Methylated purines formed in DNA by dimethylnitrosamine in rats previously exposed to hepatotoxic and hepatocarcinogenic regimes: Effects on the repair of O6-methylguanine

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METHYLATED PURINES FORMED IN DNA BY DIMETHYLNITROSAMINE IN RATS PREVIOUSLY EXPOSED TO HEPATOTOXIC AND HEPATOCARCINOGENIC REGIMES: EFFECTS ON THE REPAIR OF 06-METHYLGUANINE

J. STYLESb, C. BRADBROOKb, D.P. COOPERa, CHUa.*, P.J. O’CONNOR” and G.P. MARGISON” “Carcirlogenesis tute. Manchester Nr Macclesfield.

Section, M20 Cheshire

Paterson 9B.X and (CJ, K.)

Laboratories. bCentral

Christie Toxicology

J.D.

CHARLESWORTHb,

Hospital Laboratory,

and

Holt Radium ICI Alderley

Y.-H InstiPark,

(Received December 21st, 1983) (Revision received December 30th, 1984) (Accepted February 15th, 1985)

SUMMARY

Studies of mammalian systems for the repair of 06-methylguanine in DNA have revealed large differences in the capacities of tissues and cells to perform this function and in the case of rat liver it has been shown that the O”-methylguanine repair system can be stimulated by exposure to hepatotoxic and hepatocarcinogenic regimes (11-23). In this report an assessment is made of possible relationships between toxic liver injury, DNA synthesis, cell proliferation and DNA repair by treating Wistar rats with agents selected to provide differing degrees of liver involvement. The effects of long-term (20 week) treatments with acetylaminofluorene (15 mg/kg/day), quinoxaline 1,4dioxide (10 mg/kg/day), 4-aminobiphenyl-HCl (15 mg/kg/day) and pronethalol (20 mg/kg/day) were assessed, using the same strain of animals in which the original toxicity and carcinogenicity data were obtained. Repair of 06-methylguanine produced in liver DNA by a low, non-toxic dose (2 mg/ kg) of [‘4C]dimethylnitrosamine was increased 3-4-fold throughout the period of treatment with acetylaminofluorene, to a lesser extent by quinoxaline 1,4-dioxide and 4-aminophenyl-HCl and not at all in the case of pronethalol. No evidence was obtained to indicate a direct relationship between 06-methylguanine repair and either the induced hepatotoxicity or the ensuing increased rates of DNA synthesis which occur following exposure to these agents.

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Key words: Methylated purines - Dimethylnitrosamine regimes - Hepatocarcinogenic regimes - Oh-Methylguanine

-

Hepatotoxic

INTRODUCTION

The ability of the alkylating agents to act as mutagens and carcinogens and to alkylate macromolecules, in particular the nucleic acids, have been well documented. Many investigations have sought to elucidate mechanisms underlying these biological effects [l-5] and have indicated the importance of alkylation reactions at oxygen sites in nucleic acids [6,8]. In this context the repair of 0-alkyl lesions (e.g. 06-alkylguanine) in DNA have also been explored in some detail [ 3,4,9,10]. As investigations into the capacity of cells and tissues to repair 06-alkylguanine in DNA progressed it became evident that the process can be stimulated in rat liver by exposure of the animals to a variety of alkylating agents [ 11-131 as well as to agents from chemically different classes of compound [14--171. So far, those agents shown to be capable of eliciting this response in rats are toxic to liver, at least to some degree, but the effect is not entirely confined to liver as a weaker response has also been observed in kidney [ 181. Reports have indicated, however, that similar effects are not so readily detected in extra-hepatic rat tissues, e.g. kidney and lung [ 19-211. Previous studies on the enhancement of 06-methylguanine repair in rat liver had shown that it develops progressively over 3-4 weeks in rats exposed continuously to dimethylnitrosamine at 2 mg/kg/day [19] whereas relatively low, single doses of 2-acetylaminofluorene (2 mg/kg, [ 221) or aflatoxin B, (2 mg/kg, [ 151) caused similar increases in O”-methylguanine repair activity by 24 h. After single dose treatments the increased repair activity returns to control levels over 3-4 weeks but over a 6-week period after 3 weeks exposure to dimethylnitrosamine [ 231. In order to further characterise this response in animals treated continuously with enhancing agents over a prolonged period and at the same time, to assess whether there was a relationship between induced repair, DNA synthesis, toxic liver injury and cell proliferation, rats were treated with a variety of agents selected to provide differing degrees of liver involvement. The experiment was conducted using the same strain of animals in which the original hepatotoxicity data was obtained and the in vivo method for assay of the repair of O”-methylguanine was used as this procedure also provides an index of DNA synthesis and hence of changes in the extent of cell proliferation during the course of treatment. The following report shows that the increased level of 06-methylguanine repair, once produced, can be maintained at a constant level over many weeks during treatment with a strong enhancing agent. During the treatment with less effective agents the level can fluctuate but there was no evidence for a direct relationship between the stimulation of repair and hepatotoxicity. A preliminary account of some of these data has been given [ 211.

