Arch. Toxieol. 34, 7~--75 (1975) 9 by Springer-Verlag t975

Short Communications

Dominant Lethal Test in Mice with 6-Mercaptopurine M. S c h u h e Schencking a n d H. F r o h b e r g Institut fiir Toxikologie der Firma E. Merck, Darmstadt, Germany Received March 24, 1975

Abstract. Cytostatic 6-mercaptopurine, which is clastogenic in somatic cells of different mammals, has a slight potency for inducing dominant lethals in NMRLmice by affecting early stages of spermatogenesis. Key words: 6-Mercaptopurine -- Dominant Lethals -- NMRI-Mice. Zusammen]assung. ])as Cytostatikum 6-Mereaptopurin, das fiir Somazellen verschiedener Sauger clastogen wirkt, induzierte in friihen Stadien der Spermatogenese bei NMRI-Mi~nsen einen leiehten Anstieg dominanter Letalmutationen. 8chli~sselwSrter: 6-Mereaptopurin -- dominante Letalmutationen -- NM~I-Mause. Cytostatic 6 - m e r c a p t o p u r i n e (6-MP) p r o v e d to be clastogenic i n

man

(Nasjleti

et al., i966) a n d i n different a n i m a l species (Frohberg et al., i 9 7 3 ; H o l d e n et al., i973). Little is k n o w n however, a b o u t its c a p a c i t y to induce d o m i n a n t lethals. T h e following s t u d y was m e a n t as a n o r i e n t a t i n g i n v e s t i g a t i o n of d o m i n a n t lethal effects i n comparison to former cytogenetic studies i n rats, mice, a n d Chinese h a m s t e r s (Frohberg et al., 1975).

Methods 6-mercaptopurine (Merck No. 5742) was administered intraperiteneally %o male mice (NMRI-EMD-SPF; age i0 to i2 weeks; weight: 37 to 40 g) at the following dose levels: I • t2.5; I • 25; i • 50; i • 125 and I • 250 mg/kg with single application, and 5 • 25 and 2 • 125 mg/kg given on successive days. The injection volume was 1 ml/kg, with aequeons earboxymethyl cellulose suspension used as a carrier. Control animals received one single injection of the carrier suspension. 10 males were treated per group. 24 hrs after the last treatment, males were paired with 3 untreated virgin females each (age: 8 to i0 weeks; weight: 28 to 29 g) for I week, followed by further 7 successive pairing periods of 7 days. Females were sectioned 16 days after the first day of pairing and the number of live embryos, dead implants, and abortions were determined per animal. Except the percentage of pregnant females and the percentage of dead implants, as referred to the total number of implantations per group (mutagenie index), that were tested for significance with the X2-test, significant differences between parallel control and dose group for all further parameters were determined using the "tied linear rank test", a non-parametric test developed by Krauth (1971).

Results and Discussion A f t e r i n j e c t i o n of t h e h i g h l y toxic dose of 250 m g 6-MP/kg, 6 of 10 of t h e t r e a t e d males died. Because of t h e low n u m b e r of a n i m a l s left i n this g r o u p t h e corresponding results are o m i t t e d .

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M. Sehulze Schencking and H. Frohberg

Table I. Dominant lethal test with 6-mereaptopurine in male NMRI-miee (tip. application)

Percentageo/pregnant/emales Time of Percentage of pregnant females pairing 0 mg/kg i2.5 mg/kg 25 mg/kg 50 mg/kg 125 mg/kg 5 • 25 (week after mg/kg treatment)

2 • 125 mg/kg

1. 2. 3. 4. 5. 6. 7. 8.

80.0 91.7 b 75.0 62.5 91.7 69.6 c 62.5 66.7

70.0 86.7 83.3 73.3 76.7 66.7 76.7 76.7 a i ~ dead.

93.3 66.7 76.7 80.0 73.3 73.3 73.3 73.3 b 2 c~c~dead.

73.3 80.0 80.0 80.0 73.3 66.7 73.3 73.3

73.3 66.7 66.7 73.3 70.0 76.7 66.7 63.3

80.0 70.0 70.0 77.8" 74A 70.4 81.5 77.8

76.7 90.0 80.0 53.3 60.0 60.0 53.3 66.7

c I further c~ dead.

