BIOCHIM1E, 1982, 64, 769-773. CNRS symposium, May 1982 - TOULOUSE

Inducible responses to D N A damages

Species differences in the inducibility of hepatic 06-alkylguanine repair in rodents.

Peter J. O ' C O N N O R , Ymag-Hua C H U (*), Donatd P. C O O P E R , Girish B. M A R U (**), R a y m o n d A. S M I T H and Geoffrey P. M A R G I S O N .

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, U.K.

Mots-cl6s : r6paration induite / 06-alkylguanine / careinog6nes / h6patotoxines / proliferation du foie / comparaison entre rongeurs de laboratoire.

Key-words : induced repair / 06-alkylguanine / proliferation of liver / carcinogens / hepatotoxins / comparison of laboratory rodents.

I n t r o d u c t i o n .

I t is now well established ~ that pretreatment of rats with multiple doses of hepatotoxic, hepato- carcinogenic agems will increase the activity of the hepatic 06-alkylguanine repair system [1-8]. Toxi- city leading to regenerat ion of damaged tissue may be responsible since partial hepatectomy in the rat will p roduce a similar effect [9]. I n order to under- stand this phenomenon more fully the effect o f pretreatment with single doses of various agents has been studied in the rat [8, 10], a r~ the res- ponse of the hepatic 06~alkylguanine repair system in other laboratory rodents has been examined. The following accourJt summarises the attempts made in these laboratories to induce an increasexl level of 06-alkylguanine repair in the livers of various labora tory roAents.

M a t e r i a l s a n d M e t h o d s .

Chemicals: Di[14C]methylamine hydrochloride (55 mCi/mmol), di [14C] ethylamine hydrochloride (55 mCi/ mmol), deoxy[8-aH]adenosine 5'-triphosphate (10 Ci/ mmol) and [5-methyl-3H] thymidine (25 Ci/mmol) were from Amersham International, U.K, Dilt4C] alkylni- trosamines were synthesised from the radioactively label- led amines as described previously [11] and diluted with unlabelled dialkylnitrosamhaes obtained from Eastman- Kodak Ltd., Kirby, Lancs, U.K. [methyl-all] nitrosourea

(*) Shanghai Cancer Institute, 127 Dong An Road, Shanghai, People's Republic o] China.

(**) Cancer Research Institute, Tata Memorial Centre, Bombay 400 012, India.

(1.6 Ci/mmol) was from New England Nuclear, Dreieich, W. Germany. Untabelled deoxynucleoside 5'-triphosphates were obtained from Boe .tn'inger, Mannheim, W. Germany. Aflatoxin B~ was purchased from Sigma (London) Che- mical Co, Poole, Dorset, U.K. and 2-acetylaminofluo- rene was a generous gift from ICI, Pharmaceuticals Division, Macclesfield, Cheshire. U.K.

Animals : Males of the following species, Wistar rats (200-260 g), C57 Black mice (21-23 g) and Mongolian gerbils (Meriones unguiculatus) (65 g) were from the stock maintained in these laboratories. Syrian hamsters (Mesocricetus auratus) (100-150 g) were purchased from Wrights, Latchingford, Essex, U.K. All were allowed free access to food and water and were maintained on a pelleted diet (LCD, Standardised Rat and Mouse Diet, Labsure, Poole, Dorset, U.K., gerbils and hamsters received a fruit/vegetable supplement). In one experiment C57B1 mice were given drinking water containing dime- thylnitrosamine (10 ppm) for a period of 38 days. Partial heptatectomies (2/3) were performed under ether anaes- thesia using the method of Higgins and Anderson [12]. Except where indicated all injections were given intra- peritoneally.

Methods.

Samples of tissue were wemoved, quickly frozen and stored at - -20°C. DNA was isolated from these samples by a phenol procedure and analysed for the presence of alkylpurines as previously described [2]. [3H] thy- midine incorporation into acid soh/ble DNA of rat liver was estimated as described [2]. Subs~rate for the in vitro assay of O6-methylguanine demethylase activity was prepared by reaction of methyl-laI-I] nitrosourea with calf thymus DNA [10] ; preparation of cell free extracts and conduct of the assays ~,ere essentially as described previously [9, 10]. Assays for DNA 9olymera~ a acti- vity in cytosol preparations were carried out as descri- bed [2, 13].

