Liver DNA alkylation after a single carcinogenic dose of dimethylnitrosamine to newborn and adult CFW Swiss mice

Chem.-Biol. Interactions, 68 (1988) 259-271 Elsevier Scientific Publishers Ireland Ltd.

259

LIVER DNA ALKYLATION AF'rER A SINGLE CARCINOGENIC DOSE OF D I M E T H Y L N I T R O S A M I N E T O N E W B O R N A N D A D U L T C F W SWISS MICE

P. COCCIAa, M. SALMONA a'*, L. DIOMEDE% L. CITTIb, L. MARIANI b and M. ROMANO a ~Laboratory for Enzyme Research, Istituto di Ricerche Farmacologiche "Mario Negri", Milan, and bLaboratory for Mutagenesis and Differentiation, Consiglio Nazionale delle Ricerche, Pisa (Italy) (Received January 25th, 1988) (Revision received June 16th, 1988) (Accepted June 20th, 1988)

SUMMARY N-nitrosodimethylamine N-demethylase activity, DNA alkylation, c a p a c i t y f o r O 6 - m e t h y l g u a n i n e r e p a i r a n d cell p r o l i f e r a t i o n w e r e m e a s u r e d in livers of n e w b o r n a n d a d u l t C F W m i c e a f t e r a single c a r c i n o g e n i c d o s e of D M N A . D N A a l k y l a t i o n w a s f o u n d in n e w b o r n a n d a d u l t m o u s e livers b u t it w a s s i g n i f i c a n t l y h i g h e r in t h e n e w b o r n . 6- a n d 7 - m e t h y l s u b s t i t u t i o n s of g u a n i n e w e r e identified b y H P L C a n a l y s i s in n e w b o r n a n d in a d u l t m o u s e livers. M e t a b o l i c l a c i n c o r p o r a t i o n i n t o a d e n i n e a n d g u a n i n e w a s o b s e r v e d only in liver D N A of n e w b o r n s . O 6 - m e t h y l g u a n i n e levels w e r e h i g h e r in n e w b o r n t h a n a d u l t m i c e a f t e r a single i.p. d o s e of [laC]DMNA. L i v e r D N A r e p a i r c a p a c i t y m e a s u r e d a s O 6 - m e G - D N A m e t h y l t r a n s f e r a s e w a s h i g h e r in a d u l t s t h a n in n e w b o r n s . De n o v o liver D N A s y n t h e s i s w a s m o r e i n h i b i t e d b y D M N A p r e t r e a t m e n t in n e w b o r n t h a n in a d u l t mice. T h e r e l a t i o n s h i p b e t w e e n t h e s e p a r a m e t e r s a n d t h e g r e a t e r n e o n a t a l liver t u m o r s u s c e p tibility is d i s c u s s e d .

K e y words: D N A a l k y l a t i o n - N e o n a t a l c a r c i n o g e n e s i s - O % m e G - O 6 - m e G DNA Methyltransferase - Dimethylnitrosamine *Correspondence to: Dr. Mario Salmona, Laboratory for Enzyme Research, Istituto di Ricerche Farmacologische "Mario Negri", Via Eritrea 62, 20157 Milano, Italia. Abbreviations used: DMNA, dimethylnitrosamine; [~4C]DMNA, N-nitroso-[14C]dimethylamine; [3H]MNU, [3H]methylnitrosourea, [3H]dT, (methyl-[3H])thymidine; 7-meG, N7-methylguanine; O6-meG, Oe-methylguanine; MT, OS-methylguanine-DNA methyltransferase; RNase, ribonuclease. 0009-2797[88/$03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland

