Effect of hypophysectomy on persistence of methylated purines in rat liver deoxyribonucleic acid after administration of dimethylnitrosamine

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Biochimica et Biophysica Acta, 520 (1978) 671--678 © Elsevier/North-Holland Biomedical Press

BBA 99263

EFFECT OF HYPOPHYSECTOMY ON PERSISTENCE OF METHYLATED PURINES IN RAT LIVER DEOXYRIBONUCLEIC ACID AFTER ADMINISTRATION OF DIMETHYLNITROSAMINE

ANTHONY E. PEGG, GEORGIANA HUI and KENNETH J. ROGERS Department of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, Pa. 1 7033 (U.S.A.) (Received January 16th, 1978)

Summary The formation of methylated purines in DNA following dimethylnitrosamine administration was studied in control and hypophysectomized rats. When given the same dose of this carcinogen (in mg/kg body weight) the formation of the major product 7-methylguanine and of the minor products 1-, 3- and 7-methyladenine and 3-methylguanine was slightly greater in the livers of hypophysectomized rats than in controls. The rate of loss of these products from the DNA was not affected by hypophysectomy. O6-Methylguanine levels were significantly greater in the hepatic DNA of hypophysectomized rats compared to controls after doses of dimethylnitrosamine ranging from 1 to 20 mg/kg. This difference was due to a slower rate of loss of this purine from the DNA in the hypothysectomized rats. Growth hormone treatment increased the rate of removal of O6-methylguanine in the hypophysectomized rats but did not ;restore the activity to that found in controls. The possible significance of these results in the induction of tumors by dimethylnitrosamine is discussed.

Introduction

The potent chemical carcinogen, dimethylnitrosamine, is metabolized in target tissues to yield a highly reactive methylating agent [1--3]. Methylation of cellular components is thought to provide the stimulus for neoplastic growth [1--5]. Recent work has suggested that the formation and persistence in DNA of certain methylated bases, particularly O6-methylguanine, might be of critical importance in the initiation of tumors [4--7]. If this hypothesis becomes substantiated, the mechanisms and regulation of the repair system removing these products from DNA would be of particular interest in determining susceptibility to carcinogenesis. Conversely, physiological factors influencing the activity of this system and, hence, the methylated purine levels in DNA would be

672 expected to alter tumor incidence in animals exposed to dimethylnitrosamine if the hypothesis is correct. Although in vivo studies suggest that a number of abnormal, methylated bases are enzymatically removed from DNA in liver and kidney [5--7,9--11], little is known of the properties and regulation of the enzymes responsible for these reactions. In the present paper the effects of hypophysectomy and subsequent growth hormone treatment on the levels of methylated bases in the livers of rats treated with dimethylnitrosamine has been examined. It was found that O6-methylguanine levels in DNA were greater in hypophysectomized rats and that hypophysectomy reduced the rate of loss from DNA of this methylated base but not others. An preliminary abstract reporting this finding has been published [ 12 ]. Materials and Methods Hypophysectomized rats (weighing about 150g) and control female Sprague-Dawley strain rats were purchased from Charles River Breeding Laboratories, Wilmington, Mass., U.S.A. and maintained under controlled lighting conditions with free access to food and water. The rats were used approximately four weeks after surgery. Dimethylnitrosamine was administered by intraperitoneal injection of a solution in 0.9% (w/v) NaC1 at approximately 10:00 a.m. The concentration of dimethylnitrosamine was adjusted so that the volume injected was between 0.3 and 0.6 ml. Growth hormone was dissolved in 0.9% (w/v} NaC1 adjusted to pH 9.0 and injected subcutaneously at a dose of 1 mg/kg. DNA was isolated from rat liver and analyzed for the content of methylated bases as previously described [ 10,13]. The purity of the methylated bases after separation by column chromatography was verified by paper chromatography using the solvent systems described by Frei and Lawley [14]. The content of methylated adenines and guanines in the DNA was expressed as pmol per mol of the parent base present. Di[14C]methylnitrosamine (5.185 or 26 Ci/mol) was purchased from New England Nuclear, Boston, Mass., U.S.A., and diluted with redistilled, unlabeled dimethylnitrosamine obtained from the Aldrich Chemical Company, Milwaukee, Wisc. U.S.A. to give the required specific activity. N-[3H]methyl-N nitrosourea (123 Ci/mol) was also purchased from New England Nuclear. All other biochemical reagents were products of the Sigma Chemical Co., St. Louis, Mo., U.S.A. Bovine growth hormone, NIH-GH-B18 (0.81 I.U./mg) was a gift from the pituitary hormone distribution program of the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md., U.S.A. All other biochemical reagents were products of the Sigma Chemical Co. Results