285 MATERIALS

AND METHODS

Materials Chemicals 2-Acetylaminofluorene and 4-aminobiphenyl-HCl were prepared as described previously [ 24,251; quinoxaline 1,4-dioxide and pronethalol(d-isopropylamino)methyl-2-naphthalenemethanol, ICI 42,464) were a gift from ICI Pharmaceuticals Division and they were prepared as suspensions in Kraft-Wesson corn oil at concentrations of 1.5, 1.5, 1.0 and 2.0 mg/ml, respectively. ‘“C-Labelled dimethylamine hydrochloride (spec. act., 54 Ci/ mmol) and [methyl-“H Jthymidine were obtained from the Radiochemical Centre, Amersham, Bucks., U.K. The labelled amine was used to prepare the labelled nitrosamine by the method of Dutton and Health [ 261 and the specific activity was adjusted by the addition of unlabelled dimethylnitrosamine (Eastman-Kodak Ltd., Lanes, U.K.). N-(L-r-glutamyl)-4-methoxy2-naphthyl-amide was obtained from Bachem Fine Chemicals Inc. (Torrence, CA, U.S.A.) for use as substrate in the histochemical detection of r-glutamyl transferase (r&T). Animals Specific pathogen-free Wistar derived rats (males) from the Alderley Park stock were 5 weeks old at the beginning of the experimental period. They were exposed to daily 12-h cycles of light/darkness, maintained at 22-23°C and given food (PCD diet, BP Nutrition, U.K.) and water ad libitum. Methods Treatment of animals Administration of compounds and control vehicle was made daily by gavage on week days. The control groups were either untreated (16 animals) or given a comparable quantity or corn oil (20 animals). Dosages and numbers of animals for the experimental groups were as follows: 2-acetylaminofluorene (15 mg/kg, 20) quinoxaline 1,4-dioxide (10 mg/kg, 16) 4-aminobiphenyl-HCl (15 mg/kg, 16) and pronethalol (20 mg/kg, 16). Compounds or corn oil alone were given 5 days per week for 4,8,12,16 and 20 weeks. Half the animals in each group were then given a single injection of 14C-labelled dimethylnitrosamine (2 mg/kg) on the last day of t,he treatment period in place of the final gavage treatment. The other half were left for 1 week before receiving the same dose of labelled nitrosamine. (The middle groups, i.e. weeks 12 and 13, were omitted in the cases of animals treated with quinoxaline 1,4dioxide, 4-aminobiphenyl-HCl and pronethalol). Five hours after the nitrosamine injection the animals were killed with an overdose of halothane which was shown to have no effect upon the DNA repair systems reported in this communication. The specific activity of the nitrosamine was in the range of 3-9 mCi/mmol but at

286 week 20 a higher specific activity (33.1 mCi/mmol) was employed. On this occasion, small nodules were dissected from the liver to provide a nodular enriched sample of liver tissue for comparison with the residual liver. A separate series of treatments were made for a period of 4 weeks only. These groups of animals were used to assess the effects of pretreatment upon the metabolism of dimethylnitrosamine and upon the incorporation of thymidine into hepatic DNA. Samples of tissues from these animals were also assayed for 06-methylguanine methyltransferase activity. Tissue samples were fixed in formal-saline for the preparation of haematoxylin/eosin stained sections. The remainder was frozen on dry ice and stored at -30°C for isolation of DNA and in the case of liver, also for the preparation of frozen sections that were stained for r-GT [ 271. Isolation of DNA and analysis of methylpurines In view of the large number of samples involved the material was processed in 2 batches for the preparation of DNA. Livers from the 2-acetylaminofluorene-treated animals and the two control groups were processed together and the remaining 3 treatment groups comprised the second batch. Two animals were taken at each time point and the livers were pooled for the isolation and analysis of DNA for the methylated purine content. In analysing the DNA from the second batch some samples from the corn-oil control groups were included as a procedural control and these additional values are included in Table I. DNA was prepared from liver by a modified phenol procedure [28] hydrolysed in 0.1 N HCl (7O”C, 30 min) and analysed by chromatography on Sephadex G-10 in the presence of authentic marker methyl purines [29,30]. The amounts of methylated purines were expressed as pmol methyl/mol. parent base assuming the specific activity of the methyl group to be the same as in the injected 14C-labelled dimethylnitrosamine. Owing to the presence of rapid acting-repair processes for 06-methylguanine and 3-methyladenine, but only very slow repair or loss of 7-methylguanine, the amounts of the former products initially present in DNA were calculated from the amount of 7-methylguanine using the ratio of 0.11 and 0.12 respectively, as previously described [ 201. Assessment of DNA synthesis Earlier work has shown that radioactivity from the 1-‘“C-labelled pool derived via the metabolism of the 14C-labelled nitrosamine was readily incorporated into both guanine and adenine of RNA [31} or DNA [32]. Radioactivity incorporated into adenine was therefore expressed as dpm/ pmol base and used as an index of DNA synthesis employing precursors arising via de novo pathways. The utilisation of salvage pathways was followed in some of the animals using [ 3H]thymidine (spec. act., 25 Ci/ mmol). In this case rats were given 0.4 mCi/kg 30 min prior to sacrifice. Tissues were fixed in Carnoy’s fluid, sectioned in paraffin wax and exposed for autoradiography using Emulsion K5 (Ilford) diluted (1: 1) with distilled

287 water. The slides were developed for 4 min at 20°C using Kodak stained with Feulgen and Light Green.