260 INTRODUCTION DMNA has a relatively simple metabolic p a t h w a y [1] and is considered to be activated by a-hydroxylation to active species able to methylate nucleophilic sites of DNA. Various DNA adducts have been found but alkylation of oxygen in position 6 of guanine has been suggested as the main promutagenic event because of its mispairing potential [2-4]. Previous studies have shown that newborn CFW mice are more susceptible than adults to hepatic t u m o r s after a single i.p. dose of D M N A [5]. However newborns are more susceptible than adults to tumors induced by a broad spectrum of chemical substances [6]. This sensitivity might be attributed essentially to the presence of some kind of "modulation of initiation" in newborn animals. In fact a high rate of cellular proliferation in newborns, resulting in a higher rate of mutation fixation, amplifies DNA damage induced by chemical carcinogens before repair mechanisms are able to eliminate these modifications [7]. However, age-related changes of the P-450-dependent metabolizing enzyme systems could affect the binding of carcinogens, that require metabolic activation, to DNA and proteins modifying the metabolic rates of chemicals and the balance between activation and detoxification during development [8-10] so to render newborns more susceptible than adults to genotoxic effects. For practical reasons, only a very limited number of studies on DNA binding has been done in newborn animals. The aim of this work was to characterize the hepatic metabolic capacity of newborn and adult CFW mice and evaluate DNA damage after a single dose of D M N A in the hope of elucidating any correlation between the observed hepatic tumor susceptibility and qualitative and quantitative differences in hepatic DNA adducts. MATERIALS AND METHODS

Chemicals Unlabelled dimethylnitrosamine (DMNA) was obtained from Sigma Chemical Company, St. Louis, MO, U.S.A. N-nitroso-[14C]dimethylamine ([14C]DMNA, 344.1-518 MBq/mmol, 8-14 mCi/mmol), [3H]methylnitrosourea ([3H]MNU), 185 GBq/mmol, 5 Ci/mmol and (methyl-[3H])thy midine ([3H]dT), 185 GBq]mmol, 5 Ci]mmol were purchased from Amersham, U.K. The N7-methylguanine (7-meG) was purchased from Sigma. O6-methylguanine (O6-meG) was synthesized according to Balsiger and M o n t g o m e r y [11]. R N a s e A, specific activity 3000units/mg was obtained from Worthington Biochemicals. Animals Outbred CFW Swiss Webster 60-day-old female and pregnant mice on the 18th day of gestation were obtained from Charles River (Calco, Como, Italy), housed in plastic cages and fed standard chow (Altromin, Rieper,

261 Bolzano, Italy) with water ad libitum. The pregnant mice delivered their litters 3-5 days after arrival in our laboratory.

Determination of DMNA-N-demethylase activity Adult female and newborn mice were decapitated and liver microsomal fractions were immediately isolated according to Kato and T a k a y a n a g h i [12]. DMNA-N-demethylase activity was measured in I ml of 100 mM TrisHC1 buffer (pH 7.4) containing 5 mM MgC12, 150 mM KC1, 27 mM NADPH, 5 mg microsomal protein and I mM DMNA. The mixture was incubated at 37°C for 5 m i n . The reaction was stopped by the addition of l ml of 15% ZnSO4 and I ml of saturated Ba(OH)2. Blank samples did not contain NADPH. DMNA N-demethylation was determined by measuring formaldehyde production according to N a s h [13] and was expressed as nmol HCHO/min per mg protein. Microsomal proteins were measured according to Lowry et al. [14].

Determination of covalent binding to DNA and proteins Adult female and newborn mice, weighing respectively 28-30 g and 3-4 g, received a single i.p. injection of [14C]DMNA (7 mg/kg, 344.1518 MBq/mmol, 8-14 mCi/mmol) and were killed 2 and 4 h later. Livers were pooled (three livers per group for adult and 20 livers per group for newborn mice) and immediately frozen at -80°C. Livers were homogenized in 10 mM Tris-HC1 buffer (pH 7.8) containing 75 mM NaC1 and 10 mM EDTA; 100 pl of each sample were collected and dissolved in I m Soluene R-350 (Packard, Groningen, The Netherlands). After digestion, 10 ml toluene scintillator (Packard) were added to the samples and total hepatic radioactivity was measured in a Beckman 5800 liquid scintillation counter. Quenching was corrected by the external standardization method. For the determination of radioactivity bound to proteins, 2 ml of 700 × g s u p e r n a t a n t were precipitated with 5 ml acetone, centrifuged at 3000rev./min for 15min and washed five times, with 5 m l ethyl acetate, petroleum ether, ethanol, methanol and acetone, until no further radioactivity could be extracted. The pellet was resuspended in 5 ml of water; I ml was centrifuged and the pellet was dissolved in i ml Soluene R-350 and counted as above. A portion of 0.1 ml was used for the protein assay. To determine covalent binding to DNA, livers were processed according to Viviani and Lutz [15]. To exclude RNA contamination, isolated DNA was dissolved in 4 ml of 0.05 M Tris (pH 7). NaC1 concentration was a d j u s t e d to 0.1 M and the sample was incubated at 37°C for 30 min with 1 mg of RNase [16]. After cooling on ice, the NaC1 concentration was adjusted to 0.9 M, the DNA was precipitated with three vols. of cold ethanol and dried under vacuum. Dried DNA (3-4rag) was dissolved in l ml of 10raM sodium cacodylate (pH 7.0) for the determination of total radioactivity bound to DNA. A 0.1-ml aliquot of the sample was added to 0.1 ml of 1 M HC104 and heated for 20 min at 70°C. Ten milliliters of Insta-Gel II (Packard) were

