Mutation Research, 31 (1975) 153-161 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
N-NITROSOMORPHOLINE AND N-NITROSOBUTYLAMINE-STIMULATED DNA SYNTHESIS IN BHK-2I/CI 3 CELLS*
C. E. KIMBLE, PAULA A. GORCZYCA AND R. G. REYNOLDS
Department of Nutrition and Food Science* *, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.) (Received October 25th, 1974)
Non-semiconservative DNA synthesis was examined in BHK-2I/CI 3 cells after treatment with the aliphatic N-nitrosobutylamine (NB) and the heterocyclic Nnitrosomorpholine (NM). The extent of repair synthesis after alkylation was compared quentitatively and calculations were made of the amount of DNA damage per cell and number of bases inserted per damaged site. Of the chromosome aberrations caused by NM the most predominant were dicentrics. Other aberrations included fragments, gaps, breaks and exchanges of both the chromatid and chromosome type. Unlike NM, NB elicted a higher frequency of chromatid breaks.
The N-nitrosamines are currently of great interest as possible etiologic agents in the induction of human cancer. There has been considerable experimental evidence to support the hypothesis that tumors arise in animals treated with N-nitrosamines because of enzymatic conversion of these compounds to alkylating agents ~4. These electrophilic species then alkylate the DNA in susceptible organs. Thus, it is postulated that some proximal carcinogenic agents may exert their action by reacting with the DNA, causing somatic mutations through misreading. The differing susceptibilities of organs to a particular agent may be a consequence not only of different levels of reaction, but also of differences in the ability of cells to repair lesions. Carcinogens may also be capable of influencing normal repair processes. Repair and unscheduled DNA synthesis have been demonstrated in a number * Research supported in part by Contracts No. N.I.H. 7o-218o and No. X.I.H. 5-PO-I-ES-oo597 from the National Cancer Institute, National Institutes of Health. ** Contribution No. 2036 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology. Abbreviations: BrUdR, bromodeoxyuridine; EBME, Eagle's basal medium supplemented with Earle's salts; FUdR, fluorodeoxyuridine; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NB, N-nitrosobutylamine; NM, N-nitrosomorpholine; NMU, N-nitroso-N-methylurea; SSC, o.15 M NaCl-o.oI 5 M sodium citrate; TdR, thymidine.
c.E. KIMBLE et al.
of mammalian cell types following exposure to X-rays 26, UV light3-~,l°,al,33,aL and methyl methanesulfonate 1. Removal of alkylated groups and radiolysis products~,~, 1~ from DNA has also been demonstrated and it is presumed that the repair processes following alkylation and X-radiation have at least some steps in common with those observed after exposure to UV light. Much of the early work described hydrolytic degradation of alkylated DNA as analogous to the action of ionizing radiation~,~9, "~. Spontaneous depurination of 3- and 7-alkylpurines from DNA at neutral pH was thought to be a possible source of DNA strand breaks, and by analogy with repair of X-ray-induced lesions, a repair to such damage was considered probable. DRUCKRE¥ and co-workers 9showed that those nitrosamines convertible, by oxidative dealkylation into presumably a monoalkylnitrosamine and then to a carbonium ion, were carcinogenic; those compounds such as tert-butylmethylnitrosamine which are incapable of such conversion were not carcinogenic 19. Later work 3~ indicated that this was not true for a number of compounds. Nitrosomethyl aniline, which falls into the same class as tert-butylmethylnitrosamine, is carcinogenic. CERDA-OLMEDO AND HANAWALT2 have presented evidence that