D19 and

Metabolism of dimethylnitrosamine Animals were pretreated for 4 weeks and in the evening preceding the final pretreatment they were placed in a glass metabolism cage. On the following morning, at the time of the final pretreatment, they were given a single (i.p.) injection of 14C-labelled dimethylnitrosamine at 2 mg/kg. The exhalation of 14C-labelled CO: was followed at 30 min intervals for the first 4 h and thereafter, hourly until 8 h. Procedures were as described previously

[201. Assay of methyltransferase activity in cell-free extracts Extracts of liver were prepared from 1 g tissue by homogenisation in 3 ml buffer (50 mM Tris, 1 mM /3-mercaptoethanol and 1 mM EDTA adjusted to pH 7.8 with HCl) and sedimented at 12 000 Xg. Sonication of the 12 000 X g pellet and preparation of the 30-55s ammonium sulphate fractions were carried out essentially as described previously [ 331. Protein concentrations in the final extracts were determined by Coomassie Brilliant Blue G-250 binding (Bio-Rad Protein Assay Kit, Bio-Rad, Watford UK) using bovine serum albumin as standard. Assays of methyltransferase activity were made by incubating samples of the 30-55s ammonium sulphate fractions (0.05-l mg protein) for 30 min at 37°C with 80 pg calf thymus DNA containing 0.5 pmol 06-[methyl-3H]guanine (5.4 Ci/mmol) in 50 mM Tris, 3 mM dithiothreitol and 1 mM EDTA adjusted to pH 8.3, at a final volume of 3 ml. Reactions were stopped by transfer to ice and the addition of 1 ml 4 M perchloric acid. After centrifugation at 1200 X g for 15 min the supernatant was removed, 4 ml 1 M perchloric acid was added to the pellet and heated at 70°C for 30 min to hydrolyse the DNA. The protein was then collected by centrifugation for 15 min at 1200 X g, the supernatant was removed and the pellet was washed with a further 4 ml 1 M perchloric acid. Finally the pellet was suspended in 0.4 ml 0.1 M HCl with 4 ml Vickers scintillator AM (Vickers Laboratories, Burley in Wharfedale, Yorks. U.K.) and counted at an efficiency of 30% to determine the transfer of methyl-.‘Hgroups to protein. Measurements of the removal of methyl-3H-groups from 06-methylguanine in DNA was carried out by HPLC analysis of the residual DNA as previously described [ 331. Rationale for selection of agents The compounds employed in this study were selected to provide differing degrees of liver involvement in order to explore the extent to which hepatotoxic and hepato-carcinogenic regimes affect the activity of DNA repair functions. The doses used were the carcinogenic doses employed in the earlier studies (see below). (I) 2-Acetylaminofluorene. A well established liver carcinogen which produces a variety of liver tumours as well as tumours at several extrahepatic

288 sites when rats are exposed to a diet containing this compound [34,35]. Previous studies with the Alderley Wistar rats have shown a similar spectrum of changes (unpublished data) and recent studies in another Wistar strain have shown that similar treatments with 2-acetylaminofluorene will increase the capacity of liver for the repair of Oh-methylguanine in DNA [ 23.221. (2) Quinoxaline 1,4-dioxide. This agent has been used to promote growth in chickens and pigs. It is carcinogenic principally at 2 sites in the rat namely, the nasal cavity and the liver [36]. Earlier studies by autoradiography had shown that administration of the compound labelled in the quinoxaline ring results in labelling, mainly of the nasal cavity but also of the liver, kidney and intestine. Although no histological changes were detected in any organs during the first three months of exposure to a diet containing this compound, the liver was slightly enlarged after 6 months and at 12 months this was accompanied by the presence of eosinophilic inclusions in the hepatocytes [ 361. (3) 4-Aminobiphenyl. This was selected as an agent without marked effects upon the liver, apart from slight bile duct proliferation in the older animals, the principal tumour site being the intestine [ 371. (4) Pronethalol. A drug used initially as a blocking agent for adrenergic o-receptors and therefore potentially capable of affecting many tissues. Prolonged exposure to agents of this type e.g. propranolol, can effect P-receptor number and stimulate adenyl cyclase activity levels in sarcolemma1 membranes [ 381. In these rats pronethalol is principally a leukaemogen giving three types of leukaemia (reticulum cell, lymphatic and myeloid) which show periportal (lymphatic) and generalised (myeloid) infiltrations of the liver; mammary tumours are also produced. Earlier studies in rats has shown no signs of toxicity during the first three months of dietary treatment but from 4 months onwards considerable weight losses were observed. RESULTS

(a) Animals

pretreated

for 20 weeks

Effects on growth At the beginning of the experiment rats were 120 g body wt. Animals in each group remained in good condition and gained weight uniformly However, in the group treated with throughout the 21-week period. 2-acetylaminofluorene weight-gain occurred more slowly due to a reduced food consumption. In this case mean body weights (g ? one S.D.) at 4 weeks and 21 weeks were 218 + 17 and 303 + 31 for 2-acetylaminofluorene treated rats compared with 256 + 25 and 498 2 53 in the corn oil controls. Weight gain in the other treated groups was the same as for the control animals.