262

added and the radioactivity was measured. DNA concentration was m e a s u r e d by the method of Burton [17] using herring sperm DNA (Sigma) as a standard.

Analysis of DMNA DNA-adducts (alkylation products) The remainder of the sample dissolved in sodium cacodylate was heated at 100°C for 30 min (neutral hydrolysis) and precipitated at 0°C with 0.1 vol. 1NHC1. The s u p e r n a t a n t was removed and stored at -20°C and it represents the netural fraction of the chemical hydrolysis in which we have measured the N7-meG levels by HPLC as described below. The partially apurinic pellet was dissolved in 5 0 m M Tris (Merck, Darmstadt, F.R.G.) 1 mM MgC12 (pH 6.5). Purine-free bases were released by acid hydrolysis after addition of 0.1 vol. of 1 N HC1 and heated at 70°C for 30 rain. Unlabeled purine bases were added to the sample as chromatographic markers. HPLC separation was carried out with a Beckman 342 apparatus equipped with a M a g n u m 9 column (250 mm = 9 mm, Chrompack, Middelburg, The Netherlands) filled with Partisil 10 SCX resin (Chrompack). Alkylated products from acid and neutral hydrolysis were eluted with a flow rate of 4 ml/min using a gradient of 0.02-0.2 M a m m o n i u m formiate (pH 4.0). Elution was monitored using a Beckman U.V. detector set at 254nm. Fractions of 1 ml]0.5 min were collected and added to 10 ml of Insta-Gel II and counted as described before.

Determination of liver O6-methylguanine-DNA methyltransferase (MT) activity Nuclei were prepared from livers of newborn and adult female mice by homogenation and sucrose sedimentation as described by Craddock and Henderson [18]. Crude nuclear extracts were obtained by sonication of nuclear suspension in 10mM Tris-HCl buffer (pH 7.4) containing l mM dithiotreitol (DTT), 0. I mM EDTA and 0. I mM methyl-phenylsulfonylfluoride (MPSF). After centrifugation (700 × g]5 rain) to remove debris, the supern a t a n t was employed for determination of repair activity. [3H]Methylated substrate was prepared by reacting 2 mg of calf t h y m u s DNA (Miles) in 0.5 ml of 10 mM Tris-HC1 buffer (pH 8) with 18.5 MBq of ZH-MNU at 37°C for 60 rain according to the method developed by Wiestler et al. [19]. The alkylated [3H]-DNA substrate containing OS-meG was incubated with variable a m o u n t s of crude liver extracts; the concentrations of DTT and Tris were a d j u s t e d so t h a t the incubation mixtures were 1 . 7 m M D T T , 72 mM Tris-HCl and pH 8. The samples were incubated, either with or w i t h o u t liver extracts (control), at 37°C for 1 h, then the DNA was precipitated by adding cold 1 N perchloric acid. The DNA pellet was hydrolysed by heating at 70°C for 30 rain in 0.1 N HCl and analyzed by HPLC [20]. Hydrolysates were loaded on the Hibar RP8 (4 × 250 mm) C8 reversed-phase prepacked column (Merck) equilibrated at 1 ml/min with 4% of eluent A and 96% of B, where A is 70% (v/v) aqueous methanol (Merck), and B is 20 mM potassium phosphate buffer (pH 6.7) containing 6 mM sodium hexane sulfonate. Analyses were done following a three-step solvent program: (1)

263 linear gradient from 4 to 8% of A in the mobile phase for 5 rain; (2) isocratic elution at 8% of A for 10 min; (3) linear gradient from 8 to 45% of eluent A in the mobile phase for 20 min. Fractions of eluted material were collected and assayed for radioactivity.

