Cancer Letters, 2 (1976) 79--86 © Elsevier/North-Holland, Biomedical Press

E F F E C T OF CHRONIC ADMINISTRATION O F DIMETHYLNITROSAMINE ON THE ]EXCISION OF 96-METHYLGUANINE FROM RAT LIVER DNA

GEOFFREY P. MARGISONa, HENRIE2"PE BRESIL, JENN]:FER M. MARGISON and R:UGGERO MONTESANO International Agency for Researcl~ on Cancer, Unit o f C.he~ical Carcinogenesis, 150 cours Albert Thomas, 69008 L y o n (Franc¢)

(Received 1 July J.976}

SUMMARY

Rats were exposed chronically to unlabel[ed N,N-dimethylnitrosamme (25 p p m in the drinking water) then given a single dose of N-[ 3H] methyl-Nnitr,oseurea (10 mg/kg body weight). The rates of loss of tritium-labeled 7-methylguanine, 0~-methytguanine and 3-methyladenine from t h e liver DNA in control and dimethylnitrosamine-treated raCs were found n o t to be significantly different. Thus, under t h e conditions used, inhibition o,f the 06-methylguanine excision repair system does not seem to be a factor in ~he induction of liver turnouts by chronic DMN applicaHon.

INTRODUCTION N,N-Dimethylnitrosamine (DMN) is carcinogenic in many animal species including the rat, mouse and hamster [5,12]. In the rat, the prJ..ncipal target organ is determined mainly by the dose schedule. Large (LDsc) doses or limited exposure to fairly laxge doses produce kidney turnouts in t h e survivors; prolonged exposure to low doses results in a high incidence of liver turnouts [5,19]. The alkylation of target.organ DNA is strongly implicated ~,~ the carcinogenic effect of DIvIN and othe~ aIkylating agents [ 1~;]. Recently, attention has focused on 0 ~ -me~hylguan]ne (0~-meG) as being possibly the critical alkylation product in DNA because of its miscodlng potcntiaJ in vitro [2], but D N A synthesis is presumed to be a necessary step in enameling th~s 'pro-mutagenic' base to pioduce a permanent change in the base seojuence of the DNA [13]. The capacity o f the liver to excise 0~-meG from DNA after a .Present addl'ess: Paterson Labot,atories, Christie Hos~.ltal and Holt Rad}um Institute, Wilmslow Road, Withington, Manchester M20 9BX, UK.

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a single dose of DMN is high [1~] and, for other alkylating agents, appear,~ to exceed that of any other rat ~Lissueso far studied [3,14]. To explain the organ specificiity of DMN under different dose schedules, we explored the possibility that chronic administration of DMN produces liver tumours by inhibiting the (}6-meg excision system. This would leave the 06-meG in the DNA longer and increase the chance of miscoding during DNA rephca~;ion. DMN alkylates DNA after me~;abolism by microsomal mixed function oxidases, a process which takes place mainly in the liver. However, chronic DMN application inhibits DMN~l~aembolising enzymes [ 1,16]. ~esulting differences in the initial extent and time of maximum DNA alkylation .make comparison of the loss of alkylating products from control and DMNpretxeated rats very complex. To overcome ~his difficulty, DNA was atkylated b y administration of a single relatively nontoxic dose of N-[3H]methyl-N-nitrosourea (MNU), a compound which does not require metabolism to react wi~:h nucleic acids, and the levels of ~H-alkytation products were determined at v~'.:ious times. Thus, excision repair capacity in control and DMN-pretreated rats could be determined under conditions when the initial extent and time o f maximum DNA alkylation were identical. MATERIALS AND METHODS A stock solution (0.0025% v/v) of DMN (Schuchardt, Munich, FRG) was used. Male BD-IV rats weighing 80 g at the start of the experiment were given this solution weekdays for 81/..,weeks. Tap water was provided during the weekend and food pellets were given ad libitum. [~ H]MNU (New England Nuclear, Dreieichenhain, FRG; 46 mCi/mmol)~ was dissolved in 3 mM sodium citrate 0.9% NaC1 at pH 6.0 and diluted to 7.0 mCi/mmol using unlabeled MNU (Schuchardt, Munich, FP~G), MNU (10 mg/kg b :,dy weight} was injected into tile femoral vein under light ether anaesthesia in the morning after the eveni.ng of the final DMN treatment. Rats were killed by exsanguination under ether an.aestllesia and tissues were remc,ved and frozen in liquid ni%rogen. DNA was i~olated by a phenol p~ocedure essentially as previously described [14]. To determine the 0~-meG/7-me,G and 3-meA/7-raeG ratios after 'alkylation in vitro (which represents the initiM in vivo reaction, see [9] ) DNA (2 mg/ml in 0.4 M Tris--HC1, pH 7,4) was in:cuba'ted with [~H]MNU (0.19 rag, 01.1 mCi in 3 mM sodium citrate 0.8 M NaC1, pH 6.0) for t h at 37°C. It was then exhaustively dialysed agains~ 0.1 standard saline citrate at 3°C, precipitated with 2 vol ethanol and dried. DNA was hydrolysed in 0.1 N HCI (1 rot/5 mg DNA) at 70°C for 3,0 rain and then adjusted to p,H 2.9 after the addi'~ion of authentic 7-metnylguanine (7-meG), 3-me~hyladenine (3-meA) and 06-meG as marker compounds. Purine bases were separated by Sephadex G-10 chromatography [10] (89 X 1 cm columns) using 0.05 M amraonium formate~-0.2% w/v sodium azide, pH 6.75, as eluant, at a flow rate of 15 rnl/h.

