Studies of factors influencing the mutagenicity of EMS in mice

245

Mutation Research, 37 (1976) 245--252 © Elsevier/North-Holland Biomedical Press

STUDIES OF FACTORS INFLUENCING THE MUTAGENICITY OF EMS IN MICE

E.R. SOARES Research Triangle Park, PO box 12233, North Carolina 27709 (U.S.A.)

(Received February 13th, 1976) (Revision received June 17th, 1976) (Accepted June 25th, 1976)

Summary Three different routes of administration of ethyl methanesulphonate (EMS) (i.p., oral, i.t.) were compared for their relative efficiencies in the induction of d o m i n a n t lethal effects. Included in the comparisons between oral and i.p. injections, was a preliminary study into the existence of strain differences in sensitivity to EMS between C3D2 F1 hybrid mice and strain DBA/2J mice. No route of administration dependent effects were found between oral and i.p. injections regardless of the test animal used. I.t. injections of EMS did n o t induce d o m i n a n t lethal effects. One treatment related strain difference was observed.

Introduction In vivo mutagenicity testing in mice has been restricted to the use of random-bred mice or a few inbred strains, and with few exceptions has considered only the effects of intraperitoneal (i.p.) injections of the test compounds. S i n c e human populations are genetically heterogeneous and since exposure of humans to various compounds, either toxic or mutagenic, is usually by routes other than i.p., it is extremely important that the varying effects of route of administration and genetic background be evaluated. In experiments oriented toward the development of in vivo test systems and which employ reference mutagens as the test compound, it is important to use the most effective and practical means of delivery of the c o m p o u n d to the test animal. Additionally, such considerations are important in routine testing experiments where reference mutagens such as EMS'may be used as positive controls. Experimentation designed to determine the varying effects of such reference mutagens when delivered by different routes of administration would b e meaningful both in terms of producing relevant data and more reliable data.

246 The evaluation of the significance of genetic background to mutation induction is also important. The restriction of tests to one inbred strain or to random bred animals might cause spurious effects in terms of estimating genetic risks. Furthermore, use of genetically standardized animals would permit accurate genetic replication of results. Genetic standardization of mutagenicity tests in mice will improve our understanding of the relationship between genotype and mutation induction. In this study we have compared the relative effects, as measured by the dominant lethal test, of i.p., gavage, and intratesticular injections of EMS. In addition a preliminary investigation into the varying sensitivities to EMS of two different genotypes has been conducted. Materials and methods Oral versus intraperitoneal

Hanks' balanced salt solution (HBSS) was used as the carrier for EMS. The EMS solution was prepared immediately prior to injection. Dosage (200 mg/kg of body weight of EMS) was based on the mean weight of ten randomly selected males. Eight groups of test animals were used; four groups of 15 ten-weekold strain DBA/2J males and four groups of 15, ten-week-old C3D2 F~ (C3H/ HeJ X DBA/2J) hybrid males. Each male received 0.25 cc of HBSS either orally or by i.p. injection or 0.25 cc of the solution of EMS and HBSS either orally or by i.p. injection. On the day of treatment, males were injected and caged individually. Fortyeight hours after treatment each male was paired with two strain C57BL/6J females for seven nights at the end of which time these females were replaced by two additional females. This procedure was repeated for a third mating period. At the end of the mating periods, females were held two per cage. On day 17 they were killed by cervical dislocation and their uteri were examined for the presence of live embryos and for evidence of postimplantation deaths. Intraperitoneal versus intratesticular