The levels of methylated guanines and methylated adenines found in liver DNA of control and hypophysectomized rats at various times after administration of dimethylnitrosamine are shown in Tables I and II. As expected from previous studies [4--11], the major methylated purine present was 7-methylguanine. The next most abundant was O6-methylguanine followed by 3-methyl-

673 TABLE I F O R M A T I O N A N D P E R S I S T E N C E O F M E T H Y L A T E D G U A N I S E S IN H E P T I C D N A O F C O N T R O L AND HYPOPHYSECTOMIZED RATS TREATED WITH DIMETHYLNITROSAMINE R a t s w e r e i n j e c t e d w i t h d i [ 1 4 C ] m e t h y l n i t r o s a m i n e (26 C i / m o l for 1 m g / k g ; 5 . 1 8 5 C i / m o l f o r 10 m g / k g ) a n d killed at t h e t i m e s h o w n , T h e m e t h y l a t e d g u a n i n e s p r e s e n t in t h e D N A w e r e t h e n d e t e r m i n e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . All m e a s u r e m e n t s s h o w n r e p r e s e n t t h e m e a n of at least t h r e e s e p a r a t e d e t e r m i n a t i o n s w h i c h a g r e e d w i t h i n ±15%. A t least 70 c p m a b o v e b a c k g r o u n d w e r e p r e s e n t in e a c h m e t h y l a t e d base p e a k . W h e r e less r a d i o a c t i v i t y t h a n this was f o u n d , t h e a m o u n t p r e s e n t was c o n sidered b e l o w t h e u n i t o f d e t e c t i o n (n.d.). T y p e of r a t used

Control Hypophysectomized Control Hypophysectomized Control Hypophysectomized Control Hyp ophysectomized Control Hypophysectomized Control Hypophysectomized Control H y p o p h y sec t o m i z ed

Dose o f dimethylnitrosamine (mglkg)

Time (h)

1 1 1 1 1 1 10 10 10 10 10 10 10 10

4 4 24 24 48 48 4 4 24 24 48 48 89 89

M e t h y l a t e d g u a n i n e s p r e s e n t in D N A (#tool/tool guanine) 7-Methylguanine

3-Methylguanine

O 6 -Methylguanine

298 369 240 287 190 234 3285 3566 2248 2348 1440 1569 830 894

2.6 2.9 1.3 1.4 n.d. n.d. 30 30 19 21 11 14 n.d. n.d.

15.6 33.0 4.3 14.7 1.2 9.3 322 405 203 329 76 246 30 71

adenine, 7-methyladenine, 1-methyladenine and 3-methylguanine, respectively. The amount of 7-methylguanine present in the livers of control rats four hours after injection of dimethylnitrosamine was slightly greater in hypophysecT A B L E II F O R M A T I O N AND P E R S I S T E N C E OF M E T H Y L A T E D A D E N I N E S IN H E P A T I C DNA OF C O N T R O L AND HYPOPHYSECTOMIZED RATS TREATED WITH DIMETHYLNITROSAMINE E x p e r i m e n t a l details w e r e as in T a b l e I. n.d. i n d i c a t e s t h a t a m o u n t o f t h e m e t h y l a t e d base p r e s e n t gave less t h a n 70 c p m a b o v e b a c k g r o u n d in p o s i t i o n o c c u p i e d b y a u t h e n t i c m a r k e r b a s e . T y p e o f rats u s e d

Control Hypophy sectomized Control Hypophysectomized Control Hypophysectomized Control Hypophysectomized Control Hypophysectomized

Dose o f dimethylnitrosamine (mg/kg)

Time (h)

1 1 1 1 1 1 10 10 10 10

4 4 24 24 48 48 4 4 24 24

M e t h y l a t e d a d e n i n e p r e s e n t in D N A ( #mol/mol adenine) 7-Methyl adenine

3-Methyladenine

1-Methyladenine

6.0 6.9 n.d. n.d. n.d. n.d. 58 63 n.d. n.d.