81

The A~0 of the fractions (4.5 ml) ~as determi:..~ed (mmolar absorption, 7.~I: for guanine; 13.3 for adenine [4] ~nd the samples dried, dissolved in 0.3 m! hyamine (1.0 M solution in me~;hanol) and counted for radioactivity in .'~ Packard 3375 spectrometer a.fte~ addition of 10 ml of toluene containing 2,5-diphenyloxazole. ~ESULTS

An example of the fractionat~o~ of the purine bases in an 0.i N HCI hydrolysate of DNA isolated from rat liver 12 h afte!, injection of [~H]MNU is shown in Fig. 1. The levels of 7-meg and 0 ~ - m e g in the DNA from ~he livers of normal mxd DMN~treated animals at various times after ~ H ] MNU administration were determined by Sephadex G-10 chromatography of 0.1 N HCi bydrolysates of the DNA (Fig. 2). Animals treated with DMN for 8~A weeks had a slightly

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Fig. 1. $ephadex G-IO chromatography of a 0.1 N I~ICi hydrolysa~e o:" a ~¢ampie or :~.~7.* i:so]ated from the livers of cont, rol raL~ killed 12 h aitel" administration of [2H]MNU. ~'v.O., ?yrimidine ollgonueleotides (ratioactivity no~, routinely determined); ~..meA. 3-met.by,. adenirie; 7-meG; 7-methylgu,'minc; G, guanlnc; A, adenine; 0*-meG, 0*'-me*.hytguanine. Authentic methyJated base~ were ,'tdded as markers. (~----o----~), A l c m ; (,.~.~,--,, }, radioactivity.

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Fig. 2. Amounts o[" methyla[ed purines in liver D N A after administration of ['~ H:]MNU (10 mg/kg). Open symbols, control rats; ~.'to~ed cymbois, DMN tree.ted rats (c, ~ ), 7-meG; ( - , *), 0 ~-meG,

higher level of atkylation than the control rats, but the rat;es of loss of the various alkyla~ion products were the same in control and DMN-pretrea~ed animals. The 0a-meG/7-meG and 3-meA/7-meG ratios, which are a better indicator of excision activity since 1;hey compensate for any differences between the groups o f animals in the absolute amounts of alkylation p~oducts, are almost identical in the control and DMN-pretreated anfln'als (~fable i). TABLE 1 METI-IYLATES PURINE BASE RATIOS IN LIVER DNA AFTER ADMINI,~TRA'IqON OF [~H]MNU (10 mg/kg) TO CONTROL, A N D DMN-PRETREATED RATS Time ~ffter [~H]MNU injection (hours) (In vltro)a 3 6 12 24

