Mutation Research, 225 (1989) 143-147
143
Elsevier
MTRL 0181
The rat-liver carcinogen N-nitrosomorpholine initiates unscheduled D N A synthesis and induces micronuclei in the rat liver in vivo J. Ashby and P.A. Lefevre 1CI Central Toxicology, Alderley Park, Macclesfield, Cheshire (Great Britain) (Accepted 14 November 1988)
Keywords: N-Nitrosomorpholine, rat liver; DNA, alkylation; UDS, induction
Summary Alkylation of DNA is generally accepted as the primary event in the carcinogenicity of nitrosamines. However, the cyclic nitrosamine N-nitrosomorpholine (NMOR), a potent rat hepatocarcinogen, has been reported as binding at very low levels to the liver DNA of treated rats. This led us to investigate the activity of NMOR in two in vivo rat-liver genotoxicity assays - - for the induction of unscheduled DNA synthesis (UDS) and the production of micronucleated hepatocytes in the liver micronucleus assay (LMN). Rats treated with oral doses of NMOR (10-200 mg/kg) gave a positive liver UDS response either 2.5 h or 12 h after dosing. Similarly, treatment with oral doses of NMOR (10 or 100 mg/kg) followed by mitogenic stimulation with 4-acetylaminofluorene (4AAF) resulted in high incidences of micronucleated hepatocytes in the LMN assay. These data confirm that the genotoxicity reported for NMOR in vitro can be reproduced in vivo and that NMOR interacts with liver DNA of treated rats. Earlier reports of only very weak binding of radiolabelled NMOR to rat liver DNA in vivo are discussed within the context of these data.
N-Nitrosomorpholine (NMOR; see Fig. 1) is a potent rat-liver carcinogen (Bannasch, 1968; Lijinsky et al., 1988). In common with non-cyclic carcinogenic nitrosamines such as N-nitrosodimethylamine (NDMA), NMOR is mutagenic to Salmonella (Gomez et al., 1974) and induces unscheduled DNA synthesis in isolated rat hepatocytes treated with it in vitro (Williams and Laspia, 1979). Within this context it would be legitimate to assume that NMOR is reactive to ratCorrespondence: Dr. J. Ashby, ICI Central Toxicology, Alderley Park, Macclesfield, Cheshire (Great Britain).
liver DNA, and that this activity initiates carcinogenesis in that organ. However, Lijinsky et al. (1988) recently suggested that this assumption may be incorrect, thus -- 'but this does not appear to be the most likely mechanism of carcinogenesis by cyclic nitrosamines, which interact with cellular DNA in vivo only at infinitesimal levels, if at all'. This statement is in apparent conflict with the reported ability of NMOR to induce both singleand double-strand breaks in rat and mouse liver DNA in vivo (Stewart and Farber, 1973; Stewart, 1981; Schwarz et al., 1979). Further, it suggests that studies such as those of Dyroff et al. (1986)
0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
144
/
O
N--NO
\
CH3
/
N--NO
CH2--CH 2
NDMA
NMOR
Fig. 1. Chemical structure of N-nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR).
which have established a relationship between DNA alkylation and carcinogenicity may be restricted to the individual chemical under study (in the case quoted, N-nitrosodiethylamine, NDEA). Within the above context we decided to evaluate the activity of NMOR in two rat liver in vivo genotoxicity assays; those for the induction of unscheduled DNA synthesis (UDS) and micronuclei (MN). Materials and methods
Chemicals The source and purity of 6-dimethylaminophenylazobenzthiazole (6BT) and 4-acetylaminofluorene (4AAF) were as described previously
(Ashby et al., 1985, 1986). Both compounds were dosed in corn oil (Kraft-Wesson), 4AAF as an homogenate (Ashby, 1987). N-Nitrosodimethylamine (NDMA; Aldrich Chemical Company Ltd.) and N-nitrosomorpholine (NMOR; Fluka A.G.) were dosed as solutions in sterile, deionised water. The concentrations of all test materials were arranged such that the volume dosed to each animal was 10 ml/kg body weight. Rat-liver UDS assay The procedure was conducted as described previously (Ashby et al., 1985). Hepatocytes were isolated from male Alpk:AP rats treated by oral gavage with the agents, and at the dose levels shown in Table 1. Hepatocytes were assessed for UDS by autoradiography using the automated counting system described earlier (Lefevre and Ashby, 1985). Positive test responses were evaluated using the criteria recommended by Butterworth et al. (1987). Rat-liver micronucleus assay The assay was conducted as described by
TABLE 1 THE RESULTS FROM A P R E L I M I N A R Y AND A MAIN LIVER UDS ASSAY ARE TABULATED Expt.