289 Histological

assessment

Changes in the liljers of control animals There was little difference in histological appearance between the liver from untreated and from corn-oil control animals. A few liver cells showed a vacuolar degeneration and there were sporadic focal necroses present throughout the series. At early times (4 and 5 weeks) bile ducts were r-GT positive. A few adjacent periportal hepatocytes showed positive reactions in the bile canaliculi but these reactions diminished in intensity until only single bile duct cells showed positive by weeks 16 and 17. Changes in the livers of treated animals Only changes additional to those seen in the control animals are reported: (I) 2-Acetylaminofluorene. Apart from very slight proliferation of liver cells and of bile ducts little change occurred until week 8 when some small hyperplastic nodules and a few regenerating nodules with fibrosis (cirrhotic nodules) were detected. Proliferation of bile ducts and liver cells were more noticeable while lines of -y-GT positively staining bile ducts were proliferating to isolate areas of parenchyma. Some small areas of liver cells (50- 60 cells) were also positive but these had regressed to some extent one week after the carcinogen treatment had ceased (week 9). By the twelfth week hyperplastic nodules were well defined and persisted as did the numerous y-GT positive islands (now >lOO cells). Changes noted at weeks 12 and 13 were more extensive by the sixteenth and seventeenth weeks but these remained similar in appearance until weeks 20 and 21. At week 20, samples of nodules were dissected out for biochemical assays. Those kept for histology comprised mainly hyperplastic nodules and small areas of adhering parenchyma of near normal appearance. (2) Quinoxaline 1,4-dioxide. At week 4, increased mitotic figures were seen in parenchymal cells but otherwise, and at subsequent times, the histology was very similar to that of the controls. (Nasal cavities were not examined until the later stages, but these showed hyperplasia of the squamous epithelium, focal hyperplasia of the respiratory epithelium and some disorganisation of the olfactory epithelium. In some cases the epitheliurn appeared to be undergoing metaplastic change). (3) 4-Aminobiphenyl hydrochloride, Generally, there was slightly more degeneration of liver cells than in the controls and throughout the treatment period some sections showed a few areas of very slight focal proliferation of liver cells. Enlarged spleens were noted from week 16 onwards. (4) Pronethalol. No significant difference from the histological and histochemical appearance observed for the controls. Positive staining for -y-GT in (2), (3) and (4) was similar to that of controls. Methylated purines in liver DNA Extent of methylation (formation of 7-methylguanine). The amounts of the major alkylation product 7-methylguanine present in DNA show a degree

290 TABLE THE RATS MENT

I AMOUNTS OF METHYLATED CHRONICALLY EXPOSED WITH A SINGLE DOSE OF

\.alucs Figures Trcatmcnt

arr means in parentheses

ol

PURINES TV AGENTS [ 14CILABELLED

2 separate analvsis on arc the amounts of the

Duration

of

Kedctlon

Site

treatment

oJMOL/MOL PARENT BASE) OF VARYING HEPATOTOXICITY DIMETHYLNITROSAMINE

pooled mcthylated

liver.

*Means of purine relative

IN

THE

LIVER BEFORE

DNA OF TREAT-

3 separate analyses 011 pooled to the amounts of 7-met:~ylguanine.

(weeks) -I

5

8

9

~45.9 603 18.6 ti.3 43.5’ 580 16.5 1.9 .49.2 5-45 13.9 5.2 19.9 602 17.9 9.6 -33.4 512 13.7 21.5 36.1 510 12.8 17.8

(0.076) (0.031) (0.075) (0.028) (0.079) (0.025) (0.023) (0.030) (0.065) (0.028) (0.07 (0.026)

1)

62.6 711 23.0 5.6 JO.8’ 516 15.1 6.1 35.1 491 16.2 6.7 17.9 619 21.3 7.3 25.7 -130 13.5 7.9 13.3 533 16.6 6.2

(0.088) (0.032) (0.079) (0.029) (0.071) (0.033) (0.029) (0.034) (0.060) (0.031) (0.081) (0.031)

53.3 (0.088) 603 18.4 (0.031) 6.6 41.0 (0.085) 516 15.0 (0.029) 5.0 53.0 (0.069) 595 17.6 (0.030) 4.3 16.-l (0.022) 717 20.8 (0.029) 13.9 38.7 (0.07 1) 513 14.-I (0.027) 5.2 -10.4 (0.075) 5-11 11.8 (0.027) 5.2

50.9 615 20.3 5.7 51.1* 531 12.4 6.6 6-1.0 ti60 24.6 7.1 30.6 664 20.1 16.3 36.1 50-I 16.4 6.3 42.9 522 21 .o 20.3

(0.083) (0.033) (0.096) (0.023) (0.097) (0.037) (0.046) (0.030) (0.072) (0.033) (0.082) (0.040)

of variation throughout the series (Table I). For the two control groups (untreated and corn-oil) these values lie between 81% and 133% of the 4 week untreated control. The mean value (*l S.D.) for the combined control groups is 660.4 2 86.5 pmol/mol guanine. Loss of 7-methylguanine from liver DNA after these small doses of dimethylnitrosamine occurs only very slowly (tl/? = 72 h; [40]). The loss is partly due to chemical instability of the N-glycosylic bond (t,, at pH 7 is 6 days, [ 40,411) and also to N-glycosylase activity which is present at a very low level [42]. In the latter case the consistency of the 3-methyladenine/7-methylguanine ratio (0.031 f 0.003 for all control values) indicates that the variation in glycosylase activity is only a minor contributing factor to the overall level of 7-methylguanine and this ratio has previously been shown to be unaffected by treatment of rats with various agents (see e.g. Refs. 10 and 21). Prolonged exposure to 2-acetylaminofluorene may possibly have affected the capacity for hepatic metabolism as after the earlier stages all the values fall below that for the 4-week untreated control. In the other three treated groups in which liver involvement was either much less or negligible (see Histological and Histochemical Assessment), the extent of variation, apart from the

liver.