0 ~-meG/7-rneG ratio

3-meA/7-naeG ratio

Control

Di~fIN-t~reated

Control

DMN-treated

0.1070 0.0296 0.0268 0.0148 0.0102

0.0279 0X)258 0.0160 0.0122

0.1300 0.0365 0.0249 0.0146 0.0086

0.0372 0.02'74 0,0159 0.0092

u Refers to the rat;io:+ i'ound in D N A ulkylated in vit.'o by MNU,

83 DISCUSSION The possibility that 0~-meG might be i m p o r t a n t in the mutagenic and carcinogenic effects of alkylating agents was originally suggested by Loveless [ 1 I ] and the mi~oding properties of this base were subsequently shown in in vitro experiments [2]. Although the initial levels of 0%meg i n t h e DNA of various tissues.do not correspond to the tumour incidence, a correlation between the persistence of 0 6-meG in DNA and the tissue specificity of carcinogenic alkylating agents has been demonstrated by several ~oups [3,14, 17]. An enzyme which cleaves the N-glycosidic bonds of 06-meG and 3-meA deoxyribonucleosides in DNA has been extracted from E. coli [ 1 8 ] . This enzyme does not act on the major base alkylation product 7-meG, which seems to be lost from rat tissue DNA in vivo by a non-enzymic process (see [9] ). The loss of this base can therefore be used as an internal control for effects of cell turnover or toxicity which would affect all alkylation products to the same extent. A single dose of DMN does not produce liver tumours in normal adult rats. However, chronic administration of DMN in the drinking water or diet results in a high incidence of liver turnouts. Since the rate of excision of O¢~+meGfrom liver DNA following a single dose of DMN is high [17,18], greater persistence of this base after chronic administration of this agent could, by analogy with other systems [3,14,17], De an essential factor in the production of liver turnouts. Such auz effect could be mediated by exhaustion or overloading of the repair system [8] o~ by reduction in the amount of repair enzyme, possibly as a consequence of inhibited protein synthesis. An example of such inhibition is found in mouse liver in which the hydroxylase enzymes, including the DMN metabolising enzyme, are considerably less active during chronic DMN administration [1,16]. A: similar effect ~;akes place in rat liver (unpublished observation). For this reason, in the present experiments, DNA damage was introduced by a low, relatively non-toxic dose (10 mg/kg) of MNU, an agent which produces alkylating species exclusively by chemical breakdown [ 13] thus avoiding possible differences in the initial level and time of maximal alkylation between control -~d DMN-pretreated animals. The reaction of MNU with DNA in vitro results in an 0%meG/7-raeG ratio of 0.107 (Table !) (see also [9] ). However, as has been obsetared previousity [7], the initial ratio in rag liver is much lower (Table 1) indicating tt~at even by 3 h considerable excision of 0 ~-meG has taken place in both control and DMN.pretreated rats. Similarly, 3-meA which is also enzymica!ly excised f'rom DNA in vivo [I5}, is lost rapidly from ~;he IN'or DNA of control and DMN pretreated rats (see Table 1). Up to 24 h after [~H]MNU injection, onty minor differences .in the rates of loss of alkylation produc~s (Fig. 2) or in the ra~ios O%meG/7:meG and 3-meA/7-meG (Table 1) between norma~ and DMN-pretreated rags ,~ere observed.