Treatment
Dose
Length of
Individual animal data
(mg/kg)
exposure
NG a
(h) 1
Cumulative data
07o
NG value for NG ± S.D.
070 Repair
NG value for
Repair b
cells in repair
_+ S.D.
cells in repair
NMOR
10 50 100 200
2.5 2.5 2.5 2.5
1.29 6.18 7.96 7.66
29 52 45 52
9.72 13.02 19.84 15.60
1.29 6.18 7.96 7.66
29 52 45 52
9.72 13.02 19.84 15.60
Water NDMA NMOR
10 ml 10 100
2.5 2.5 2.5 2.5 2.5
-3.86 13.98 -0.03 7.37 6.68
0 74 27 56 63
18.58 9.15 13.79 10.17
-3.86 13.98
0 74
18.58
4.67±4.09
49± 19
11.04+2.44
12 12 12 12 12
-3.63 14.30 2.94 2.39 1.16
0 94 33 28 22
15.04 12.58 13.43 11.41
-3.63 14.30
0 94
15.04
2.16+0.91
28+5
12.47±1.01
Water 6BT NMOR
10 ml 40 100
a NG, net grain count (nuclear grain count minus cytoplasmic grain count). b Cells in repair have an NG count of />5.
145 B r a i t h w a i t e a n d A s h b y (1988). M a l e A l p k : A P rats were t r e a t e d with the c o m p o u n d u n d e r s t u d y b y o r a l g a v a g e o n D a y 0. H e p a t o c y t e d i v i s i o n was s t i m u l a t e d o n D a y 3 b y o r a l g a v a g e with 4 A A F (1000 m g / k g ) a n d h e p a t o c y t e s were i s o l a t e d o n D a y 5. T h e selection a n d scoring o f h e p a t o c y t e s f o r the presence o f m i c r o n u c l e a t e d h e p a t o c y t e s ( M H ) , using c o d e d slides to a v o i d o b s e r v e r bias, were c o n d u c t e d using t h e criteria r e c o m m e n d e d b y B r a i t h w a i t e a n d A s h b y (1988). Results
The UDS data from a preliminary dose-ranging e x p e r i m e n t (Expt. 1) a n d a r e p e a t e x p e r i m e n t
( E x p t . 2) are s h o w n in T a b l e 1. I n d i v i d u a l a n i m a l net g r a i n ( N G ) values o f g r e a t e r t h a n 5 were o b s e r v e d in the m a j o r i t y o f rats e x p o s e d to N M O R for 2.5 h, t h u s fulfilling one o f the criteria for a p o s i t i v e r e s p o n s e d e s c r i b e d b y B u t t e r w o r t h et al. (1987). L o w e r doses o f N M O R gave i n d i c a t i o n s o f a U D S d o s e r e s p o n s e (Expt. 1). N G values o f g r e a t e r t h a n zero, t o g e t h e r with increases in the p e r c e n t a g e o f cells in r e p a i r , were o b s e r v e d 12 h a f t e r t r e a t m e n t with N M O R (100 m g / k g ; E x p t . 2) i n d i c a t i n g , in c o n j u n c t i o n with the 2.5-h e x p o s u r e d a t a , a w e a k b u t c o n f i r m e d positive r e s p o n s e (cf. B u t t e r w o r t h et al., 1987). T h e test d a t a for the liver m i c r o n u c l e u s assay, s h o w n in T a b l e 2, i n d i c a t e a s t r o n g positive
TABLE 2 THE RESULTS FROM TWO SEPARATE LIVER MICRONUCLEUS EXPERIMENTS ARE TABULATED Expt. Treatment Dose (mg/kg) 1
2
Number of slides assessed per animal
Number of cells assessed per animal
Individual animal mean (MH/1000 H)
Cumulative data (mean _+ S.D.)