8.9 10.3 0.9 1.1 n.d. n.d. 104 125 n.d. n.d.

3.8 4.9 2.3 2.5 n.d. n.d. 35 42 19 15

674 tomized rats. The difference was small {only 10--25%) but a similar effect was observed in a number of experiments at all doses of dimethylnitrosamine tested. The difference was statistically significant when all experiments were compared (P < 0.05). The rats in the present experiment were compared following similar doses of the dimethylnitrosamine in mg/kg b o d y weight. The h y p o p h y s e c t o m i z e d rats had slightly smaller livers {4.94 + 0.15 g per 150 g b o d y weight) than the control rats (6.45 + 0.1 g per 150 g b o d y weight) as previously reported by others [15,16]. Dimethylnitrosamine is metabolized to an alkylating agent in the liver at a rate sufficient to ensure that very little is excreted and up to 25 mg/kg can be completely metabolized in 4 h [17,18]. Therefore, in h y p o p h y s e c t o m i z e d rats the metabolism of the nitrosamine by the smaller a m o u n t of liver generates a greater concentration of the alkylating species within the liver. This could account for the small increase in the extent of alkylation when the h y p o p h y s e c t o m i z e d rats are compared to controls. The levels of all three methylated adenines (Table II) and 3-methylguanine (Table I) were also slightly higher in the hypophysectomized rats. (Since the amounts of these purines formed were much lower than the a m o u n t o f 7-methylguanine this increase was not as reproducible as with the major product.) All of these bases and 7-methylguanine were lost from the liver DNA so t h a t the levels declined from those seen 4 h after administration of the dimethylnitrosamine. As shown in Tables I and II there was no significant difference in the rate of loss for each of these methylated bases when normal and h y p o p h y s e c t o m i z e d rats were compared. 7-Methyladenine and 3-methyladenine were lost quite rapidly from the DNA and were at or below the limit of detection within 24 h. 1-Methyladenine and 3-methylguanine were lost more slowly but were below the limit of detection by 48 h. 7-Methylguanine was lost at a rate comparable to 3-methylguanine and 1-methyladenine but because of the much larger amounts of this product it could readily be detected even 89 h after injection of the dimethylnitrosamine. In contrast to the products described above, O6-methylguanine levels were substantially greater in the hypophysectomized rats than in the controls. Thus, 4 h after injection of 1 mg/kg of dimethylnitrosamine the a m o u n t of O6-methylguanine present in the liver DNA of hypophysectomized rats was more than twice that found in the liver DNA of control rats. This difference became even greater with time because O6-methylguanine was lost more slowly from the DNA of the hypophysectomized rats (Table I). Therefore, by 24 h the difference was more than 3-fold, and by 48 h it was almost 8-fold. When the dose of dimethylnitrosamine was increased to 10 mg/kg, the a m o u n t of O6-methylguanine in the DNA of h y p o p h y s e c t o m i z e d rats was only 30% greater than in control rats at 4 h, but this difference increased to more than 3-fold by 48 h because of the slower rate of loss from the hypophysectomized rats. It is likely that the difference in levels seen at the earliest time point was also due to a reduced rate of loss of the O6-methylguanine in the hypophysectomized rats. In previous experiments, O6-methylgnanine was removed quite efficiently from normal rat liver after low doses of dimethylnitrosamine so that up to 60% of the expected level of this product was lost in 4 h after a dose of 1 mg/kg [13]. When the dose of dimethylnitrosamine was increased to 10 mg/kg, much less was lost in the first 4 h partly because the removal system