291

13

12

60.3 767 24.2 6.1 55.4 679 19.9 4.2 -

(0.079) (0.032) (0.082) (0.029)

17.6 (0.030) 578 16.0(0.028) 14.3 -

64.3 760 26.2 6.1 53.9 637 17.6 6.0 -

(0.085) (0.032) (0.085) (0.028)

26.3 573 18.9 14.3 -

-

(0.046) (0.033)

16

17

-

-

-

-

56.8 706 22.0 6.6 45.1 637 22.9 4.3 17.8 553 15.1 29.3 32.4 672 26.7 6.2 23.5 619 24.5 14.0

(0.080) (0.031) (0.071) (0.036) (0.032) (0.027) (0.048) (0.040) (0.038) (0.040)

73.3 802 24.7 4.5 51.0 676 33.5 4.8 28.4 488 14.3 21.9 28.7 521 43.4 8.5 28.5 560 33 2.5

21

20

(0.091) (0.031) (0.075) (0.050) (0.058) (0.029) (0.055) (0.083) (0.051) (0.059)

64.0 677 21.4 6.6 65.9* 742 23.5 5.1 58.4 650 34.5 11.5 17.7 585 16.9 30.2 53.5 814 32.1 4.8 26.8 664 33.3 25.2

(0.095) (0.032) (0.089) (0.032) (0.090) (0.053) (0.030) (0.029) (0.066) (0.039) (0.040) (0.050)

68.0 719 24.6 10.8 55.6' 720 27.5 5.4 82.4 890 48.6 6.0 19.7 405 12.5 14.4 35.4 701 32.7 3.2 47.3 771 36.9 3.7

(0.095) (0.034) (0.077) (0.038) (0.092) (0.055) (0.049) (0.031) (0.050) (0.047) (0.061) (0.048)

lower value for quinoxaline 1,4-dioxide at 5 weeks, is more or less comparable to that observed in the two control series. This is true also in the case of pronethalol for which some slowing of nitrosamine metabolism was observed (Table III). Overall, there are no major differences in the extent to which liver DNA was alkylated following the single pulse of dimethylnitrosamine. Changes in the amounts of other methylated purines in the liver DNA. In the comparison of the amounts of 06-methylguanine and 3-methyladenine in the DNA of the various groups and pretreatment intervals (Table I), the slightly different extents of initial DNA alkylation due to variations in DMN metabolism which are indicated by the 7-methylguanine levels (see above) must be taken into account. This is conveniently achieved by expressing 06-methylguanine and 3-methyladenine relative to the amounts of 7-methylguanine, these ratios being interpreted as indicating the activity of the systems responsible for the repair of these adducts. The validity of this in vivo method of assay using low doses of DMN in rats is now well established. It is possible because 7-methylguanine is lost from liver DNA only very slowly (see above) and the dose response for 7-methylguanine formation by

292

DMN in liver DNA is linear, even after extremely low doses of DMN [43,44] thereby providing an appropriate base line. Moreover, assay of the repair activity for O”-methylguanine made from changes in the 06/7-methylguanine ratios have been substantiated in many cases by direct measurements of O”-methylguanine repair activity in in vitro assays [ 19,22,43,45]. They also have the advantage of measuring repair activity in those cells which metabolise the nitrosamine in contrast to the tissue average values given by methyltransferase assays performed on cell free tissue extracts. Thus in the case of the animals given 2-acetylaminofluorene, changes in the relative amounts of Of’-methylguanine to 7-methylguanine indicate that the capacity of liver to repair O”-methylguanine was enhanced significantly at all times over that for the control livers. When this estimate of repair capacity was made on the last day of each treatment period, the increase was approx. 4-fold but in the animals rested for one week this was reduced to 2-3-fold (Fig. la). In contrast, pronethalol (Fig. Id) an agent without detectable histological or histochemical effects on the liver showed none of these increases: data for the other two compounds were intermediate between those for 2-acetylaminofluorene and the combined control and pronethalol series. In the case of quinoxaline 1,4-dioxide there was a tendency for the repair of 0”-methylguanine to be raised above the control level reaching 2-fold at the later stages of treatment (Fig. lb). 4-Aminobiphenyl increased the repair of O”-methylguanine 3-fold at 16 and 20 weeks after which times the l-week rest period reduced the extent of repair as in the case of the animals treated with 2-acetylaminofluorene (see above and Fig. lc). The ratio of 3-methyladenine to 7-methylguanine remains constant at 0.031 % 0.003 in the control series and following treatment with 2-acetylaminofluorene, indicating that the repair of 3-methyladenine is unaltered. There was a tendency, however, for repair of this base to proceed more slowly in the latter half of the treatment period after exposure to pronethalol, quinoxaline 1,4-dioxide and 4-aminobiphenyl (see Table I). Methylated purines in the DNA of 2-acetylaminofluorene-induced liver nodules. Nodules were dissected from the livers of 20 week-treated animals after they had been given an injection of high specific activity dimethylnitrosamine and the amounts of 7-methylguanine found in this DNA were lower than in control liver but comparable to that of the 21 weeks liver from this treatment group. The 7-methylguanine to 06-methylguanine ratios (Table II) indicate that the capacity for the repair of 06-methylguanine in these nodules was higher (2-fold) than that for liver tissue from control animals but lower than in the liver of animals given the continuous 2-acetylaminofluorene treatment. It was, however, very similar to the repair capacity of the liver from the 21-week animals which had been rested from the 2-acetylaminofluorene-treatment for one week. (b) Animals