Water
10 ml
3 3
6000 6000
7.3 4.5
5.9
NDMA
10
3 3
3000 3000
90 93.7
91.9
NMOR
10
3 3
6000 6000
26.3 48.5
37.4
100
3 3 3 3
3000 6000 6000 6000
62 29.1 36.8 28.3
39.1 +_ 15.8
3 3 2
6000 6000 4000
4.7 6.7 7.0
6.1 + 1.3
3 2 3
3000 2000 4000
54.3 58 51.8
54.7 _ 3.1
2 3 3
2000 5000 3000
58 43.2 55
52.1 +_ 7.8
3 2 3
3000 4000 6000
56 26.3 35.5
39.3 +_ 15.2
Water
NDMA
NMOR
10 ml
10
10
100
Between 1 and 2000 hepatocytes (H) were assessed per slide for micronucleated hepatocytes (MH) according to the criteria listed earlier (Braithwaite and Ashby, 1988).
146
response for NMOR in two separate experiments. Control data were at the highest level reported by Braithwaite and Ashby (1988), but the positive control (NDMA) also gave similarly elevated values. Variation in the magnitude of both negative and positive control values has been observed before (Braithwaite and Ashby, 1988).
Discussion The present data confirm that NMOR is genotoxic to the rat liver in vivo. Its ability to initiate hepatic UDS and to induce micronuclei in the rat liver add to earlier reports of its ability to induce strand breaks in hepatic DNA of the rat in vivo (Stewart and Farber, 1973; Stewart, 1981). In these respects, NMOR is similar to alicyclic dialkyl nitrosamines such as N D M A and NDEA, and this argues for a similar genotoxic mechanism of carcinogenic action. This conclusion would be unexceptional were it not for the fact that Lijinsky et al. recently came to the opposite conclusion, namely, that NMOR, unlike NDMA and NDEA, probably does not act via the formation of an electrophilic alkylating species (Lijinsky et al., 1988). Lijinsky et al. based their conclusion on the earlier observation that only very low levels of DNA binding occurred in the liver of rats dosed intraperitoneally with 3H-NMOR (Lijinsky, 1976). Similarly, Stewart et al. (1974) found low levels o f radioactivity associated with rat-liver DNA 24 h after an intraperitoneal injection of [3-~4C]nitrosomorpholine. Lijinsky et al. (1988) recently used the expression 'cyclic nitrosamines interact with cellular DNA in vivo only at infinitesimal levels, if at all', to describe the earlier findings. In order to reconcile these divergent observations it is necessary to speculate. First, let it be assumed that the present data confirm that NMOR is genotoxic to the rat liver in vivo. Second, that some form of covalent interaction between DNA and NMOR or a metabolite, must have initiated these genotoxic effects. With these two assumptions made, the failure o f Lijinsky to detect significant DNA binding was probably due to one of two causes; either
the DNA adducts were repaired before they were assayed, or they were too labile to survive the process of DNA isolation. The first of these is possible because the wave of UDS following exposure to nitrosamines is rapid (typically, UDS is at a maximum after 2 h, as opposed to - 12 h for most liver genotoxins). The very weak binding reported by Lijinsky (1976) was observed at 12-16 h. The second prospect, the chemical lability of the DNA adducts, is feasible on two counts. First, no suggestions as to the nature of these adducts have yet been made, so their stability cannot be estimated. Second, unstable adducts are not without precedent, as illustrated by the early detection of DNA N-7 alkylguanine adducts for simple alkylating agents and the much later detection of the corresponding 0-6 alkyl adducts (Loveless, 1969). The present considerations indicate that caution is required when using DNA adducts as a measure o f carcinogenic initiating potency. Such data can be highly enlightening for a given chemical (e.g. D y r o f f et al., 1986, when studying NDEA), but they should not be extrapolated too readily between chemicals. To emphasize this point it is interesting to note that data from Lijinsky (1976) on this subject was used by Lutz (1979) to calculate a covalent binding index (CBI) of 44 for NMOR, of 7650 for NDMA, and of only 125 for NDEA. These figures do not correspond with the relative potency of these 3 agents as liver carcinogens, a discrepancy noted by Lutz (1979). Further, the CBI of 125 reported for the potent liver carcinogen NDEA is close to the figure of 44 reported for NMOR, the latter figure being regarded by Lijinsky (1979) as providing little evidence of DNA interaction, and by Parodi et al. (1982; quoting a review by Lutz, 1979) as providing some evidence of DNA interaction.