675 was less efficient after this higher dose and partly because metabolism to produce the methylating species takes a greater proportion of the 4 h before measurement was made [13]. Therefore, the results shown in Table I suggest that alkylation by dimethylnitrosamine occurs to only a slightly greater extent in the hypophysectomized rats but that the rate of removal of O6-methylguanine is substantially slower in these animals. Since the rate of loss of the other methylated purines was not affected by hypophysectomy, these products were only slightly higher in the DNA of hypophysectomized rats, whereas the O6-methylguanine levels were significantly greater. Results consistent with this were also observed after injection of other doses of dimethylnitrosamine (data not shown). At all times, levels of other alkylated purines were only slightly greater in DNA of hypophysectomized rats after injection of 2.5 or 20 mg dimethylnitrosamine/kg body weight. However, O6-methylguanine levels were up to 4-fold greater 48 h after 2.5 mg/kg and 2-fold higher 48 h after 20 mg/kg. Since O6-methylguanine is stable in DNA in the absence of enzymatic excision [19] and the content of this product did decline in the liver DNA of hypophysectomized rats (Table I) it appears that the activity catalyzing this removal is reduced, but not absent, in these animals. In agreement with this, it was found that when the initial amount of O6-methylguanine present in the liver DNA was very low there was substantial removal in the hypophysectomized as well as in the control rats. The rats were given N-methyl-N nitrosourea, a direct alkylating agent which does not require metabolic activation, at a dose of 0.5 mg/kg body weight. This produced an initial (measured 10 min after administration) level of alkylation of hepatic DNA amounting to about 14 pmol 7-methylguanine and 1.6 pmol O6-methylguanine per mol of guanine in DNA. More than 90% of this O6-methylguanine was removed in 24 h from both the control and the hypophysectomized rats (data not shown).

TABLE III EFFECT

OF GROWTH

ADMINISTRATION

HORMONE

TREATMENT

ON METHYLATED

OF DIMETHYLNITROSAMINE

GUANINES

TO HYPOPHYSECTOMIZED

IN DNA AFTER

RATS

Hypophysectomized rats w e r e i n j e c t e d d a i l y w i t h g r o w t h h o r m o n e (1 m g / k g ) o r s a l i n e as i n d i c a t e d f o r six days. Two hours after the sixth injection, di[14C]methylnitrosamine w a s g i v e n as d e s c r i b e d in T a b l e I. The daily injection of growth hormone were continued. At the time shown after the dimethylnitrosamine a d m i n i s t r a t i o n , t h e rats w e r e k i l l e d a n d t h e c o n t e n t o f m e t h y l a t e d g u a n i n e s m e a s u r e d as in T a b l e I. T h e v a l u e s s h o w n a r e e i t h e r t h e m e a n o f t h r e e e s t i m a t i o n s o r t h e m e a n ± S.E. f o r f o u r o r five e s t i m a t i o n s . Growth hormone treatment

-+ -+ -+

Dose of dim ethylnitrosamine (1 m g / k g )

1 1 1 1 10 10

Time (h)

4 4 24 24 48 48

* Not significantly different from -- growth hormone. ** S i g n i f i c a n t l y d i f f e r e n t f r o m growth hormone, P ~ 0.05.

Methylated guanines in D N A (/~mol/mol guanine) 7-Methylguanine

06-Methyl guanine

369 377 270 276 1595 1402

33.0 25.7 14.2 8.1 286 164

± 24 ± 38" ± 243 ± 79 *

± 3.1 ± 0 . 9 ** ± 49 ± 2 4 **

676 When hypophysectomized rats were treated with growth hormone in amounts sufficient to induce a maximal weight gain, the levels of O6-methyl guanine detected in the DNA after injection of dimethylnitrosamine were decreased. There was no effect on the levels of 7-methylguanine (Table III). It, therefore, appears that growth hormone treatment increased the activity of the enzymatic system removing O6-methylguanine from DNA. On comparison of the O6-methylguanine levels found in hypophysectomized rats treated with growth hormone (Table III) with those found in control rats which had not had their pituitaries removed (Table I) there was still a significant difference. This suggests that other factors affecting the activity are also changed in the hypophysectomized rats.