In this test distilled water rather than HBSS was used as a solvent for the EMS. EMS solutions were prepared immediately prior to injection. I.p. injections were carried out as was described above. Intratesticular (i.t.) injections were done in the following manner. The test animals were anesthetized with Avertin and a microsyringe was surgically inserted into the testes. Dosages were based on testicular weight and were calculated using the mean weights of the testes from 10 randomly selected males. The EMS was administered in a volume of 10 pl to each testis. This constituted a dose of 200 mg/kg of testis weight or 0.021 mg of EMS per testis. In total, six groups of 15, 12-week-old strain DBA/2J mice were used in the study. Of these six groups, four constituted the controls: Group I, distilled water i.p. ; Group II, Avertin i.p., and distilled water i.t.; Group III, Avertin i.p. ; and Group IV no treatment. Groups V and VI received 200 mg/kg of body weight of EMS (i.p.) and 200 mg/kg of testis weight of EMS (i.t.) respectively. Following injection, males were caged individually for 48 h. At the end of this period each male was paired with two, ten-week-old strain C57BL/6J females

247

for seven nights. Females were replaced by additional females at weekly intervals. Males of Groups II, III, IV and VI were paired with females during weeks 1, 2, 4 and 6 after treatment. Groups I and V received females during weeks 1 and 2 after treatment. All other experimental procedures were carried out as outlined above. Statistical procedures used in comparisons between control and treated series animals and between routes of administration were similar for the two experiments. In each experiment, preliminary analyses showed no significant male effects and hence, the individual females were taken as the experimental units. Three basic criteria were used in evaluating these data: (1) the percent of fertile matings based on the total number of paired females; (2) the percent of fetal deaths per pregnant female; and (3) the number of implantations per pregnant female. Differences in fertility were analyzed using Fisher's exact test for 2 X 2 tables. The remaining two criteria were analyzed using the Mann-Whitney U test. In addition, analysis of variance techniques were employed in the first experiment to determine how fetal death and implants were affected by route of administration, strain, treatment and interactions among these factors. Differences in fertility were tested by a direct analysis of proportions. Results Oral versus intraperitoneal

In the analyses of the effects of EMS on fertility, neither DBA/2J nor C3D2 F, showed any significant reduction in the percent of fertile matings (Table I). Table II shows the means and standard errors for the number of implants per pregnant female. Treatment dependent reductions in implants were noted in the C3D2 F1 groups for weeks l a n d 2. Mann-Whitney U Test analyses of these results showed the observed reduction in week two for the i.p. group to be significant (P < 0.01). All other differences were not statistically significant. Corn-

TABLE I THE P E R C E N T OF F E R T I L E M A T I N G S BASED ON THE T O T A L NUMBER O F P A I R E D F E M A L E S Strain

C3D2 F l

DBA/2J

Treatment

a

Route

0

oral

200

oral

0

i.p.

200

i.p.

0

oral

200

oral

0

i.p.

200

i.p.

a EMS 200 m g / k g o f b o d y w e i g h t .

Week post-treatment

1

2

16 53.3(~-6) 7 0 . 0 ( 21 ~)

3

18 62.1(~) 13 43.3(~-6)

21 70.0(~--~) 22 73.3(g-~)

14 46.7(~-~) 12 4o.o(T6)

21 ) 75.0(~-~ 25 83.3(~-d)

'

7 6 . 7 ( 23 ~) 17 58.6(~-~)

60.0(~-~)

20 71.4(~) 21 70.0(~) 18

64.3(~-~) 18

13

44.8(~--~-)

15 50.0(~-6) 15 50.0(~) 14

46.7(~) 16 59.3(~-~) 23 76.7(~) 19 63.3(~-6) 17 56.7(~)

248 T A B L E II THE MEAN NUMBER PLANTS) Strain

C3D2 F 1

DBA/2J

± S.E. O F I M P L A N T S P E R P R E G N A N T

Treatment a

Route

0

oral

200

oral

0

i.P.

200

i.p.

0

oral

200

oral

0

i.p.

200

i.p.