770 P. 1. O 'Connor and coll.

Results.

E]]ects o] single doses in the rat.

Single doses of aflatoxin B1 (2 m g / k g ; 1/4 LDs0) will erdaance the hepatic 06-methy~gua - nine repair system [8]. T he maximum effect is

repair was assayed by pulsing the animals with a single dose of di r[l~] methylnitrosamine (2 rag/ kg) at various times after the pretreatment and measuring the amounts of methylpurines in hepatic D N A 5 h later. Durirrg tiffs irrterval all the dime- thylnitrosamine wotdd have been metabolised to yield the reactive chemical species to allow alkyla-

DNA Synthesis

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06-methylguanine repair and DNA synthesis in rat liver. Animals were given the mycotoxin suspended in 1.75 per cent gum acacia in 0.15 M sodium chloride and were killed at intervals from 6 th to 35 days after this pretreat- m ent. Control animals received vehicle alone. Five hours before sampling, each animal received a purse of di[X4Clmethylnitrosamine (2 mg/kg) in order to estimate Or-methylguanine DNA repair capacity (see text). Each group comprised 2 animals and the data are the means of 2 separate analyses made on ~he liver DNA of individual animals. The percentage ~ O6-methylguan~ne removed is based on the amoun.t irdtially farmed in DNA and is calculated from the amount of 7-methylguanin¢ measured using the O¢/7-methylguanine ratio (0.11) as described [2]. DNA synthesis was determined from the [14C]1abelled guanine eluting from Scphadex G10 gel columns used to analyse acid DNA hydrolysates for the presence of methyl pm'ines [1, 2]. (redrawn from Ref. No. 8).

obtained by 24 hours after admin,~tration of the mycotoxin and the response remained elevated for many days (see fig. 1). In these experimerLts

B1OCHIMIE, 1982, 64, n ° 8-9.

tion of D N A so that repair reactions at specific sites in D N A would already be in progress. This early time in the repair process was therefore opti-

Inducibility of hepatic 06-alkylguanine repair in rodents. 771

mal for demonstrating differences between the constitutive and, enhanced levels of repair activity. In the same experiments DNA syitthesis was deter- mined f rom the metabolic incorporation of di [~4C] methylnitrosamine breakdown products into hepa- tic D N A and this was also increased, by these pre- treatments. However, at later times DNA syrtthesis returned to control values whilst 06-methylguanine repair activity was still elevated. Confirmation of this enhancement of repair activity in response to

week after the pretreatment by pulsing the ani- mals with di [~4C] methylnitrosamine (see Methods and above) it was found that a single ½ LDs0 dose (100 mg/kg) elicited a response of similar magnitude to that produced by 15 daily pretreat- merits at 1/2o of the LDso dose (10 mg/kg) , while the response to this lower dose given as a single injectiort was somewhat less (see fig. 2). In these experiments, DNA synthesis was measured by the metabolic incorporation of radioactive breakdown

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FIG. 2. - - The effects of single and multiple doses of diethybfftrosamine on O6-methylguanine repair and DNA synthesis in rat liver. Unlabelled diethylnitrosamine was administered in 0.15M sodium chloride at 10 mg/kg (1/20 LI~) either as a single dose (acute treatment) or as 15 daily (chronic) treatments. A third group received a single 1/2 L I ~ dose (100 mg/kg). Control animals were given an equivalent volume of saline. In each single dose experiment there were 4 animals per group and 2 per group for the chronic pretreatment. Animals were pulsed with di [x4C] methyhaitrosamine in order to estimate O6-methylguanine repair (see legend to fig. 1 )either, 1 day after completion of the chronic treatment or one week after the single dose of unlabeIIled diethylnitrosamine and were then allowed to survive for 6 h .before removing the livers which were pooled for analysis of the DNA. Ratios of O6/7-methylguanine indi- cate the proportion of O6-methylguanine remaining in DNA relative to the control animals and DNA synthesis was determined from [14C] labelled adenine as indicated under figure 1. (redrawn from Ref. No 14).

single doses of aflatoxin B1 has since been obtained by in vitro assay of this ftmctior~ in liver extracts prepared from pretreated arid control animals. As observed in vivo, pretreataTlent with doses of aria- toxin B1 (2 mg/kg) increased this repair activity but it was reduced again to control values after the higher dose of 1 mg /kg [13].