T h e percentage o/pregnant animals was n o t influenced b y 6-1VIP; t h e v a l u e s o f t h e t e s t g r o u p s c o r r e s p o n d e d to those o f t h e controls w i t h i n t h e r a n g e of biological v a r i a t i o n (Table 1). F o r t h e number o/ implants p e r fertile female a significant r e d u c t i o n in c o m p a r i s o n to t h e p a r a l l e l control was f o u n d for t h e dose g r o u p s l • 50 a n d l • 125 m g / k g in t h e 6 t h week. H o w e v e r , w i t h r e s p e c t to t h e high control v a l u e (12.55 i m p l a n t a t i o n s p e r fertile female) a n d to t h e lack o f dose d e p e n d e n c e , t h e biological relevance of this finding for this p a i r i n g p e r i o d a n d for a d d i t i o n a l s t a t i s t i c a l significance in t h e dose g r o u p i • 125 m g / k g in t h e second week is d o u b t f u l . P a r a l l e l t o this, a s t a t i s t i c a l l y significant r e d u c t i o n of t h e number o/ living embryos p e r fertile female a p p e a r e d in t h e second week for t h e dose g r o u p i • ~25 m g / k g a n d in t h e 6th week for t h e dose g r o u p l • 50 m g / k g . F o r t h e s a m e reasons m e n t i o n e d , t h i s finding also seems t o be w i t h o u t biological relevance. I n t h e 5 t h week o f p a i r i n g in t h e dose groups with single a p p l i c a t i o n of 6-MP a t e n d e n c y t o w a r d a d o s e - d e p e n d e n t increase in t h e number o/dead implants per ]ertile/emale is t o be seen (Table 2). A s t a t i s t i c a l l y significant increase for this p a r a m e t e r was o b s e r v e d a l r e a d y in t h e 4 t h week for t h e dose g r o u p I • 25 m g a n d in t h e 5 t h week for t h e dose groups t • 125, 5 • 25, a n d 2 • 125 m g / k g . A d d i t i o n a l s t a t i s t i c a l significance in t h e 3 r d week for t h e dose g r o u p 2 • 125 mg/kg, however, seems to be w i t h o u t biological relevance, since t h e corresponding control v a l u e ( z 0.2 d e a d i m p l a n t s p e r fertile female) is v e r y low as c o m p a r e d to t h e control v a l u e o f o t h e r p a i r i n g periods. I n all dose groups h i g h n u m b e r s of d e a d i m p l a n t a tions o n l y a p p e a r after d a y 23/24, i n d i c a t i n g t h a t t h e corresponding stages of s p e r m a t o g e n e s i s a l r e a d y are affected b y 6-M-P. A f t e r 2 injections of t 2 5 m g 6 - M P / k g t h e m e a n v a l u e of d e a d i m p l a n t a t i o n s p e r fertile female in t h e 5th week o f p a i r i n g was n e a r l y two times t h e one f o u n d in t h e same p e r i o d a f t e r one single a p p l i c a t i o n o f 125 m g / k g . On t h e o t h e r h a n d t h e effect o f 5 injections of 25 m g / k g was m u c h m o r e p r o n o u n c e d t h a n t h a t of one single injection of 25 m g / k g a n d even s u r p a s s e d t h e effect seen after one single i n j e c t i o n o f t h e s a m e t o t a l dose of i25 mg/kg.

Dominant Lethal Test in Mice with 6-Mercaptopurine

73

Table 2. Dominant lethal test with 6-mercaptopurine in male NMRI-mice (i.p. application) Time of mean number of dead implantations per fertile female pairing (week affcr (~mg/kg 12.5 mg/kg 25 mg/kg 50 mg/kg 125 mg/kg 5 • 25 treatment) mg/kg

2 x t25 mg/kg

t. 2. 3. 4. 5. 6. 7. 8.

0.88 0.55 0.72~ 0.67 2.18 a 0.50 0.53 0.56

1.00 0.42 0.20 0.50 0.6t 0.80 0.57 0.83

0.71 0.80 0.I7 0.7t 0.73 0.91 0.77 0.59

0.68 0.50 0.42 0.88 ~ 1.05 0.60 0.73 0.86

0.95 0.55 0.40 0.73 tA0 1.17 0.40 0.68

0.67 0.33 0.24 0.76 t.30 a 0.37 0.82 0.43

0.43 0.59 0.50 0.69 t.83 a 0.50 0.44 1 A0

Time of % dead implantations per group (mutagenie index) pairing (weekafter 0mg/kg 12.5mg/kg25mg/kg 50mg/kg t25mg/kg 5• 25 treatment) mg/kg

2 x t25 mg/kg

1. 2. 3. 4. 5. 6. 7. 8.