F E M A L E ( T O T A L N U M B E R O F IM-

Week post-treatment 1

2

7.81 -~ 0.51 (125) 6 . 8 6 ± 0.51 (144) 7.95 ± 0.60 (167) 7.00 ± 0.54 (154)

8.50 + (153) 7.31 • (95) 9.21 + (129) 6.42 ± (77)

8 . 4 8 -+ 0 . 2 4 (178) 6.60 ~ 0.50b (165) 8 . 5 7 -+ 0 . 4 8 (197) 6.35 • 0.74b (108)

8.50 + (170) 6.24 ± (131) 9.33 + (167) 5.83 + (105)

3 0.54 0.74 0.39 0.88 b

0.43 0.51b 0.21 0.60b

8.85 ± 0.30 (115) 9 . 2 0 -+ 0 . 3 8 (138) 8.33 ± 0.56 (125) 7 . 6 4 ~- 0 . 6 3 (107) 9.25 ± 0.30 (148) 7.30 + 0 . 4 6 b (168) 9.16 ± 0 . 2 4 (174) 7.82 ~ 0.55 c (133)

a EMS 200 m g / k g of b o d y weight. b p ~ 0.01. c p ~ 0.05.

parisons between groups among DBA/2J males also showed an EMS related reduction in implants. These reductions occurred in all three weeks post-treatment and in all cases were statistically significant. Mean values and standard errors for the percent of fetal mortality are shown in Table III. Both C3D2 Fl and DBA/2J mice showed statistically significant, EMS related increases in postimplantation fetal mortality in weeks 1 and 2 post treatment. None of the observed differences for week 3 proved statistically significant. Although comparisons of differences between routes of administration revealed no significant route-dependent effects, one strain effect, and one strain by treatment effect was noted. In weeks 2 and 3 significant straiu effects on fertility were apparent. In these weeks, C3D2 F, males impregnated significantly fewer females (P < 0.01, P < 0.05 respectively) than DBA/2J males. The strain by treatment interaction was revealed in analyses of total implantations. In week 3, the DBA/2J group showed a significantly larger (P < 0.06) treatment related reduction in implants than did the C3D2 F, group. All other strain by treatment, treatment by route, or route by strain differences were not significant. Intraperitoneal versus intratesticular No statistically significant differences were found between any of the control groups for any of the parameters tested. Hence, data from all four controls have been combined. Table IV shows the mean percent of fertile matings for the three test groups

249 T A B L E III T H E M E A N P E R C E N T -+ S.E. O F P O S T I M P L A N T A T I O N D E A T H S PER P R E G N A N T F E M A L E (TOT A L NUMBER OF DEAD IMPLANTS) Strain

Treatment a

Route

Week post-treatment 1

C3D2 F 1

DBA/2J

0

oral

200

oral

0

i.p.

200

i.p.

0

oral

2

14.97 (18) 37.84 (50) 14.13 (18) 28.76 (38)

± 6.16 ± 6.1 5 b -+ 4.89 -+ 4 . 6 4 b

6 . 8 4 -+ 1 . 8 4

(12) 200

oral

0

i.p.

200

i.p.

37.98±6.15 b (51) 5.92 +- 1.57 (12) 38.69 ± 7.05 b (42)

10.79 (17) 49.53 (46) 10.19 (14) 41.71 (30)

3 +- 3.56

10.19 (12) 9.15 (12) 16.28 (21) 13.50 (14)

-+- 7.55 b ± 2.54 -+ 9 . 0 0 b

1 1 . 6 7 +- 3.20 (19) 3 3 . 0 7 ± 5.67 b (39) 7.00 ± 1.85 (12) 4 1 . 5 3 ± 5.61 b (39)

± 3.35 ± 3.25 ± 2.61 ± 4.89

1 1 . 3 5 +- 2.71 (17) 7.52±2.07 (13) 1 3 . 4 7 +- 2.86 (23) 16.98 ± 4.05 (14)

a EMS 2 0 0 m g / k g of b o d y w e i g h t . b P < 0.01.