Determination o f de novo hepatic DNA synthesis Adult and newborn mice were given a single i.p. injection of DMNA (7 mg/kg) 3, 7, 23 and 71 h before a single dose of (methyl-[aH])thymidine (37 kBq/g, 185 GBq/mmol) and were killed 1 h later. Livers were removed and frozen at -80°C. DNA was isolated according to Matsui e t a ] . [21]. Thymidine incorporation was determined by liquid scintillation counting using Insta-Gel II cocktail as above. Statistical analysis Statistical analysis was performed using Duncan's test for multiple comparisons or Student's t-test, depending on the experimental design. RESULTS

Bioactivation o f DMNA Table I shows microsomal DMNA-N-demethylase activity in newborn and adult mouse liver. Adults had almost three times higher enzymatic activity t h a n newborns. De novo DNA synthesis Table II shows the effects of the single i.p. dose of DMNA on liver weight, DNA content and incorporation of (methyl-[3H])thymidine in newborn and adult mice. The rate of [3H]dT uptake into DNA was approximately 25 times faster in newborn t h a n in adult control mice. In newborns [3H]thymidine incorporation was significantly inhibited after DMNA pretreatment. In adult mice DMNA did not affect DNA [3H]dT incorporation up to 24h. P r e t r e a t m e n t with DMNA did not affect liver weight or DNA content in

TABLE I IN VITRO DMNA-N-DEMETHYLASE ACTIVITY IN LIVER MICROSOMES FROM UNTREATED CFW NEWBORN AND ADULT MICE Experimental group

Total protein concentration (mg/g liver)

DMNA-N-demethylase activity (nmol HCHO/min per mg protein)

Newborn Adult

160 ± 2.5 261.5 ± 28.5**

3.25 ± 0.17 9.23 ± 1.04"*

DMNA incubated concentration was i raM. Each point represents the mean ± S.D. of three determinations: pools of twelve newborn and three adult mice were used for every determination. **P ~ 0.01 by Student's t-test.

N.D. N.D. N.D.

1.15+0.1 1.83 + 0.08 27.19 + 4.06

Liver weight (g) Hepatic DNA content (mg/g liver) [3H]thymidine incorporation (DPM × 10E-03/mg DNA)

Adult

1.15±0.1 1.92 + 0.18 23.80 ± 4.99

115.4 + 12.8 3.33 ± 0.38 111.0 + 5.2**

8

1.16+0.05 1.58 + 0.23 29.79 + 4.14

109.5 + 10.5 3.14 + 0.02 63.0 + 7.1"*

24

AND KILLED

For [3H]thymidine incorporation, mice received a single i.p. injection of (methyl[3H])thymidine (37 kBq/g) I h before killing. Each point represents the mean ± S.D. of triplicate determinations of livers. Five newborn mice and three adult mice were used for each determination. **P < 0.01 vs. controls by Duncan's test for multiple comparisons. N.D., not determined.

111.1 + 4.0 3.38 + 0.41 97.1 ± 2.0"*

110.6 + 10.8 3.44 + 0.38 727.0 ~ 236.0

4

Hours after DMNA t r e a t m e n t

Liver weight (mg) Hepatic DNA content (mg/g liver) [SH]thymidine incorporation (DPM x 10E-O3/mg DNA)

Controls

A N D A D U L T M I C E T R E A T E D i.p.W I T H 7 mg/kg D M N A

Newborn

Experimental group

D E N O V O D N A S Y N T H E S I S IN T H E LIVERS O F C F W N E W B O R N 4, 8 A N D 24 H A F T E R T R E A T M E N T

T A B L E II

b~

265 T A B L E III TOTAL HEPATIC DISTRIBUTION, TOTAL DNA AND PROTEIN ALKYLATION 2 AND 4 h A F T E R A S I N G L E i.p. D O S E O F [~4C]DMNA (7 m g / k g , 3 4 4 . 1 - 5 1 8 M B q / m m o l ) IN N E W B O R N AND ADULT MICE Experimental group