8.57 4.96 6.449 5.65 18.39~ 4.50 4.91 4.86

8.75 3.57 t.75 5.16 5A3 6.37 5.24 7.60

6.45 6.75 1.53 5.74 6.40 8.00 6.23 5.88

6.82 4.23 3.72 7.07 8.68 4.94 6.t8 7.79

8.33 4.66 3.57 6.43 9.39 1t.30 4.06 5.83

5.80 3.14 2.20 6.90 tl.26~ 3.24 7.00 3.46

4.29 5.08 4.67 5.85 15.35~ 4.15 4.22 9.09

Time of mean number of dead implantations per fertile female pairing mean number of implantations per fertile female (weekafter 0mg/kg 12.5mg/kg25mg/kg 50mg/kg 125mg/kg 5• 25 treatment) mg/kg

2• 125 mg/kg

I. 2.

0.09 0.03

0.06 0.07

0.I0 0.04

0.08 0.05

0.05 0.03

0.05 0.05

0.09 0.05

3. 4. 5. 6. 7. 8.

0.02 0.06 0.06 0.06 0A0 0.08

0.02 0.06 0.06 0.11 0.06 0.08

0.04 0.07 0.09 0.05 0.07 0.08

0.03 0.07 0.09 0.14 0.04 0.05

0.03 0.07 0AI 0.03 0.07 0.03

0.04 0.07 0A5a 0.04 0.04 0.t0

0.06~ 0.06 0.21~ 0.05 0.04 0.05

a significant for p ~ 5 %.

E x c e p t for t h e 3rd p a i r in g period, w h e r e a statistical significance w i t h o u t biological r e l e v a n c e was f o u n d for t h e dose g r o u p r ecei v i n g 2 x i 2 5 m g 6-M-P/kg, t h e mutagenic index was significantly increased o n l y for t h e 5 t h week o f p ai r i n g for t h e dose groups t x t25, 5 x 25, a n d 2 x 125 m g / k g (Table 2). Si m i l ar t o t h e p a r a m e t e r " n u m b e r o f d e a d i m p l a n t s p e r fertile f e m a l e " h o w e v e r , in t h e 5 t h week a t e n d e n c y o f d o s e - d e p e n d e n t increase of t h e m u t a t i o n i n d e x was seen for single application, b e g i n n i n g a l r e a d y w i t h t h e dose o f i2.5 m g / k g .