(control, i.t., i.p.). Analyses of differences in fertility revealed only one significant difference (week 2, EMS i.p.). Tables V and VI show the means and standard errors for total implantations and percent post-implantation deaths. Comparisons made with respect to the number of implants showed a significant reduction in weeks 1 and 2 for the group injected with EMS i.p. No clearcut reduction was apparent for the i.t. injection group. Similarly, the only obvious treatment effect regarding post-implantation deaths was among the EMS i.p. injection group. Here a significant increase (P < 0.01) is found in both weeks 1 and 2. A slight increase in fetal death was observed in week 1 for the i.t. injection group, but was not statistically significant. T A B L E IV T H E P E R C E N T OF F E R T I L E MATINGS BASED ON T H E T O T A L NUMBER OF P A I R E D FEMALES Treatment a

0 c

Route b

i.p., i.t. d

Week post-treatment 1

2

4

6

82.6(~6)

6S) 73.9(~ 20 74.1(~-~)

200

i.t. d

98 83.8(1-T~) 23 76.7(~-6 )

lOS 89.0(1-T~) 24 80.0(~-~)

200

i.p.

83.3(~0)

50.0(~-~)1S e

21 70.0(~-~)

a EMS 2 0 0 m g / k g o f b o d y w e i g h t . b i.p., i n t r a p e r i t o n e a l ; i.t., i n t r a t e s t i c u l a r . c T h e s e f i g u r e s i n c l u d e f o u r g r o u p s ; (1) distilled w a t e r (i.p.); (2) distilled w a t e r (i.t.) a n d A v e r t i n (i.p.); (3) A v e r t i n (i.p.) ; (4) No t r e a t m e n t . d F o r all i.t. i n j e c t i o n s , d o s e s are b a s e d o n t e s t e s w e i g h t . e p < 0.01.

250 TABLE V T H E M E A N N U M B E R + S.E. O F I M P L A N T S P E R P R E G N A N T PLANTS) Treatment a

o c

Route b

i.p., i.t. d

200

i.t d

200

i.p.

F E M A L E ( T O T A L N U M B E R O F IM-

Week post-treatment 1

2

4

6

7.89 ± 0.19 (773) 7.74 ± 0.52 (178) 6.08 ± 0.52 e (152)

8.27 ± 0.22 (868) 8.29 ± 0.30 (199) 5.53 -+ 0 . 5 4 e (83)

8.01 ± 0 . 2 6 (569) 7.71 ± 0 . 3 0 (162)

8.00 + 0.24 (520) 7 . 7 5 -+ 0 . 3 5 (155)

a EMS 200 mg/kg of body weight. b i.p., i n t r a p e r i t o n e a l ; i.t., i n t r a t e s t i c u l a r . c T h e s e f i g u r e s i n c l u d e f o u r g r o u p s : (1) d i s t i l l e d w a t e r ( i . p . ) ; (2) d i s t i l l e d w a t e r (i.t.) a n d A v e r t i n (i.p.); (3) A v e r t i n ( i . p . ) ; ( 4 ) N o t r e a t m e n t . d F o r all i.t. i n j e c t i o n s , d o s e s are b a s e d o n t e s t e s w e i g h t . e p < 0.01.

TABLE VI T H E M E A N P E R C E N T ± S.E. O F P O S T I M P L A N T A T I O N TAL NUMBER OF DEAD IMPLANTS) Treatment a

0 c

Route b

i.p., i.t. d

200

i.t. d

200

i.p.

DEATHS PER PREGNANT

FEMALE (TO-

Week post-treatment 1

2

4

6

9.14 ± 1.18 (73) 13.56 + 3.36 (21) 3 6 . 5 6 +- 3 . 8 8 e

1 3 . 7 6 + 1.47 (111) 9 . 1 6 ± 2.11 (19) 49.28 ÷ 8.17 e

10.51 + 1.54 (61) 11.00 ± 3.00 (17)

10.60 + 1.58 (56) 11.19 ± 4.32 (16)

(46)

(39)

a EMS 200 m g / k g of b o d y weight. b i.p., i n t r a p e r i t o n e a l ; i.t., i n t r a t e s t i c u l a r . c T h e s e f i g u r e s i n c l u d e f o u r g r o u p s : (1) d i s t i l l e d w a t e r ( i . p . ) ; (2) d i s t i l l e d w a t e r (i.t.) a n d A v e r t i n ( i . p . ) ; ( 3 ) A v e r t i n (i.p.) ; ( 4 ) N o t r e a t m e n t . d F o r all i.t. i n j e c t i o n s , d o s e s a r e b a s e d o n t e s t e s w e i g h t . ep< 0.01.