T i m e a f t e r t r e a t m e n t (h)

Total hepatic radioactivity content (nmol[14C]-methyl]g liver) Newborn

4

141 ± 11.0

131.5 ± 3.5

Total protein binding (nmol[14Cl-methyl/g proteins)

0.20 ± 0.01

0.35 ± 0.01"*

Total D N A binding (nmol114C]-methyl/g DNA)

0.80 ± 0.02

1.94 ± 0.12"*

Total hepatic radioactivity content (nmol[14C methyl/g liver) Adult

2

Total protein binding (nmol[14C]-methyl/g proteins)

153.0 ± 7 . 0

135.2 ± 2.9*

0.33 ± 0.01

0.30 ± 0.01

0.59 ± 0.02

0.55 ± 0.19

Total D N A binding (nmoi[14C]-methyl/g D N A )

E a c h p o i n t represents the m e a n + S.D. of t r i p l i c a t e d e t e r m i n a t i o n s of pooled livers f r o m t w e n t y n e w b o r n m i c e a n d t h r e e a d u l t m i c e for e a c h d e t e r m i n a t i o n . * P < 0.05 a n d * * P < 0.01 vs. 2 h a f t e r t r e a t m e n t by S t u d e n t ' s t-test.

newborn or adult mice. Newborn DNA content, expressed as mg/g of liver, was double the content of adult mice.

Hepatic distribution o f [14C]DMNA in adult and newborn mice Table III shows the total radioactivity content and the binding to DNA and proteins, 2 and 4 h after a single i.p. dose of [14C]DMNA. Total hepatic radioactivity levels in newborn and adult mice were comparable at both times. Protein binding was also similar in newborn and adult livers 4 h after DMNA. In adult liver, total DNA binding was similar at 2 and 4 h after the treatment. In contrast, in newborns, it increased from 2 to 4 h after DMNA treatment. At 4 h, the total DNA binding was in newborn three times higher than in adult.

Formation and persistence o f DNA adducts in adult and newborn mice Figure 1 shows the radioactivity HPLC profile of the acid hydrolysis of alkylated liver DNA of newborn and adult mice after [14C]DMNA pretreatment. The methylated bases eluted by HPLC were identified as 7-meG (B) and Oe-meG (D) using synthetic standards. The peaks A and C corresponded respectively to guanine and adenine. They represent the metabolic incorporation of 14C following the metabolism of [14C]DMNA. The contribution of the radioactivity derived from the (14C)-metabolic guanine incorporation to the N7-meG and O6-meG adducts collected in the acid fraction of the newborn livers was negligible.

266

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Fig. 1. HPLC profile of the acid hydrolysis of alkylated liver DNA of newborn and adult CFW mice after I14ClDMNA pretreatment. Each bar represents the mean of three different injections. A = Guanine; B = N7-methylguanine; C = Adenine; D = O6-methylguanine. Table IV s h o w s the c o n c e n t r a t i o n of the liver D N A a d d u c t s in n e w b o r n a n d a d u l t mice a f t e r a single i.p. dose of D M N A . In n e w b o r n livers, the level of O6-mG w a s r e s p e c t i v e l y f o u r a n d five t i m e s t h a t in adult mice liver a t 2 a n d 4 h. 7 - m e g w a s the a d d u c t p r e s e n t in the h i g h e s t c o n c e n t r a t i o n . The time c o u r s e of 7 - m e G f o r m a t i o n w a s different in the two g r o u p s of a n i m a l s . In a d u l t m i c e the liver D N A 7 - m e g c o n t e n t w a s the s a m e a t all the i n t e r v a l s of t i m e c o n s i d e r e d b u t in n e w b o r n mice t h e r e w a s a s i g n i f i c a n t r a i s e f r o m 2 to 4 h. A t 4 h 7 - m e G w a s 50% h i g h e r in n e w b o r n t h a n a d u l t mice. U n p u b lished d a t a s h o w e d no difference b e t w e e n the levels of D N A a d d u c t s inc u b a t e d or n o t w i t h R N a s e r e s p e c t i v e l y in n e w b o r n or a d u l t liver. Eluted r a d i o a c t i v i t y w a s 70 a n d 80% of the total r a d i o a c t i v i t y e x t r a c t e d f r o m D N A of n e w b o r n a n d a d u l t m o u s e liver.