N-methylN'-nitro-N-nitrosoguanidine (MNNG) initiates a repair process similar to that induced by ultraviolet irradiation. However, information on the metabolism of the potent liver carcinogen, NM 3s is extremely limited. It has been found to be mutagenic for bacteria only under the conditions of a host-mediated assay 38. This suggests that in contrast to N-nitroso-N-methylurea (NMU) and MNNG, NM requires metabolic conversion for mutagenic activity29, 38. In order to adapt NM into the activation scheme for aliphatic nitrosamines, a ring-opening mechanism would have to be devised. An alternative to a ring-opening mechanism would be a metabolic activation of the nitroso group. It is important, therefore, that more is known about the effects of this chemical on the DNA of mammalian cells in order to elucidate its mode of action. This paper deals with nitrosomorpholine-stimulated DNA synthesis in cultured cells and the types of chromosome aberrations elicited by it as compared to the alkylnitroso compounds NB and MNNG. MATERIALS AND METHODS
Chemicals MNNG was obtained from Eastman Organic Chemicals, Rochester, N.Y. N-Nitrosomorpholine and N-nitrosobutylamine were kindly supplied by S. R. Tannenbaum (Cambridge, Massachusetts). Cell culture B H K - 2 I , clone 13 derived from an established line of pseudo-diploid Syrian hamster cells were used throughout the experiments. This cell line exhibits vigorous growth and high plating efficiency. In addition, the relatively low chromosome number and morphologically distinguishable individual chromosomes facilitate cytogenetic investigations. Stock cultures were grown as a monolayer either on glass or Falcon plastic in Eagle's basal medium supplemented with Earle's salts (EBME), non-essential amino acids, IO~o heat-inactivated calf serum, and antibiotics (50 units penicillin/ml and 50 /~g streptomycin/ml). This medium was prepared from commercial component
D N A DAMAGE BY N-NITROSAMINES
solutions as supplied b y Grand Island Biological Co., Grand Island, N.Y. Both experimental and stock cultures were maintained at 37 ° in humidified 5~o CO2 in air.
Density gradient centrifugation Repair replication was determined by the method of PETTIJOHN AND HANAWALT adapted for use in mammalian cells ~6. Freshly transferred cells were grown in EBME for 17 h and then grown in bromodeoxyuridine (BrUdR, 3/~g/ml) and fluorodeoxyuridine (FUdR) (io-6M) for 2 h to begin the formation of BrUdR-substituted DNA. The cells were then incubated in [~HIBrUdR (3/~Ci/ml), F U d R (Io-~M) and hydroxyurea (2. lO-13 M) for 4 h. Hydroxyurea was used to suppress semiconservative DNA replication and improve resolution of repair replication. The amount of repair replication is unaffected by hydroxyurea in mammalian cells1, 4. After labelling, DNA was isolated and analysed in isopycnic gradients. Gradients were calibrated using Micrococcus luteus DNA (1.731 g/cm3). Relative levels of repair replication were determined on the basis of the cpm//~g in DNA purified from isopycnic gradients. Calculations of number-average molecular weights (M~) for complete profiles were made according to LETT et al. ~1. After labeling the cells were lysed with o.2~o sodium dodecyl sulfate. The lysate was treated with a concentrated ribonuclease (Sigma) solution that had been heated to 80 ° for IO rain. The RNAase was made to a final concentration of 60 #g/ml and incubated for I h. Pronase (Calbioehem) was added to a final concentration of 500 #g/ml and the incubation continued for another hour. This was followed by two treatments with chloroform-amyl alcohol (24:1) to remove extraneous protein. The solution was then dialyzed against several changes of o.15 M NaCl-o.oI 5 M