84 It t h u s a p p e a r s t h a t a l o w e r rate o f excision repair o f 0 6 - m e g (c,r 3-meA) f r o m liver D N A o f D M N - p r e t r e a t e d animals c a n n o t explain t h e hi~rlh t u m o u r incidence in t h i s organ during c h r o n i c a d m i n i s t r a t i o n o f DMN. Figure 2 a n d Table 1 s h o w t h a t t h e rate o f less o f 06-meG (and 3-meA) f r o m liver D N A decreases w i t h t i m e a f t e r MNU injection. This m a y result f r o m a r e d u c t i o u in t h e initial r a t e o f excision o f 06-meG f r o m t o t a l liver D N A or f r o m a l o w e r ex.cision ~apacity o f a small p o p u l a t i o n o f I i v e r cdlls. This gradual r e d u c t i o n in t h e rate o f excision o f 06-meG c o u l d allow a n a c c u m u l a t i o n o f this base d u r i n g c h r o n i c a d m i n i s t r a t i o n o f DMN. This w o u l d b e analogous t o ~he a c c u m u l a t i o n o f 0 .~-meG in t h e D N A o f t h e b~ain durin~i w e e k l y a p p l i c a t i o n o f MNU [14] a n d is p r e s e n t l y u n d e r investigation. REFERENCES 1 Den Engelse, L. and Emmelot, P. (1971~[972) Efl~ectsof feeding the carcinogen dimethylnitrosamine on its metabolism and methylation of DNA in the mouse. Chem. Biol. Interactions, 4, 321--327. 2 Gerchman, L.L. and Ludium, D,B. (1973)Properties or" 0 ~-methylguanine in templates for RNA polymerase. Biochim. Biophys. Aeta, 308, 310--316. 3 Goth, R. and Rajewsky, M.]?. (1974} Persistence of 0 d -ethylguanine in rat brain DNA. Prec. Natl. Acad. Sci. USA, 71,639-.643. 4 Handbook of Biochemistry (1970) Editor: H.A. Sober, CRC Press. 5 International Agency l:or Research on Cancer, Monograph on the Evaluation of the Carclnogc~ic Risk go .,i,n (1972) 1, -q5--144, Lyon. 6 Kirtikar, D. and Goldthwait, D.A. {1974) The enzymatic release of 0 k -methylguanine and 3-methyladenine from DNA reacted with the carcinogen N-methyi-N-nitrosourea. Prec. Natl. Acad. Sci. USA, 7t, 2022.-2026. 7 Kleihues, P. and Margison, G.P. (1974) Careinogenicity of N-methyt-N-nittosourea: possible role of repair excision of 06-methylguanine from DNA. J. Natl. Cancer Inst., 53, 1839--184:[. 8 Kleihues, P, and Margison, G.P. (1976) Exhaustion and recovery of repair excision of 0 n-methylguanine from rat liver DNA. Nature (London), 259, 153--155. 9 Lawley, P. (1976) Methylation of DNA by carcinogens: some application.~ of chemical analytical methods. In: Screening Te~ts ia Chemical Carcinogenesis, pp. 181--208. Editors: R. Montesano, H. Bartsch and L. Tomatis, IARC Scientific Publications No. 12, Lyon~ 10 Lawley, P.D. a~d Shah, S.A. (1972) Methylatioa of RNA by the carcinogens dimethylsulphate, N-methyPN-nitrosourea and N-methyl-N~ni~rosoguanidine. Compariaons of chemical analyses at the aucleosidc and base levels. Biochcm. J., 128,117--132. l I Loveless, A. (1969) Possible relevance of 0-6 atkylation of deoxygaanosine to mutagenlcity of nitrosamines and nitrosamides. Nature (London), 223, 206--207. 12 Magee, P.N. and Barne~, J.M. (1967) Carcinogenic nitro~o compounds. Adv. Cancer Re~., lO, 163--246. 13 Magee, P.N., Pegg, A.E. and Swann, P F~ (1975) Molecular Mechanisms of Chemical Carcinogenesis. in: Handbuch dcr Allgemeinen Pathologic pp. 329~419, Springer-Verlag, Beriin, Heidelberg. 14 Margison, G.P. and Kleihues, P. (1975) Chemical carciqogenesis in the nervous system Preferential accumulation of 0 ~-methylguanJne in rat brain DNA during repetitive administration of N-methyl-N-nitroz, Ourea. Biochem. J., 148, 521--525. 15 Macgison, G.P. ~,nd O~Conno~-, P J. (1973) Biological implications of the instability of the .N'.glycosidic bond of 3-methyl deoxya~Jenosine in DNA~ Biochim. Biophys~ Aeta, 331, 34-9--356.

85 15 Nemoto, N. and Takayama, S. (1974) Rapid loss of 7-methylguanine from liver nucleic acids in mice during the initial stage of liver carcinogenesis induced by dimethylnitros. amine. Bi*~chem. Biophys. Res. Commun., 58,242--249. 17 Nicholls, J.V,, Swann, P.F. and Pegg, A.E. (1975) Effects of dimethylnitro~amine on persistence of methylated guanines in rat liver and kidney DNA. Nature (London}, 254, 261--262. 18 O'Connor, P.J., Capps, M.J. and Craig, A.W. (1973) Comparative studies of the hepatocarcinogen N,N-dimethylnitrosamine in vivo: reactior~ sites in rat liver DN.~Land the significance of their relative :stabilities. Brit. J. Cancer, 27, 153--166. 19 Swann, P.F. and Magee, P.N. (1968) Nitrosamine-indueed carcinogenesis. The aikylatinn of nucleic acids of the rat by N-methyI-N-nitroso urea, dimethylnitrosamin.e, dime~.hyi sulphate and methylmethane sulphonate. Biochem. J., I10, 39--47.