pretreated

for 4 weeks

Effects on nitrosamine metabolism The metabolism of 14C-labelled dimethylnitrosamine, as determined by exhalation of labelled COz, was similar in control and pretreated animals

293

Fig. 1. Repair of 06-methylguanine from the liver DNA of control and pretreated Wistar rats in relation to the rate of DNA synthesis in the liver DNA of the same animals. 06-Methylguanine repair was assayed by pulsing the animals with a single dose of “C-Labelled dimethylnitrosamine (2 mg/kg) 5 h before they were killed for the analysis of the methylated purine content of hepatic DNA. The extent of repair is shown as the percentage of 06-methylguanine lost from DNA based on a calculation of the initial amounts of the methylated purine initially present when the alkylation reaction was complete (see text for details). The rate of DNA synthesis is determined as the incroporation of radioactively labelled breakdown products derived from the metabolism of “C-labelled dimethylnitrosamine into hepatic DNA and is expressed as dpm/pmol adenine. The treatment groups are indicated as follows: , untreated controls; x , corn-oil controls; I, animals given ‘%labelled dimethylnitrosamine at the end of the pretreatment preiod; n , animals given the labelled nitrosamine 1 week after pretreatment for (a) 2-acetylaminofluorene, (b) quinoxaline 1,4-dioxide, (c) 4-aminobiphenyl and (d) pronethalol.

294 TABLE

II

THE AMOUNTS OF METHYLATED PURINES IN DNA FROM AND RESIDUAL LIVER OF RATS CHRONICALLY EXPOSED FLUORENE FOR 20 WEEKS BEFORE TREATMENT Treatment mmol) Tissue

was

sample

a single

dose

Amounts (pmol/mol

of

of methylated parent base

N-7meG

Liver

nodules

Residual

liver

= S.D. b Amount

relative

443.0

f

584.9

* 20.1

to the

“C-1abelled

8.5a

amount

THE LIVER NODULES TO 2-ACETYLAMINO-

dimethylnitrosamine

(spec.

purines f S.D.)

act.,

33.;

mCi/

Metabolic incorporation (dpm/pmol adenine/ spec. act.)

Ob-meG

N-3meA

20.05 f 3.15 (0.045)b 17.65 + 0.25 (0.030)b

24.35 f 0.35 (0.056)b 16.90 20.10 (0.029)b

104.0

+ 4.04

30.2

f 0.25

of 7-methylguanine.

with the exception of those exposed to pronethalol (Table effect of pretreatment with pronethalol is significant (i.e. 0.07 h for all other groups combined) it did not detectably alkylation of DNA measured 5 h after injection of the above and Table I). In agreement with previous observations exposed to a similar level of 2-acetylaminofluorene (26 diet) and then given different doses of dimethylnitrosamine there was no effect on dimethylnitrosamine metabolism

III). Although the 2.20 h vs. 1.28 + affect the overall nitrosamine (see [ 201 on animals mg/kg/day in the (1-9 mg/kg) in this group.

Effects on O”-methylguanine methyltransferase activity Assay of cell-free extracts of liver tissue taken from animals exposed to pretreatment for 4 weeks, apart from those from the 2-acetylaminofluorene treated group, exhibited a similar or slightly increased methyltransferase capacity compared to those of the control groups, irrespective of whether TABLE

III

EFFECT OF PRETREATMENTS LOW DOSE OF *‘C-LABELLED

ON THE CAPACITY DIMETHYLNITROSAMINE

OF

RATS TO (2 mg/kg)

METABOLISE

Treatment

Time (hf

Treatment

Time (h)

Untreated controls Pronethalol 2-Aeetylaminoflurorene

1.25a 2.20 1.20

Corn-oil controls Quinoxaline 1,4-dioxide 4-Aminobiphenyl

1.27 1.27 1.40

a Data over

are presented as the time taken to reach 50% 8 h (For details see Methods). Pairs of animals

of the total were used

“C-labelled CO,exhaled in each treatment group.