267 TABLE IV LIVER DNA ALKYLATION OF CFW NEWBORN AND ADULT MICE 2 AND 4 h AFTER A SINGLE i.p. INJECTION OF [14C]DMNA (7 mg/kg, 344.1-518MBq/mmol). THE ANIMALS WERE KILLED 2 AND 4 h AFTER THE TREATMENT (pmol/ftmol guanine) Time after treatment (h) 2

4

Newborns

7-meG O6-meG

760.0 ± 20.4 99.4 ± 19.3

1561.0 ± 175.0"* 159.0 ± 23.0**

Adults

7-meG O6-meG

1070.0 ± 72.0 25.9 ± 0.1

937.0 ± 184.0 34.2 ± 3.9**

**P ~0.01 vs. 2 h by Student's t-test.

Determination of liver 06-methylguanine-methyltransferase (MT) T a b l e V r e p o r t s t h e e x - v i v o d e t e r m i n a t i o n of M T a c t i v i t y a n d t h e 0 6m e t h y l a t i o n l e v e l s i n h e p a t i c D N A of D M N A p r e t r e a t e d n e w b o r n a n d a d u l t m i c e . B a s a l M T a c t i v i t y w a s a l m o s t f o u r t i m e s h i g h e r i n a d u l t a n i m a l s in r e s p e c t t o n e w b o r n a n i m a l s . D M N A p r e t r e a t m e n t c a u s e d a d e c r e a s e of M T activity in both animal groups. DISCUSSION The present study reports quantitative d i f f e r e n c e s in h e p a t i c D N A d a m a g e i n n e w b o r n a n d a d u l t m i c e a f t e r a s i n g l e c a r c i n o g e n i c d o s e of DMNA. T h e i n v i t r o m i c r o s o m a l a c t i v i t y of D M N A - N - d e m e t h y l a s e , considered as a p a r a m e t e r of t h e l i v e r ' s c a p a c i t y t o p r o d u c e m e t h y l a t i n g s p e c i e s f r o m DMNA, was three times higher in adult than in newborn livers. In contrast, total liver DNA binding was three times higher in newborns than in adults. TABLE V EX-VIVO DETERMINATION OF O6-METHYLGUANINE-DNA METHYL TRANSFERASE (MT) ACTIVITY IN LIVER NUCLEAR PREPARATIONS FROM DMNA-TREATED NEWBORN AND ADULT MICE 4 h AFTER TREATMENT Experimental group

Newborn Adult

Methyltransferase (pmol O6-meG removed/mg DNA) Controls

Treated

1.5 ± 0.3 5.9 ± 1.3"*

0.4 ± 0.3 0.4 + 0.2

Each value is the mean + S.D. of at least three animals for adults and twenty for newborn mice. **P. : 001 by Student's t-test.