74

M. Sehulze Schencking and H. Frohberg

A significant increase of the quotient dead implantations per fertile female also total implantations resulted in the 5th week of pairing for the dose groups 5 • 25 and 2 • i25 mg/kg (Table 2). As is the case for the parameters mentioned before, a tendency of doserelated increase was observed for this week with single application of 6-MP, too. For 6-MP, t h a t in a dose of i • 25 mg/kg i.p. showed clear clastogenic action in bone marrow cells of rats, mice, and Chinese hamsters, with single application under the circumstances given, statistically significant effects as far as the induction of dominant lethals are concerned, were only demonstrated with a dose of 125 mg/kg for week 5. A tendency toward ~ dose-dependent increase of dominant lethals, however, was observed for the parameter ~ number of dead implantations per fertile female and for the mutation index, beginning with the dose of t2.5 mg/kg and for the parameter ~ dead implants per fert. $ from 25 mg/kg total implants per fert onwards. Due to the low number of animals in these orientating tests this tendency could not be ascertained b y statistical methods. 5 injections of 25 mg/kg, however, resulted in a clear, statistically significant increase of dominant lethal mutations in week 5. The findings with single application of 6-MP partly agree with those of R a y et al. (t973) who after i.p. injections of 6-MP doses from 50 to 200 mg/kg reported significant effects in the 5th and 6th week in CDl-mice. In our experiments with NMRI-mice, however, dominant lethal effects were nearly exclusively confined to the 5th pairing period. I n contrast to m a n y known mutagens such as T E P A (Epstein et al., 1969), M E T E P A (Epstein et al., i969 ; Wallenstein et al., i974), MMS, and EMS (Ehling et al., 1968) t h a t for instance induce dominant lethals b y affecting spermatozoa and spermatids, or like TEM mainly b y affecting mid-spermatids (Arnold et al., 1974; Matter et al., 1973), 6-MI~ in the doses tested mainly causes damage to the early stages of spermatogcnesis, namely in primary spermatocytes according to Oakberg's timing of spermatogenesis in mice (1960). A more precise definition of the stage is not possible, since Oakberg's investigations were done in another strain of mice and also because a retardation of spermatogenesis might be caused b y the cytostatic action of 6-M-P. The dominant lethal mutations observed b y R a y et al. (1973) in CDl-mice for the 6th week also indicate that, similar to the case of T F P (Petersen et al., 1973), spermatocytes and spermatogonia as well might be sensitive to 6-MP. I n contrast to an oral treatment with 40 mg 6-MP/kg on 7 successive days, t h a t did not result in dominant lethal effects in CDt-mice (Ray et al., i973), 5 intraperitoneal injections of 25 mg/kg induced clear, statistically significant effects in the 5th week of pairing in the NMRI-mouse strain studied. Abbreviations. 6 - M P - 6-mercaptopurine, T E P A = tris(l-aziridinyl)-phosphine oxide, M E T E P A ~. tris(2-methyl-i-aziridinyl)-phosphine oxide, M M S = methyl methanesulfonate. E M S ~ ethyl methanesulfonate, T E M = triethylenemelamine, T F P = triflupromazine. References Arnold, D.W.,Kennedy, G. L.,Keplinge~M. L.: Compa~son of the mutagenic effects of triethylenemelamine on rats and mice in the dominant lethal assay. Mutation Res. 96, 459--460(1974)

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Ehling, U. H., Cumming, R. B., Malling, H. V. : Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5, 417--428 (1968) Epstein, S. S., Bishop, J. : Mutagenic effects of TEPA and METEPA in mice. Genetics 61, t6 (1969) Frohberg, H., Bauer, A.: Mutagenicity testing under toxicological aspects. ArzneimittelForsch. (Drug Res.) 23, 230--236 (i973) Frohberg, H., Schulze Schencking, M.: In vivo cytogenetic investigations in bone marrow cells of rats, Chinese hamsters and mice treated with 6-mercaptopurine. Arch. Toxikol. 33, 209-- 224 (1975) Holden, H. E., Ray, V. A., Wahrenburg, M. G., Zelenski, J. D. : Mutagenicity studies with 6-mercaptopurine: I. Cytogenetic activity in vivo. Mutation Res. 20, 257--263 (1973) Krauth, J. : A locally most powerful tied rank test in a Wilcoxon situation. Ann. Math. Star. 42, 1949--1956 (1971) Matter, B. E., Generoso, W. M.: Dose-response studies on the induction of dominant lethal mutations in post-spermatogonial stages of the mouse treated with triethylenemelamine (TEM). Mutation Res. 21, 41--42 (1973) Nasjleti, C. E., Spencer, H. H. : Chromosome damage and polyploidization induced in human peripheral leukocytes in vivo and in vitro with nitrogen mustard, 6-mercaptopurine, and A-649. Cancer Res. 26, 2437--2443 (1966) Oakberg, E. 1%: Irradiation damage to animals and its effects on their reproductive capacity. J. Dairy Science, Suppl. 43, 54--67 (1960) Petersen, K. W., Legator, M. S.: Dominant lethal effects of Triflupromazine in Hybrid CsD2Fz/J Mice. Mutation Res. 17, 87--92 (1973) Ray, V. A., Holden, H. E., Ellis, J. E., Hyneck, M. L. : The mutagenic activity of 6-mercaptopurine in host-mediated and dominant-lethal assays. Mutation Res. 21, 231 (i973) Wallenstein, S., Brobst, J. Bagdon, R. E.: Establishment of a positive control and statistical techniques for the dominant lethal test in mice. Mutation Res. 26, 458--459 (t974) Dr. Harald Frohberg E. Merck Institut ffir Toxikologie D-6100 Darmstadt 2, P.O. Box 4119 Federal Republic of Germany