Acknowledgements We are grateful to Peter Schmezer for stimulating discussions and to Gill Barber for technical assistance.
147
References Ashby, J. (1987) The efficient preparation of corn oil suspensions, Mutation Res., 187, 45. Ashby, J., P.A. Lefevre, B. Burlinson and M.G. Penman (1985) An assessment of the in vivo rat hepatocyte DNArepair assay, Mutation Res., 156, l-18. Ashby, J., P.A. Lefevre, B. Burlinson and B. Beije (1986) Potent mitogenic activity of 4-acetylaminofluorene to the rat liver, Mutation Res., 172, 271-279. Bannasch, P. (1968) The cytoplasm of hepatocytes during carcinogenesis, Electron and light-microscopical investigations of the nitrosomorpholine intoxicated rat liver, Recent Results Cancer Res., 19, l-100. Braithwaite, I., and J. Ashby (1988) A non-invasive micronucleus assay in the rat liver, Mutation Res., 203, 23-32. Butterworth, B.E., J. Ashby, E. Bermudez, D. Casciano, J. Mirsalis, Cl. Probst and G. Williams (1987) A protocol and guide for the in vivo rat hepatocyte DNA-repair assay, Mutation Res., 189, 123-133. Dyroff, MC., F.C. Richardson, J.A. Popp, M.A. Bedell and J.A. Swenberg (1986) Correlation of ti-ethyldeoxythymidine accumulation, hepatic initiation and hepatocellular carcinoma induction in rats continuously administered diethylnitrosamine, Carcinogenesis, 7, 241-246. Gomez, R.F., M. Johnston and A.J. Sinskey (1974) Activation of nitrosomorpholine and nitrosopyrrolidine to bacterial mutagens, Mutation Res., 24, 5-7. Lefevre, P.A., and J. Ashby (1985) Investigations into the reported ability of cimetidine to initiate UDS in rat hepatocyte primary cultures, Environ. Mutagen., 7,833-837. Lijinsky, W. (1976) Interaction with nucleic acid of carcinogenic and mutagenic N-nitroso compounds, in: W.E. Cohn (Ed.), Progress in Nucleic Acid Research and
Molecular Biology, Vol. 17, Academic Press, New York, pp. 246-269. Lijinsky, W., R.M. Kovatch, C.W. Riggs and P.T. Walters (1988) Dose response study with iV-nitrosomorpholine in drinking water of F-344 rats, Cancer Res., 48, 2089-2095. Loveless, A. (1969) Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides, Nature (London), 223, 206-207. Lutz, W.K. (1979) In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis, Mutation Res., 65, 289-356. Parodi, S., M. Taningher, P. Boero and L. Santi (1982) Quantitative correlations amongst alkaline DNA fragmentation, DNA covalent binding, mutagenicity in the Ames test and cardinogenicity for 21 compounds, Mutation Res., 93, l-24. Schwarz, M., J. Hummel, K.E. Appel, R. Rickart and W. Kunz (1979) DNA damage induced in vivo evaluated with a nonradioactive alkaline elution technique, Cancer Lett., 6, 221-226. Stewart, B.W. (1981) Generation and persistence of carcinogeninduced repair intermediates in rat liver in vivo, Cancer Res., 41, 3238-3243. Stewart, B.W., and E. Farber (1973) Strand breakage in rat liver following administration of cyclic nitrosamines, Cancer Res., 33, 3209-3215. Stewart, B.W., P.F. Swann, J.W. Holsman and P.N. Magee (1974) Cellular injury and carcinogenesis, Evidence for the alkylation of rat liver nucleic acids in vivo by Nnitrosomorpholine, Z. Krebsforsch., 82, l-12. Williams, G.M., and M.F. Laspia (1979) The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test, Cancer Lett., 6, 199-206. Communicated
by F.H. Sobels