sodium citrate (SSC) and added to CsC1. The solutions were then centrifuged at 38 ooo rev./min in a SW 5o.1 rotor (Beckman) for 38 h. After centrifugation, fractions were collected from the bottom of the tubes on filter discs and radioactivity measurements made on a Beckman model LS-23 o liquid scintillation counter. Chromosomal analysis In order to estimate chromosome aberrations, dividing cells were arrested at metaphase with colchicine (0.06 ml of o.oI ~o solution per ml of EBME). The colchicine was added 4 h prior to sampling. The cultures were pretreated for 15 min in 1°/0 sodium citrate solution at 37 °, fixed in three changes of ethanol:acetic acid (3:I), 15 rain each, air dried, stained in 2% aceto-orcein, dehydrated and mounted. Frequency of chromatid aberrations and incidence of metaphase plates were estimated on the same preparations. For each sample well-spread metaphase plates were analyzed for chromatid and chromosome breaks, exchanges and fragmentation. RESULTS
The results with B H K - 2 I cells (Figs. i and 2, Table I) indicate that repair replication does occur after treatment with NB and the cyclic NM. The peak fractions from the parental density peaks were pooled and rebanded isopycnicly. In the second reband it was important that there was a minimum of trailing of the hybrid control peak into the parental light density region. The trailing of the hybrid peak into the
2800~ 2400 F
2000 ~ ' ~ k
30 40 50 60 70
0.20 1 010
80 90 Io ° 0.70 o
40 50 60 70 80 Fraction Number
Fig. I. D e n s i t y g r a d i e n t profiles, w i t h B e c k m a n S W 5o.1 rotor, of D N A f r o m B H K - 2 I cells grown for 2 4 h in B U d R (4 # g / m l ) , t r e a t e d w i t h n i t r o s a m i n e s for 2 h, a n d labeled for 4 h in [ 3 H ] B U d R (3 #Ci: i m]). A. Initial g r a d i e n t s f r o m cells t r e a t e d w i t h N M (IOO # g / m l ) .....  ..... , a n d N B (5 ° # g / ml), - - e - - . T h e control is r e p r e s e n t e d b y - - - O - - - . B. Profiles of g r a d i e n t s m a d e f r o m n o r m a l d e n s i t y regions of original gradients. B a r s ( ) indicate p o r t i o n s of g r a d i e n t s t a k e n for r e b a n d i n g a n d specific a c t i v i t y m e a s u r e m e n t s . 1400 -A
~0 ~0 ,oo
600 450 300
I o-J~20 50 40 50 60 70 80 Fraction Number
I 0 90 100
Fig. 2. A. Profiles of g r a d i e n t s m a d e f r o m n o r m a l d e n s i t y regions of first r e b a n d (B) after h e a t i n g to c o n v e r t D N A to single s t r a n d . B. Profiles of g r a d i e n t s m a d e f r o m second r e b a n d (single s t r a n d s ) . S y m b o l s as Fig. I.
DAMAGE BY N'-NITROSAMINES
CHROMOSOME ABERRATIONS AFTER NITROSAMINE TREATMENT OF B H K - 2 I
Compound Number of cells andconcen-Scored Aberrant a tration (t'g/ml)
Type and frequency of aberrations/cell ChroChroChromo- Dicen- Rings matid matid some trics gaps breaks breaks
Chromo- Total somal changes
NM IOOOO IOOO ioo IO o
391 4o7 400 4o0 400
186 193 135 90 15
(47.6) (47.4) (33.8) (22.5) (3.8)
o.o84 0.096 0.025 o.o15 0.075
o.o69 o.o42 0.025 O.OLO 0.075
o.o56 0.037 o.o13 O.OLO o
o.223 o.211 o.193 0.083 0.025
o.141 0.o66 0.028 0.005 0.0o3
o.o54 0.o49 o.175 o.o2o 0.003
o.627 o.5ol o.3ol o.143 0.o45
192 2o0 200 3o0
79 77 53 7
(41.2) (38.5) (26.5) (2.34)
0.o73 o.o80 o.o3o o
o.151 O.lO 5 0.055 O.OLO
O.lO 4 0.o85 o.o4o 0.003
o.141 o.o70 o.o55 o.007
0.o885 o.o65 0.050 o.oo 7
0.042 0.04o 0.035 o.oo 3
0.599 0.445 0.265 o.030
NB IOO io I o MNNG IOOO ioo io i
2o 5 230
127 (62.o) i o o (43.4)
a Figures in parentheses TABLE
no mitoses no mitoses o.161 o.o88 0.074 0.026
SPECIFIC ACTIVITY OF SINGLE-STRANDED D N A OBTAINED FROM THIRD REBAND OF NORMAL DENSITY REGION OF CsC1 GRADIENT