Discussion Significant changes in the metabolism of certain drugs by the liver has been noted in response to diabetes, h y p o p h y s e c t o m y , insulin and growth hormone [20--22]. However, hepatic dimethylnitrosamine metabolism is known to be atypical in a number of respects [2,23,25] and it appears unlikely that hypop h y s e c t o m y substantially affects the activation of dimethylnitrosamine to the alkylating species. As discussed above, the slightly greater production of 7-methylguanine in DNA of hypophysectomized rats which was observed in the present experiments would be expected because of the difference in liver size for rats of the same b o d y weight. 7-Methylguanine production in DNA and RNA of hypophysectomized rats was comparable to that in controls in earlier experiments in which the dose of dimethylnitrosamine was much higher and probably not all metabolized before measurement was made [25]. Studies from a number of laboratories have indicated that enzymatic removal of 1- and 3-methyladenine and of 3- and O6-methylguanine from DNA occurs in rodent livers [4--11]. 7-Methyladenine and 7-methylguanine are lost from DNA by spontaneous depurination at neutral pH but could also be enzymatically removed [5,26]. Recent evidence suggests that the enzyme required for the removal of O6-methylguanine is unlikely to act on the other methylated purines [27]. The present studies support this hypothesis. The greater levels of O6-methylguanine in the DNA of hypophysectomized rats indicate that the system for the excision of this product is reduced in these animals whereas none of the other methylated products showed a similar increase. Since growth hormone treatment partially restored the activity of the O6-methylguanine excision system it appears that this hormone is able to affect the activity but that other factors which may also be hormonal are also needed for full activity. H y p o p h y s e c t o m y results in a deficiency of a number of hormones including corticotropin, but other experiments in this laboratory have not revealed a loss of O6-methylguanine excision capacity in adrenalectomized rats. The possibility that thyroid hormones might effect the excision rate has not yet been examined. Endocrine influences on t u m o r induction by chemical carcinogens are well k n o w n [28--31]. As discussed in a recent review [31] such effects can be due to hormonal influences on the metabolic activation of the carcinogen or on the