Discussion The dominant lethal and mutagenic effects of intraperitoneal and gavage injections of EMS have been reported earlier [2,5,14,15]. The present investigation has considered the relative efficiencies of these two modes of injection, as well as the effect of intratesticular injections of EMS in inducing dominant lethal events. In addition we have examined the relative sensitivities to EMS of two different mouse genotypes. The results obtained in these studies have demonstrated that both oral and i.p. delivery of EMS are equally effective in inducing increased frequencies of dominant lethals. Furthermore, although the present study differs in many respects (strain of mice used, batch of EMS used and time and place of experiment) with other studies of the dominant lethal ef-

251 fects of i.p. injections of EMS, for both oral and i.p. injections our results appear to be consistent with those of others [2,5,14,15]. In contrast, intratesticular injections of EMS gave no evidence of an induced dominant lethal effect. Cumming et al. [1], have described in detail the metabolic fate in mice of EMS when injected i.p. Their data indicate a rapid uptake of EMS by the circulatory system and a subsequent rapid dispersal of the mutagen to various tissues including the germ cells. Similar data for EMS administered either orally or by i.t. injection are not available. Since our data indicate no difference between oral and i.p. injections, it may be that oral injections allow for an equally rapid uptake and distribution of the EMS to the germ cells. Furthermore, since EMS is base labile [12] it might be presumed that it would not be subjected to rapid breakdown while in the acid environment of the stomach. Hence, absorption of the EMS through the stomach, soon after injection, and prior to its entrance into the more basic environment of the small intestine could allow for a rapid dispersal of the mutagen throughout the body. The present studies gave little evidence of EMS-induced effects following i.t. injections. Here, dosage was based on testicular weight and hence, only small amounts of EMS were injected in necessarily small volumes (10 pl) of solvent. In light of the results obtained, it is possible that the EMS was removed from the testes and distributed throughout the animal in extremely small concentrations. If this is so, the germ cells of interest (sperm or spermatids) may have been exposed to a concentration of mutagen, the effects of which were not detectable by the dominant lethal methodology. Two examples of strain differences were apparent in these experiments. The first of these differences was in fertility and was neither treatment nor route of administration related. Such variations between strains are c o m m o n and were not unexpected [6]. In contrast to these, the significant differences observed regarding mean number of implantations were treatment related and were indicative of the trend toward increased sensitivity on the part of DBA/2J males to EMS for this parameter. Strain and species differences or variations in sensitivity to toxicants, including radiation have been well documented [8,9,11,13]. Green and Meier [7] and, more recently, Roberts and Vogel [10] and Festing [3] have discussed the importance of the relationship between the genotype of an animal and the response of that animal in pharmacological, toxicological and mutagenicity testing. Clearly such a relationship is important to the development of reliable and efficient, in vivo mammalian test systems for environmental mutagens. However, only a few comparisons between strains of mammals with respect to their differing susceptabilities to mutagenic damage have been reported [4,16]. Although only preliminary in nature, our data could reflect such differences and emphasize the complexities involved in the development of such assays. Acknowledgement The author wishes to express his appreciation to Dr. J.K. Haseman for advice concerning the statistical treatment of the data. I thank Dr. W. Sheridan for his review of the manuscript and his thoughtful suggestions. Portions of this investigation were conducted at the Jackson Laboratory with the technical assis-

252

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