Diethylmtrosamine, given as sil~gle high or low doses will also enhance hepatic 0%methylguanine repair [14]. When this repair was assayed one

BIOCH1MIE, 1982, 64, n ° 8-9.

products (see above) aBd was increased by both the chronic and high single dose pretreatments but not by the lower dose.

The aromatic amide 2-acetylaminoriuorene, given as various single doses (0.74-60.0 mg/kg) has also been shown to enhance hepatic repair of 06-methylguanine at some of the higher dose levels [10]. When 06-methylguanine repair was assayed by the in vitro method, increased repair cart easily b e detected at doses of 2.22 m g / k g and

772 P. 1. O 'Connor and coll.

above in a quasi-dose dependent manner, but when assayed in vivo by pulsing with di [14C] methylni- trosamine (see Methods and above), this response was no t detected below 20 mg/kg. DNA synthesis was estimated from the metabolic incorporation of di [14C] methylnitrosamine breakdown products and in separate e:~periments, by the incorporation of [3H] thyrrfidine. These two parameters gave consistent results indicating that DNA synthesis was increased (3-4 fold) by these treatments but not in a dose c~eperrdent manner.

EJfects of pretreatments and partial hepatectomy in other rodents.

Attempts have beerr made to enhance the hepatic repair of 06-methylguanine by a variety of treat- ments irr mice .[18]. Single toxic doses of either dimethylnitrosamine (0.1-10.0 mg/kg) or AFB~ (0.1-100.0 mg/kg) given to C57B1 mice failed to stimulate the repair system as did exposure to dimethylnitrosamine in the drinking water (10 ppm) for 38 days. The effects of partial hepatec- tomy were also examined. Whereas DNA polyme- rase ~ activity was irrd~ced over a time scale simi- lar to that followed by the increase of DNA syn- thesis, both reaching a peak at about 36 hours, the activity of the 06-methylguanine repair system assayed in liver extracts was initially depressed and thert returned to control values during the post- operative period (0-4 days) when the majority of the cells have passed through at least one cell cycle. The effects of partial hepatectomy on 06- methylguanine repair in the Mongolian gerbil were almost identical to those observed in C57B1 mice [15].

In Syrian hamsters chronic administration of dimethylnitrosamine as 14 daily doses of either 2 mg/kg or 0.25 mg/kg or, once weekly ttoses of 3 mg/kg (s.c.) for 4 or 12 weeks were all ineffec- tive in inducing increased hepatic Or-methylgua - nine repair [16]. Similarly, single doses of 2-ace- tylaminofluorene (20 mg/kg) failed to induce an increased level of this repair activity and 14 daily doses of diethylnitrosamine (10 mg/kg) had no enhancing effect on the repair of 06-ethylguanine.

D i s c u s s i o n .

The experiments described above show that various hepatotoxic treatments are very effective in inducing the 06-methylguani~e repair system in the rat whereas similar treatments are apparently without effect in mice and hamsters. The mecha- nisms involved irr this irrduction process in the rat

are not known but the fact that partial hepatecto- my will also produce this reponse has indicated that the phenomenon may be linked in some way to liver cell proliferation ,[9, 18]. On the other hand the lack of a uniformly close association of increased DNA synthesis and 06-methylguanine repair (see above) may argue to the contrary. Tht~s, in an attempt to resolve some of these uncertainties and to explore the phenomenon fur- ther, the effects of partial hepatectomy have been examined in mice and gerbils and the activity of their 08-methylguanine repair systems was not increased but was indeed reduced during the period when many of the cells were in cycle. These experiments do not exclude the presence of very transient phases of increased repair activity during the cell cycle or a temporal displacement of an enhanced repair process from the period of exten- sive cell proliferation. However, it does appear from our data that in at least 3 rodent species other than the rat, cell proliferation in the liver is not intimately associated with enhanced 06-methyl - guanine repair activity.

Acknowledgements .

This work was supported by grants to the Paterson Laboratories #ore the Cancer Research Campaign. Our thanks are due to Mr J. ,4. Bailey [or preparation of di [14C] methylnitrosamine, Mr B. C. Keenan [or tech- nical assistance and to Ms Gillian ,4. Simpson 1or pre- paration o/ the manuscript.

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Inducibility of hepatic 06-aIkylguanine repair in rodents. 773

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BIOCHIMIE, 1982 , 64, n ° 8-9 .