N M (5 ° # g / m l ) NB (ioo/zg/ml) Control
Counts per zo min/ml
Specific activity (counts/mini#g)
i334 4355 2o5
12.i 13.3 12.4
i i.o 32.7 1.7
parental density region would significantly increase the apparent extent of repair replication. The radioactivity found at parental density in the second neutral CsC1 gradient represents incorporation into repair patches. Further analysis of nonconservatively synthesized DNA in BHK-2I cells was carried out by pooling the fractions from the light density region from the first reband. These fractions were denaturated and rebanded in CsC1. Table n gives the specific activities of the DNA from the cells after the third reband. The specific activities are in (cpm//~g) : control, 1.7; NB (50/zg/ml), 32.7; NM (IOO #g/ml), n . o . In the alkaline CsC1 gradients of the third reband a slight skewing of the repair peak to the heavy side of the prelabel peak was seen. The reband does demonstrate clearly that repair occurs, however. The aberrations caused by NM included fragments, gaps, breaks, and exchanges of both the chromatid and chromosome type. Of the aberrations the most predominant were the dicentrics followed by rings, chromosome breaks and chromatid gaps. Unlike NM, MNNG (our positive control) elicited a higher frequency of chromatid breaks than dicentrics (o.21o and o.161 at IO #g/ml). Concentrations of IOO #g/ml of MNNG suppressed mitosis completely despite the presence of colchicine. 62 % of those cells treated with IO/~g/ml MNNG were aberrant compared to 43.4% of the cells
C . E . KIMBLE et al.
treated with I #g/ml. The frequency of aberrations for the io and I #g/ml concentrations were o.741 and 0.360, respectively. This indicated significantly fewer spreads with multiple aberrations at tile lower concentrations of the test compound. With NB chromatid breaks also occurred at the highest frequency followed by dicentrics and chromosome breaks. The average incidence of metaphases of the control cells showing chromosome aberrations was 3.0%. DISCUSSION
Our data show that after multiple rebandings of the normal density regions of equilibrium density gradients of DNA from treated and untreated ceils that a peak of nonsemiconservative DNA synthesis does occur both with the aliphatic NB and the cyclic NM. The incorporation of label indicates that a repair process is taking place in response to chemical damage. It is probable that a "cut and patch" type of repair is operative for both NB and NM induced lesions similar to that reported for UV3, 5. The single-strand breaks we observed after treatment with the two agents may be induced by the alkaline treatment used to separate the 2 strands of DNA and may not necessarily exist iu situ. 7-Methylguanine is a major product of the action of most methylating agents on DNAT,18,~°. The ability to alkylate 3'- and 7-ring N-atoms of purine bases in nucleic acids is common to most alkylating agents. It makes the molecule highly susceptible to depurination at that site in alkaline solutions and may result in strand breakage2, TM. It is unlikely that all depurinations will be spontaneous chemical hydrolyses. It has been observed that 3-methyladenine residues in all cells examined are lost more rapidly from DNA than would be expected from spontaneous hydrolysisiS,2°,2'~,~7. In fact, the depurination-strand-breakage may be enzymatically mediated to a great extent. The data with BHK-21 cells (Table I) showed that specific activity of control cultures was 1. 7 cpm/#g. With a counting efficiency of 2o% this would be approximately 8.5 dpm//~g. The decay constant for tritium in minutes is about io -7. Thus there were approximately 8. 