268

However, in newborns some of the radioactivity bound to DNA derived from the incorporation of laC-C-1 fragments into newly synthesized DNA purines as already described by Magee et al. [22]. These radioactive purines differ substantially from the ones in which the radioactivity derives from the binding of methyl groups to already existing DNA. Figure 1 shows t h a t there is a considerable metabolic labelling of DNA in newborns but none in adult livers. If we subtract the contribution of the metabolic 14C-incorporation in guanine and adenine to liver DNA binding, the methylation of DNA liver in newborns was still significantly different from adults. The present results suggest t h a t there is not a positive correlation between in vivo DNA binding and in vitro liver capacity to a-hydroxylate DMNA. Other authors reported data, not conclusive and contrasting, in which modulation of DMNA a-hydroxylation, the total DNA binding and the induced hepatocarcinogenicity were tentatively correlated [23-26]. The liver content of total radioactivity was similar in both groups at the two times studied, therefore differences in binding cannot be ascribed to a different distribution of DMNA in newborns and adults. The differences in total DNA binding might arise from qualitative differences in chromatin structure in adults and newborns. In this regard F a u s t m a n - W a t t s and Goodman reported that, after in vivo t r e a t m e n t with DMNA, r a t euchromatic DNA was more methylated t h a n heterochromatic DNA [27]. In newborns, on account of the active cell replication, euchromatic DNA is more despiralized than in adults. Thus, greater availability of nucleophilic DNA sites could be one of the causes of the higher alkylation. An equally plausible explanation might be t h a t the newborn liver has a different cell composition when compared with the adult liver. In fact it is well known t h a t the main cell type in the newborn liver was differentiating hemopoietic cell while in adult livers it was hepatocyte cell. Different authors [28,29] reported t h a t the principal characteristic of non-parenchymal hepatic cells is the inability to remove DNA adducts. Due to the difficulties in separating parenchymal from non-parenchymal cells in the newborn livers we could not differentiate the O6-meG derived from hepatocytes or non-parenchymal cells. However differentiating hemopoietic cells might be an explanation for the relatively high DNA content of the newborn liver on weight basis (see Table II). The O6-meG adduct, considered responsible for the mutagenic and carcinogenic properties of m a n y alkylating agents [2-4], was from two to five times higher in newborn t h a n in adult livers. Due to the presence of high a m o u n t s of RNA in newborn liver, the possibility t h a t some of the O6-meG adduct t h a t we measured derived from RNA alkylation was taken into consideration. To test this hypothesis, DNA extracted from the livers were incubated with RNase before chemical hydrolysis. The pattern of adducts formed and the levels of the methylated bases obtained were not different from samples not incubated with RNase (data not published). Therefore we can exclude t h a t the O6-meG and N7-meG t h a t we measured was of RNA origin. The O%meG: N7-meG ratios 2 and 4 h after t r e a t m e n t with DMNA were

269

respectively 0.13 and 0.11 for newborn and 0.024 and 0.034 for adult mice. The 061N7 ratio that we have measured in newborn mouse liver come close to the ratio observed by several authors [30,31] after "in vitro" DNA alkylation by methylnitrosourea. These "in vivo--in vitro" similarities m a y support the hypothesis that DNA in newborn is more accessible than in adult liver. At both times, in adult liver, the 06/N7 ratio was three to four times lower than in other species or mouse strains treated with the same dose of DMNA [30,32,33]. Thus the resistance of CFW adult livers to O%meG formation could be an important clue in explaining their low tumor susceptibility to DMNA. In addition, MT, the specific protein responsible for alkyl excision of the 0 8 position of guanine, was five times higher in adult than newborn mouse liver. This indicates that the neonatal liver has a limited capacity to repair DNA damage caused by DMNA. At a relatively high dose of DMNA, repair capacity is extremely reduced [33]. This is confirmed by the inhibition of MT that we observed 4 h after DMNA in both newborns and adults liver. This reduction further limits the already low repair capacity observed in control newborns. The inhibition of the repair capacity of the specific N 7 - m e G glycosylase after DMNA treatment can be hypothesised on the basis of the higher N7-meG content in the newborns in respect to adults. The metabolic 14C-incorporation in guanine and adenine observed in newborn but not in adult livers indicates that in the newborn liver, despite the lower rate of DNA synthesis after DMNA treatment, the stimulus of the replicative transcription of DNA persists. The difference in metabolic 14Cincorporation could be explained by the larger amount of DNA/unit of liver weight and by the higher rate of DNA synthesis in newborns than in adults (almost 30 times in control animals and two to four times in treated animals). Replication of alkylated DNA during the rapid cell proliferation in newborn livers could be the principal event in the carcinogenic process. In fact, when DMNA is administered in adult animals, during DNA synthesis after partial hepatectomy or exposure to toxic compounds, liver tumors are induced [33]. Preliminary data indicate that the same metabolic 14C-incorporation occurs in guanine and adenine in newborn and adult lung. This correlates well with the susceptibility to lung adenoma in newborn and adult as reported by Frey [5]. In conclusion, in newborn liver the higher rate of DNA synthesis related to a greater availability of DNA nucleophilic sites increases the probability of damage to guanine sites that could potentiate the tumorigenic effect. The lower repair capacity for O6-meG in newborn livers and the increase of these adducts resulting from impairment of the specific DNA repair systems might possibly facilitate the "fixation" of the damage to DNA and correlate with the reported higher susceptibility of newborn livers to hepatic tumors. ACKNOWLEDGEMENTS

The generous contribution of the Italian Association for Cancer Research, Milan, Italy is gratefully acknowledged.

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