Mutagenesis, Vol. 15, No. 2, 133-136,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
1-Aminobenzotriazole inhibits acrylamide-induced dominant lethal effects in spermatids of male mice
GSF–Institute of Mammalian Genetics, Ingolstaedter Landstraße 1, D-85764, Neuherberg, Germany and 1 University of Medicine and Dentistry of New Jersey, Newark, NJ 07424, USA
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Abstract |
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Acrylamide (AA) is a germ cell mutagen and induces clastogenic effects predominantly in spermatids of mice. The mechanism of AA clastogenicity has been a matter of dispute. Since the reactivity of AA with DNA is low but is high with proteins containing SH groups, it was suggested that protamine alkylation could be the mechansim of clastogenicity by AA in spermatids. This was substantiated by the observation that the time course of protamine alkylation and dominant lethal effects in spermatids of mice induced by AA was strictly parallel. Another suggestion was that AA may be metabolized by cytochrome P-450 to the epoxide glycidamide (GA), which is then the ultimate DNA-reactive clastogen. This suggestion was based on the similarity of the stage specificity pattern for dominant lethality and heritable translocation induction by AA and GA. To test this latter assumption, 1-aminobenzotriazole (ABT), an inhibitor of P-450 metabolism, was used in the present experiments. Male mice were pretreated with ABT (3x50 mg/kg) on three consecutive days followed by AA treatment (125 mg/kg) on day 4. Parallel groups of animals were treated with AA (125 mg/kg), ABT (3x50 mg/kg) or with the solvent double-distilled water. The experiment was repeated once with slightly varied mating parameters. The results of both experiments showed that ABT inhibited or significantly reduced the AA-induced dominant lethal effects. Thus, the present data support the hypothesis that the AA metabolite GA is the ultimate clastogen in mouse spermatids.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Several reports indicate that acrylamide is a mammalian germ cell mutagen. Dominant lethal mutations were induced by acrylamide (AA) in spermatids of mice and rats (Shelby et al., 1986
; Smith et al., 1986) Heritable translocations were induced by AA in the same germ cell stage as dominant lethal mutations (Shelby et al., 1987; Adler, 1990; Adler et al., 1994). These clastogenic effects were restricted to spermatids while mutagenic effects were also induced by AA in stem cell spermatogonia (Ehling and Neuhäuser-Klaus, 1992).
The stage specificity of the clastogenic response to AA in male germ cells was confirmed by measurement of chromosome breakage by an alkaline elution procedure (Sega and Generoso, 1990). Whole body autoradiography for the distribution of [14C]AA showed an accumulation of radioactivity in the mouse testis and the progressive disappearance of radioactivity from the testis to the epididymis paralleled the movement of the spermatids from the testes to the epididymis as mature sperm (Marlowe et al., 1986). It was demonstrated that the stage specificity coincided with protamine alkylation by AA, rather than DNA binding (Sega et al., 1989). Recently, it was shown that the epoxide glycidamide (GA), which can be formed from AA by cytochrome P-450-mediated metabolism, shows the same pattern for induction of dominant lethal effects and heritable translocations as AA (Generoso et al., 1996). Thus, the authors suggest that GA is the DNA-reactive moiety of AA clastogenicity in mouse spermatids and not AA itself. To test this hypothesis, dominant lethal experiments were performed with pretreatment of male mice with 1-aminobenzotriazole (ABT) followed by treatment with acrylamide. In rats, ABT was reported to destroy hepatic and renal P-450. If indeed AA is metabolized by P-450 to GA, which in turn is the ultimate clastogen in mouse spermatids, one would expect pretreatment with ABT to inhibit AA-induced dominant lethal effects in these germ cell stages. This expectation was confirmed by the present study.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The experiments were conducted with (102/ElxC3H/El)F1 mice bred in the colony of the GSF (Neuherberg, Germany). Throughout the experiment, the animals were kept in plastic cages and fed with pellet food and chlorinated water ad libitum. Animal quarters were maintained at 20°C, 50% relative humidity on a 12 h light/dark cycle.
