homologs, Mutation Res., 99, 129-242. Goth-Goldstein, R. (1977) Repair of DNA damaged by alkylating carcinogens is defective in xeroderma pigmentosum derived fibroblasts, Nature (London), 267, 81-82. Gruenert, D.C., and J.E. Cleaver (1981) Repair of ultraviolet damage in human cells and also exposed to agents that cause strand breaks, crosslinks, monoadducts and alkylations, Chem.-Biol. Interact., 33, 163-177. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) D N A repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Haynes, R.H. (1966) in General discussion, led by M. Delbruck, Radiation Res., 6 (Suppl.), 231-232. Jostes Jr., R.F. and R.B. Painter (1980) Evidence for repair of premutational damage from split X-ray doses, Radiat. Res., 84, 586-590. Lawley, P.D., D.J. Orr, S.A. Shah, P.B. Farmer and M. Jarman (1973) Reaction products from N-methyl-N-nitrosourea and DNA-containing thymidine residues, synthesis and identification of a new methylation product O4-methylthymidine, Biochem. J., 135, 193-201.
83 Park, S.D., and J.E. Cleaver (1982) Inactivation of nucleotide excision repair in Chinese hamster ovary cells by exposure to an alkylating agent, Photoehem. Photobiol., 35, 419-421. Park, S.D., K.H. Choi, S.W. Hong and J.E. Cleaver (1981) Inhibition of excision repair of ultraviolet damage in human ceils by exposure to methyl methanesulphonate, Mutation Res., 82, 365-371. Roberts, J.J. (1978) The repair of DNA modified by cytotoxic, mutagenic and carcinogenic chemicals, Adv. Radiat. Biol., 7, 211-414. Roberts, J.J., J.M. Pascoe, J.E. Plant, J.E. Sturrok and A.R. Crathorn (1971) Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells, 1. The effect on Hela and Chinese hamster cell survival of alkylation of cellular macromolecules, Chem.-Biol. Interact., 3, 29-47. Sasaki, M.S. (1973) DNA repair capacity and susceptibility to chromosome breakage in xeroderma pigmentosum cells, Mutation Res., 20, 291-293.
Singer, B. (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents, Nature (London), 264, 333-339. Stich, H.F., R.H.C. San and Y. Kawazoe (1973) Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagen, Mutation Res., 17, 127-137. Topal, M.D., and M.S. Baker (1982) DNA precursor pool: A significant target for N-methyl-N-nitrosourea in C3H/101 clone 8 cells, Proc. Natl. Acad. Sci. (U.S.A.), 79, 2211-2215. Warren, W., A.R. Crathorn and KS. Shooter (1979) The stability of methylated purines and of methylated phosphotriesters in DNA of V79 cells after treatment with N-methyl-N-nitrosourea, Biochim. Biophys. Acta, 563, 82-88. Wolff, S., B. Rodin and J.E. Cleaver (1977) Sister-chromatid exchange induced by mutagenic carcinogens in normal and Xeroderma pigmentosum cells, Nature (London), 265, 347-349.