A

295 the assay was made by transfer of the labelled nethyl group to protein or by removal from the guanines in DNA (Table IV). These results, in comparison with the data for the in vivo removal of 06-methylguanine in Fig. 1 show that the repair of 06-methylguanine can be assessed either by in vitro or by in vivo procedures as previously indicated [ 22,451. The enhancement of 06methylguanine repair in response to treatment with the aromatic amide was about 4-fold in both cases (Table IV and Fig. la). DNA synthesis in the livers of control and treated animals DNA synthesis estimated from the extent of incorporation of radioactivity derived from the breakdown of labelled DMN via the 1-‘“C-pool has been shown to give results similar to those obtained by measuring the incorporation of [“Hlthymidine into DNA [ 221. Results obtained here indicate changes in DNA metabolism following some of these treatments. For example, 2-acetylaminofluorene caused a progressive increase in DNA synthesis which reached a maximum at weeks 16 and 20 but declined again slightly in each case after the week during which treatment was stopped (Fig. la). These results were in contrast to the uniformly low, resting rates of DNA synthesis observed in the untreated, corn-oil treated and pronethalol treated rats (Fig. Id) with mean values (51 S.D.) for each group of 6.7 + 1.6, 5.4 + 0.8 and 6.2 + 2.2 dpm/lmol adenine, respectively. Exposure to 4-aminobiphenyl (Fig. lc) also caused increases in DNA synthesis but these did not follow a consistent pattern. In the case of quinoxaline 1,4dioxide (Fig. lb) the only change detected was an increase at 4 weeks when mitotic figures could also be seen in sections of these livers (see Histological Assessment). By far the largest change in DNA synthesis (>lO times control values) was observed in the hyperplastic nodules taken from the livers of the 20 week 2-acetylaminofluorene treated animals (Table II). Results similar to those outlined above (see also Table I and upper parts of Figs. la--c) were obtained when the uptake of labelled thymidine was TABLE EFFECT FERASE

IV OF PRETREATMENTS ON THE 06-METHYLGUANINE ACTIVITY IN CELL-FREE EXTRACTS OF LIVER

Pooled samples from a pair of animals in each treatment Treatment

Activitya bmohv9

Untreated control Pronethalol

0.394b 0.366

0.31oc 0.318

2-Acetylaminofluorene

2.106

2.260

a Activity is expressed as pmol methyl transferred b By transfer of methyl to protein. c By removal of methyl from DNA guanine.

METHYLTRANS-

group.

Treatment

Activity (PmoUmg)

Oil control Quinoxaline 1,4-dioxide 4-Aminobiphenyl

0.465b 0.474

0.356’ 0.430

0.448

0.400

per mg protein/h

296 assessed by autoradiography of livers from animals pretreated for 4 weeks indicating the presence of increased numbers of cycling cells in some treated groups. Virtually no labelled cells were detected in liver sections from the two control groups or from animals pretreated with pronethalol. By contrast, a sporadic distribution of labelled hepatocytes was obtained in animals exposed to quinoxaline 1,4-dioxide, 2-acetylaminofluorene and 4-aminobiphenyl. Although quantitation was difficult due to the uneven distribution of labelled cells, the heaviest labelling (>200 grains/nucleus) was seen after quinoxaline 1,4dioxide treatment. These heavily labelled cells were relatively few in number and cells carrying 20-100 grains/nucleus were more common; this pattern of lighter labelling was also observed in hepatocytes after treatment with 2-acetylaminofluorene or 4-aminobiphenyl. DISCUSSION

The effects of 2-acetylaminofluorene on body weight is in keeping with earlier observations in which rats were exposed to a diet containing this compound [ 20,461. However, as the carcinogen was administered by gavage in the present experiments, palatability of the carcinogen diet can be ruled out as the factor responsible for the reduced food intake observed in the present study and in earlier experiments [ 201. The methylation of liver DNA after a single dose of dimethylnitrosamine as judged by the formation of the major product 7-methylguanine was not markedly affected by treatment with the agents used in this study (Table I). A greater degree of individual variation is to be expected when the nitrosamine is given over an extended period to animals of different ages as compared with the treatment of a uniform batch of animals on the same day. Nevertheless, some of the values probably fall outside the normal range of variation. For example there was a tendency towards lower levels of alkylation in the nodular livers of animals exposed to 2-acetylaminofluorene for prolonged periods, although an earlier report had indicated that 2-acetylaminofluorene-induced liver nodules were as readily alkylated by dimethylnitrosamine when compared with normal liver [47]. Overall, however, no marked effects on DNA alkylation were encountered which could be ascribed to metabolism as would occur when, for example, the hepatotoxin carbon tetrachloride is administered [ 481. When the amounts of 06-methylguanine initially formed in DNA are calculated from the amounts of 7-methylguanine present at 5 h (see Methods) the values for the whole series fall within the range 42--98 pmol/ mol guanine. Comparison of the in vivo 06-methylguanine repair activity with these individual substrate levels (pmol/mol guanine) indicates that over this relatively narrow range, substrate concentration did not influence the livers’ capacity for the repair of this promutagenic base (calculated from Table I, data not shown). Moreover, the uniformity of the data for the repair of 06-methylguanine in the two control groups and the pronethalol series (Fig. 1) indicate that the repair capacity did not alter significantly during the period of growth from the young adult (9 weeks; 250 g body wt.) to the