ABT (CAS no. 1614-12-6; Sigma) was dissolved in physiological saline. AA (CAS no. 79-06-1; Sigma) was dissolved in double-distilled water. The chemical solutions were prepared daily at concentrations to ensure correct dosing with injected volumes of 10 ml/100 g body wt. Animal treatment was always started at 10:00 am. The treatment regimens are listed in Table I.
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In the first dominant lethal experiment, 40 males were i.p. injected with 50 mg/kg body wt ABT on three consecutive days. Of these males, 20 were i.p. injected with 125 mg/kg body wt AA on day 4. Parallel groups of 20 males were i.p. injected on the same day with 125 mg/kg body wt AA alone or with the solvent.
In the second experiment 68 males were treated with 50 mg/kg body wt ABT on three consecutive days. Of these, 34 males were injected with 125 mg/kg body wt AA on day 4. Parallel groups of 34 males were treated with 125 mg/kg body wt AA alone or with the solvent.
In the first experiment, all males were mated 6 h after treatment at a ratio of 1:2 to untreated virgin females of the same stock, aged 12–16 weeks. In the second experiment 30 males of each treatment group were mated 6 h after treatment at a ratio of 1:1 to untreated virgin females of the same stock and age. Females were replaced every week for a total of 4 weeks in the first experiment and every 4 days for a total of four mating intervals in the second experiment.
Females were inspected for the presence of a vaginal plug every morning. Females with plugs were removed from the mating pans. At pregnancy days 14–16 the females were killed and uterus contents were inspected for live and dead implants (Bateman and Epstein, 1971; Ehling et al., 1978). The numbers of dead implants were compared between control and treated mating groups using the 
2 test (Sachs, 1974). Dominant lethality (DL) was expressed as % 
Four males of each treatment group were killed 1 week after treatment and testes and epididymes were dissected for spermiograms. Both caudae epididymes from each male were isolated and several incisions were made. The epididymes were then placed in Eppendorf cups with 300 µl of fetal calf serum and the sperm were allowed to actively leave the epididymes for 1 h at 35°C. Sperm concentration, mobility and and morphology were determined under the microscope using a hematocytometer according to the WHO manual for the examination of human semen (WHO, 1992). Per animal, 100 sperm each from two slides were oberserved for their mobility over five or six micro-windows per slide. Data for individual animals were averaged. Spermiogram parameters were compared between different treatment groups and the solvent control group using the t-test (Sachs, 1974).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The results of the first dominant lethal experiment are shown in Table II
and Figure 1A and B. Significant increases in the percentage of dead implants or the rate of dead implants per female were found during the first and second mating week after treatment of the males with AA. This confirms the previous data (Shelby et al., 1986), even though the magnitudes of the effects were not identical. The quantitative difference may be due to different mouse stocks and different stocks of AA being used by Shelby et al. and in the present experiment. In the ABT + AA treatment group, no dominant lethal effect could be observed. Thus, our expectation that ABT pretreatment would inhibit the AA-induced dominant lethal effect was confirmed. The data from the first experiment fully support the hypothesis by Generoso et al. (1996) that GA is the genetically active metabolite of AA.
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A second experiment was performed to confirm these results. To obtain a better coverage of the critical sensitive days, the mating ratio in the second experiment was 1:1 and the mating intervals were 4 days. Furthermore, four animals per treatment group were randomly selected to perform spermiograms 1 week after treatment to elucidate the reduced fertility rates observed in the first experiment for the AA and ABT + AA treatment groups.