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subsequent: growth and development of tumors after the initial production of a neoplastic cell. The present experiments raise a third possibility, that hormonal influences might affect the repair of D N A damaged by chemical carcinogens. If the persistence of O6-methylguanine in D N A throughout cell division is of importance in carcinogenesis by dimethylnitrosamine as has been proposed [2--7], our results suggest that h y p o p h y s e c t o m y might increase sensitivity to tumor induction. Treatment with 0.0035% dimethylnitrosamine in the drinking water for 30 weeks produced a similar incidence of liver cancer in control and hypophysectomized Wistar strain rats [25,28]. However, the incidence of liver cancer was almost 100% in all the rats given this dose of dimethylnitrosamine [28] and a further experiment with a lower dose of the carcinogen is required before a conclusion can be drawn as to their relative susceptibility. Thyroidect o m y increased the incidence of tumors in both the kidney and the liver of NZR/Gd rats after a single dose of 20 mg dimethylnitrosamine/kg body weight [32]. The investigation of the possible changes in persistence of O6-methyl guanine in D N A of liver and kidney in such rats would, therefore, be of considerable interest. Acknowledgement This research was supported by grants C A 1 8 1 3 7 and 1P 30 C A 1 8 4 5 0 awarded by the National Cancer Institute, DHEW. Anthony E. Pegg is an Established Investigator of the American Heart Association. References 1 Magee, P.N. and Barnes, J.M. ( 1 9 6 7 ) Adv. C a n c e r Res. 10, 1 6 3 - - 2 4 6 2 Magee, P.N., M o n t e s a n o , R. and P r e u s s m a n , R. ( 1 9 7 6 ) in C h e m i c a l C a r c i n o g e n s (Searle, C.E., ed.), ACS M o n o g r a p h Series No. 173, pp. 4 9 1 - - 6 2 5 , A m e r i c a n C h e m i c a l S o c i e t y , W a s h i n g t o n . D.C. 3 Magee, P.N., Pegg, A.E. a n d S w a r m , P.F. { 1 9 7 5 ) in H a n d b u c h der A l l g e m e i n e n P a t h o l o g i e (Grundm a n n , E., ed.), pp. 3 2 9 - - 4 2 0 , Springer-Verlag, Berlin 4 L a w l e y , P.D. ( 1 9 7 6 ) in C h e m i c a l C a r c i n o g e n s (Searle, C.E., ed.), ACS M o n o g r a p h Series No. 173, pp. 8 3 - - 2 4 4 , A m e r i c a n C h e m i c a l S o c i e t y , W a s h i n g t o n , D.C. 5 Pegg, A.E. ( 1 9 7 7 ) Adv. C a n c e r Res. 25, 195---269 6 G o t h , It. and R a j e w s k y , M. ( 1 9 7 4 ) Z. K r e b s f o r s c h . 82, 3 7 - - 6 4 7 Margison, G.P. and Kleihues, P. ( 1 9 7 5 ) B i o c h e m . J. 148, 5 2 1 - - 5 2 5 8 Singer, B. ( 1 9 7 7 ) J. T o x i c o l . E n v i r o n m e n t a l H e a l t h 2, 1 2 7 9 - - 1 2 9 5 9 C r a d d o c k , V.M. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . Acta 312, 2 0 2 - - 2 1 0 10 Nicoll, J.W., S w a n n , P.F. a n d Pegg, A.E. ( 1 9 7 5 ) N a t u r e 254, 2 6 1 - - 2 6 2 11 Margison, G.P., Margison, J.M. and M o n t e s a n o , R. ( 1 9 7 6 ) B i o c h e m . J. 157, 6 2 7 - - 6 3 4 12 P e g g A.E., Hui. G. and Rogers, K.J. ( 1 9 7 7 ) Proc. A m . Assoc. C a n c e r Res. l S , 13 13 Pegg, A.E. { 1 9 7 7 ) J. Natl. C a n c e r Inst. 54, 6 8 1 - - 6 8 7 14 Frei, J.V. and L a w l e y , P.D. ( 1 9 7 5 ) C h e m . - B i o L I n t e r a c t . 10. 4 1 3 - - 4 2 7 15 S i m p s o n , M.E., Evans, M.H. a n d Li, C.H. ( 1 9 4 9 ) G r o w t h 13, 1 5 1 - - 1 7 0 16 T o l m a n . E.L., S c h w o r e r , C.M. and J e f f e r s o n , L.S. ( 1 9 7 3 ) J. Biol. C h e m . 248, 4 5 5 2 - - 4 5 6 0 17 H e a t h , D.F. ( 1 9 6 2 ) B i o c h e m . J. 85, 7 2 - - 8 1 18 S w a r m , P.F. a n d Magee, P.N. { 1 9 6 8 ) B i o c h e m . J. 110, 3 9 - - 4 7 19 L a w l e y , P.D. a n d Orr, D.J. ( 1 9 7 0 ) C h e m . - B i o l . I n t e r a c t . 2, 1 5 4 - - 1 5 7 20 Wilson, J . T . ( 1 9 6 9 ) B i o c h e m . P h a r m a c o l . 18, 2 0 2 9 - - 2 0 3 1 21 C o o k , D.F. and Phares, C.K. ( 1 9 7 7 ) E x p e r i e n t i a 33, 1 1 1 0 - - 1 1 1 1 22 R e i n k e , L.A., R o s e n b e r g , H., S t o h s , S.J. a n d R y a n , C.F. ( 1 9 7 7 ) Fed. Proc. 36, 9 1 4 23 Davies, D.L., B r y a n t , G.M., Arcos, J.C. a n d Argus, M.F. ( 1 9 7 6 ) J. Natl. C a n c e r Inst. 54, 1 0 5 7 - - 1 0 5 8 24 L a k e , B.G., Cottrell, R.C., Phillips, J.C., Gangolli, S.D. and L l o y d , A.G. ( 1 9 7 7 ) B i o c h e m . Soe. Trans. 5, 1 0 1 3 - - 1 0 1 5 25 Lee, K.Y. a n d G o o d a l l , C.M. ( 1 9 6 8 ) B i o c h e m . J. 106, 7 6 7 - - 7 6 8

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