The rat-liver carcinogen N-nitrosomorpholine initiates unscheduled DNA synthesis and induces micronuclei in the rat liver in vivo

The rat-liver carcinogen N-nitrosomorpholine initiates unscheduled DNA synthesis and induces micronuclei in the rat liver in vivo

Mutation Research, 225 (1989) 143-147 143 Elsevier MTRL 0181 The rat-liver carcinogen N-nitrosomorpholine initiates unscheduled D N A synthesis an...

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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

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by F.H. Sobels