5.1o 7 E3HlBrUdR molecules//~g of DNA. The specific activity of the [~HIBrUdR was 2 Ci/mmole. There are approximately 15 times as many BrUdR molecules as tritium-labeled ones. This amounts to 12.75. lO 8 BrUdR molecules per #g DNA. The specific activities of cells treated with NM and NB were II.O cpm//,g and 32. 7 cpm//~g, respectively. This means 5.5" lO8 BrUdR molecules//~g were incorporated into DNA after NM treatment and 16.3-1o 8 molecules/~g after NB treatment. This means that NM and NB induced 4.65.1o 8 and 15.4.1o 8 more BrUdR molecules than were incorporated by controls. Assuming damage to bases is random there will be approximately 3.3 times as many total bases inserted into DNA (ref. 26). Therefore, the dose of NM induced the insertion of 15. 3 •lO 8 bases//~g DNA; whereas, NB induced 5o.8-lO 8 bases/#g DNA. Possibly NB is activated by an ~.-C oxidation similar to DMN. The methylating species would be a methyldiazohydroxide, CH3NH •NO ~- CH3" N : N. OH, or the ionized form CH3N2+~6,~8,2~,~5. The latter is an SNI agent and source of CHa+ ion, which is transferred intact in methylations 18. There is little evidence on the activation of NM~a,29,~8. Activation of this compound in a manner similar to the aliphatic compounds must first be preceded by a ring-opening step. The B H K - 2 I cells were most susceptible to the toxic effect of the nitroso com-
D N A DAMAGE BY N-NITROSAMINES 100
2 4 6 8 I0 12 Hour of Treatment After Plating Mitotic Cells
Fig. 3. Cell cycle stage sensitivity of BHK-2I cells following treatment with NM (&), IOO/zg/ml; MNNG (O), I #g/ml; NB (O), IO/~g/ml; and nitrosopyrlolidine (•), IO/~g/ml.
pounds during G1 (Fig. 3). MNNG served as the positive control and the degree of synchrony was monitored by pulse labeling at various times with F3Hlthymidine (TdR) (I #Ci/ml, 1. 9 Ci/mM) to determine the percentage of cells in S phase and by scoring the mitotic index. The fact that NM produces chromosomal aberrations at the concentrations used without appreciable toxicity is of considerable interest. It elicited fewer chromatid and chromosome breaks than NB and MNNG but a higher proportion of dicentrics. The latter may be a manifestation of double polynucleotide chain breaks. Since these scissions are present prior to chromosome replication, they are themselves replicated. This results in chromosome-type aberrations (dicentrics) at the subsequent metaphase. ACKNOWLEDGEMENT
We would like to thank Dr. GEORGE WRIGHT of the University of California, Berkeley for many helpful discussions during the course of this work. We are very grateful to Miss EUNICE HARRIS for helping us prepare this manuscript. REFERENCES I AYAD, S. R., M. Fox, AND B. W. Fox, Non-semiconservative incorporation of labelled 5-
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D N A DAMAGE BY N-NITROSAMINES
34 ROBERTS, J. J., J. M. PASCOE, B. A. SMITH AND A. R. CRATHORN, Quantitative aspects of the repair of alkylated DI~A in cultured mammalian cells, II. N'on-semiconservative DNA synthesis ("repair synthesis") in HeLa and Chinese hamster cells following treatment with alkylating agents, Chem-Biol. Interact., 3 (1971) 49-68. 35 STEWART, B. W., AND E. FABER, Strand breakage in rat liver DNA and its repair following administration of cyclic nitrosamines, MutaZion Res., 33 (1973) 32o9-3215. 36 STICH, H. F., AND R. H. C. SAN, DNA repair and chromatid anomalies in mammalian cells exposed to 4-nitroquinoline 1-oxide, Mutation Res., io (197 o) 389-4o4 . 37 WALKER, I. G., AND D. F. EWART, Repair synthesis of DNA in HeLa and L cells following treatment with MNUA or UV light, Can. J. Biochem., 51 (1973) 148-157. 38 ZEIGER, E., AND M. S. LEGATOR, Mutagenicity of N-nitrosomorpholine in the host-mediated assay, Mutation Res., 12 (1971) 469-471.