The results of the second experiment are shown in Table III and Figure 2A and B. Significant increases in the percentage of dead implants and the rates of dead implants per female were observed during the second, third and fourth mating intervals after AA treatment of the males. Similar to the first experiment, there was no dominant lethal effect in the second mating interval after pretreatment of the males with ABT. During the third mating interval, the rate of dead implants per female and the percentage of dead implants in the ABT + AA group was significantly increased over the concurrent solvent control and the ABT group. However, it was also significantly lower than in the AA group. In the fourth mating interval, both the AA and the ABT + AA group showed dominant lethal effects which were of the same order of magnitude. Thus, some dominant lethality was induced despite the ABT pretreatment, suggesting that the inhibition of P-450 metabolism was not complete. Alternatively, an additional mechanism may be involved in AA-induced clastogenicity in spermatids, such as protamine alkylation by AA itself, as postulated by Sega et al. (1989).
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The results of the spermiograms are shown in Table IV
and Figure 3. It became obvious that the percentage of fast moving sperm was dramatically reduced by AA treatment and this effect was not inhibited by ABT pretreatment. In fact, ABT alone significantly reduced the percentage of fast moving sperm compared with the solvent control. Concomitantly, the percentages of immobile sperm were increased. Sperm counts and sperm morphology remained unaffected.
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Reduced pregnancy rates and reduced numbers of total implants were observed during the first mating intervals in both dominant lethal experiments. The reduced fertility can be explained by mobility impairment of sperm by AA, ABT + AA and ABT treatments. This indicated a third mechanism of AA reactivity, namely inhibition of motor proteins by AA itself.
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Notes |
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2 To whom correspondence should be addressed.Tel: +49 89 3187 2302; Fax: +49 89 3187 2210; Email: adler{at}gsf.de
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Adler,I.-D. (1990) Clastogenic effects of acrylamide in different germ-cell stages of male mice. Banbury Rep., 34, 115–131.
Adler,I.-D., Reitmeir,P., Schmöller,R. and Schriever-Schwemmer,G. (1994) Dose response for heritable translocations induced by acrylamide in spermatids of mice. Mutat. Res., 309, 285–291[Medline]
Bateman,A.J. and Epstein,S.S. (1971) Dominant lethal mutations in mammals. In Hollaender,A. (ed.), Chemical Mutagens. Principles and Methods of Their Detection. Plenum Press, New York, NY, Vol. 2, pp. 541–568.
Ehling,U. and Neuhäuser-Klaus,A. (1992) Reevaluation of the induction of specific-locus mutations in spermatogonia of the mouse by acrylamide. Mutat. Res., 283, 185–191.[Medline]
Ehling,U.H., Machemer,L., Buselmaier,W., Dycka,J., Frohberg,H., Kratochvilova,J., Lang,R., Lorke,D., Müller,D., Peh,J., Röhrborn,G., Roll,R., Schulze-Schenking,M. and Wiemann,H. (1978) Standard protocol for the dominant lethal test on male mice. Arch. Toxicol., 39, 173–185.[Medline]
Generoso,W.M., Sega,G.A., Lockhardt,A.M., Highes,L.A., Cain, K.T., Cacheiro,N.L.A. and Shelby,M.D. (1996) Dominant lethal mutation, heritable translocations and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide. Mutat. Res., 371, 175–183.[Web of Science][Medline]
Marlowe,C., Clark,M.J., Mast,R.W., Friedman,M.A. and Waddell,W.J. (1986) The distribution of [14C]acrylamide in male and pregnant Swiss-Webster mice studied by whole-body autoradiogaphy. Toxicol. Appl. Pharmacol., 86, 457–465.[Medline]
Sachs,L. (1974) Angewandte Statistik, 6th edn. Springer, Berlin, Germany.
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Sega,G.A., Valdivia Alcota,R.P., Tangcongco,C.P. and Brimer,P.A. (1989) Acrylamide binding to the DNA and protamine of spermatogenic stages in the mouse and its relationship to genetic damage. Mutat. Res., 216, 221–230.[Web of Science][Medline]
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WHO (1992) Manual for the Examination of Human Semen Samples and Sperm–Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK, pp. 3–27.
Received on August 9, 1999; accepted on October 19, 1999.
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