297 fully mature animal (24 weeks; 490 g body wt.). A similar result was obtained when methyltransferase levels were measured in liver extracts from animals aged 6 and 52 weeks [49]. In comparison with the controls, treatment with 2-acetylaminofluorene resulted in an approx. 4-fold increase in the capacity for the removal of 06-methylguanine (Fig. la) which was confirmed by the 4--5-fold increase in the methyltransferase activity in the in vitro assay (Table IV). This increased activity persisted virtually unaltered throughout the entire period of treatment despite considerable histological changes which included the formation of -y-GT positive islands of hepatocytes and effects of toxicity that progressed during the treatment. As the data are based on the analysis of whole liver samples, this implies that a substantial proportion of the liver cell population had undergone changes, one at least of which affects the extent to which 06-methylguanine repair is stimulated. The effect of stopping the treatment is also of interest. This did not alter 06-methylguanine repair after the first 4 weeks of exposure to 2-acetylaminofluorene but after longer treatment periods allowed some return towards the rate seen in the control animals, indicating the emergence of cells with a more limited capacity to sustain the enhanced repair response. The partial loss of the repair response during the one week rest period may in part be associated with areas of liver which eventually give rise to the nodules since repair in the nodules was slightly lower than in the residual tissue (Table II). This observation is in keeping with an earlier report [20] on the repair of 06-methylguanine in the nodular livers of rats exposed to periods of 2-acetylaminofluorene feeding. Treatment schedules leading to a much lower degree of toxicity also gave rise to an enhanced capacity for 06-methylguanine repair, but to a much lesser degree. Thus, an increased capacity for repair did not occur until the later stages of treatment with 4-aminobiphenyl whereas in the case of the quinoxaline 1,4dioxide similar increases were observed without any detectable signs of hepatotoxicity other than an increased mitotic index at 4 weeks. The rate of DNA synthesis was also affected by these treatments (Fig. 1, upper curves) and the labelling patterns determined by autoradiography indicate that these changes were due predominantly to cells in cycle. The persistent hepatotoxic changes seen during exposure to 2-acetylaminofluorene lead to a gradual rise in the rate of DNA synthesis throughout the treatment period whereas the repair of 06-methylguanine remained at a constantly elevated level throughout. At the later stages of exposure to the aromatic amide the rate of DNA synthesis decreased after cessation of treatment for one week. Changes in DNA synthesis after treatment with 4-aminobiphenyl were much more variable and in the case of quinoxaline 1,4-dioxide, DNA synthesis remained at the base level observed for the two control and pronethalol series except for a brief rise at 4 weeks which was associated with an increased number of mitoses (see Histological Assessment) and cycling cells with moderate to heavily labelled nuclei. This early rise in DNA synthesis is of interest in view of the growth promoting effects of this agent in domestic animals [36] which are apparently due to bacteriocidal

298 activity rather than to a direct effect on cells. There was, however, no effect on 06-methylguanine repair at this time. Throughout treatment with quinoxaline 1,4-dioxide or 4-aminobiphenyl there was no evidence of any direct relationship between the levels of DNA synthesis and the repair of this adduct. Similarly, there was no obvious evidence for a direct relationship between this parameter and 06-methylguanine repair during exposure to 2-acetylaminofluorene. In summary, it appears that neither the hepatotoxic effects associated with these treatments, nor the ensuing increased rate of DNA synthesis were obviously related to the enhanced repair of 06-methylguanine. This was true even in the case of hyperplastic nodules (Tables II) where incorporation of radioactivity from the l-carbon pool was greatly increased and the already increased repair of 06-methylguanine showed evidence of declining. Although not fully conclusive, the data discussed here offer no support for a direct association of increased rates of 06-methylguanine repair with the onset of the proliferative state induced by these toxic changes even though partial hepatectomy in the rat will increase the rate of 06-methylguanine repair several fold [45,50]. Moreover, methyltransferase activity is low in neonatal liver [ 181 and in other laboratory rodents e.g. mice [ 45,511, gerbils [33] and hamsters [ 521, neither partial hepatectomy nor hepatotoxic treatments are able to induce the 06-alkylguanine repair response which is so readily initiated in the rat. Thus, whilst there is no clear evidence so far in rodent species for a specific association of the increased repair of 06-methylguanine with hepatotoxic changes or the ensuing proliferative response, the data in this study show that an elevated level of 06-methylguanine repair is maintained throughout an hepatocarcinogenic regime of 2-acetylaminofluorene treatment. There are indications that some cell populations may be affected differentially and further studies are in progress to determine the relevance of these observations for mechanisms in carcinogenesis. ACKNOWLEDGEMENTS

We thank Dr. Iona Pratt (ICI Alderley Park) for advice on pathology, Mr. Brian Berlinson (ICI Alderley Park) for management of the animals and Mr. Brian Keenan (Paterson Laboratories) for assistance with the analysis of the DNA samples. We are indebted to Mrs. Caroline Chadwick (Paterson Laboratories) for the preparation of autoradiographs and to Mr. J.A. Bailey (Paterson Laboratories) for measurements of nitrosamine metabolism and for methyltransferase assays. Financial support to the Paterson Laboratories from the Cancer Research Campaign and from the World Health Organisation to Dr. Y.-Y. Chu is gratefully acknowledged. REFERENCES

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41 42

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46 47

48

49

50

51

52

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