Mutagenesis, Vol. 15, No. 1, 39-43,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
A comparison of enzyme activity mutation frequencies in germ cells of mice (Mus musculus) and golden hamsters (Mesocricetus auratus) after exposure to 2 + 2 Gy 
-irradiation
GSF–National Research Center for Environment and Health, Institute of Mammalian Genetics, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany
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Abstract |
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The radiation-induced germ cell mutation rate has been investigated in two species of mammals. Mice and golden hamsters of both sexes were exposed to 2 + 2 Gy
-irradiation with a 24 h fractionation interval and mated to untreated partners. In mice, specific locus mutations were examined as positive controls and the obtained mutation rates (per locus and gamete x10–5) were 51.4, 10.1, 13.6 and 17.4 for irradiated post-spermatogonia, spermatogonia and 1–7 and >7 days post-treatment oocytes, respectively. Offspring of mice and golden hamsters were screened for activity alterations of 10 erythrocyte enzymes coded by at least 14 loci. The observed mutation rates per locus per gamete x10–5 for treated post-spermatogonial stages, spermatogonia and oocytes 1–7 and >7 days post-treatment were 6.5, 1.5, 8.8 and 7.0, respectively, for mice and 16.7, 0, 7.6 and 0, respectively, for golden hamsters. There is a significant difference for mutation rates in mouse oocytes 1–7 days post-treatment compared with the control. No differences in the frequencies of mutations in the various germ cell stages could be observed between mice and golden hamsters. A critical assumption for the extrapolation of experimental mutagenesis studies to humans is that no species effects exist in sensitivity to mutation induction by irradiation. Our results do not contradict this assumption. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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For the human population it is essential to make estimates of the genetic risk arising from exposure to harmful noxa to orientate the frame of legislative measures for preventing an increase in inherited diseases. Such estimates are possible by extrapolating experimental animal data to the human situation. They are limited by certain assumptions, i.e. that there is no difference in sensitivity of germ cells from different mammalian species to irradiation or chemically induced mutations. Essentially all experimental data for the estimation of radiation-induced genetic risk are from the laboratory mouse. The mouse as a mammal shares various similarities with man in germ cell development. However, differences are known, such as sensitivity of oocytes to killing by irradiation (Russell,W.L., 1977
). Hence there are various dissimilarities due to ontogenesis between mouse and man. Therefore, it is important to investigate whether another experimental animal species can serve as a surrogate for man.
An intraspecific comparison between different mouse strains (Favor et al., 1987; Pretsch et al., 1994) showing some strain to strain variability in the frequency of radiation-induced mutations could not demonstrate an effect of genotype on the radiation-induced frequency of mutations in germ cells. The method employed was the screen for specific locus, dominant cataract and enzyme activity mutations. Screening for enzyme activity mutations offers a number of advantages. It has a comparative sensitivity to the mouse specific locus test which allows statistically robust conclusions to be made. The screen is not dependent on precisely matched strains or species, allowing comparative studies to be designed. Finally, the mutational end-points are models of human inborn errors of metabolism. Therefore, this method fulfils the prerequisites for studying radiation-induced mutation frequencies in different species, i.e. the mouse and the golden hamster.
In this study we report results of experiments screening for enzyme activity mutations in F1 offspring from male and female mice and hamsters exposed to 2+2 Gy -irradiation. In mice, fractionating the radiation dose results in an optimal yield of induced mutations (Russell,W.L., 1963). The experiments with mice were carried out together with a screen for specific locus mutations as a positive control for the radiation treatment.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Treatment
All animals used were obtained from the colonies maintained in Neuherberg. Eight- to 10-week-old (102/ElxC3H/El)F1 mice (Mus musculus) and approximately 3-month-old golden hamsters (Mesocricetus auratus) were exposed to a dose of 2 + 2 Gy with a 24 h fractionation interval. Mice were irradiated with 137Cs
-rays (acute irradiation with 0.4 Gy/min); hamsters were exposed to 60Co -rays (acute irradiation with 0.3–0.4 Gy/min). The experiments had been officially approved by the Regierung von Oberbayern (ref. nos 211-2531-60/90 and 211-2531-61/90). Immediately after the second irradiation, the animals were mated with an untreated partner. Irradiated male or female (102/ElxC3H/El)F1 mice were mated with a test stock partner, 10–13 weeks old. The test stock is homozygous recessive at seven loci: agouti, a; tyrosinase-related protein 1, Tyrp1 (formerly brown); tyrosinase, Tyr (formerly albino); pink-eyed dilution, p; myosin 5a, Myo5a (formerly dilute); bone morphogenetic protein 5, Bmp5 (formerly short ear); endothelin-B receptor, Ednrb (formerly piebald). These seven loci are distributed among five autosomes. The Tyr and p loci are linked on chromosome 7 and the Myo5a and Bmp5 loci are linked on chromosome 9.
Specific locus test with mice
The specific locus test was performed essentially as described by W.L.Russell (1951) and Ehling (1978). Offspring were categorized as to treated spermatogenic or oogenic stage, sexed and inspected for recessive phenotypes characteristic of mutations from wild-type allele of one of the seven specific loci. Presumed specific locus mutations were subjected to an allelism test. In those cases where presumed mutants were sterile or died prior to confirmation, they were included as mutants when they exhibited a definitive phenotype.
Enzyme activity mutations
F1 offspring were screened for genetic activity variants of 10 enzymes coded by at least 14 loci (Table I). The procedures for sample preparation and enzyme activity determinations have been described in detail elsewhere (Charles and Pretsch, 1986, 1987). Individuals with enzyme activities (expressed as units per g haemoglobin) deviating by at least 3 SD of the mean of the wild-type population were considered as outliers. If this enzyme activity alteration was confirmed in a second blood sample, the presumed mutant was outcrossed to inbred strain C3H/El mice or wild-type hamsters and the offspring screened for enzyme activity alteration for genetic confirmation. The enzyme activity mutations were maintained by back-crossing to inbred C3H/El mice or wild-type hamsters. To determine homozygous viability, mutant heterozygotes were crossed inter se and offspring were classified for enzyme activity. Individuals which expressed an enzyme activity alteration more extreme than the heterozygotes were suspected of being homozygous for the mutant allele and genetically tested by out-crossing to wild-types. If no offspring expressing a phenotype more extreme than the heterozygote were recovered in inter se crosses, the mutation was suspected to be either homozygous lethal or completely dominant. To distinguish between these two alternatives, 20 offspring expressing the mutant phenotype were recovered from the inter se crosses of heterozygotes and genetically tested to determine if they were homozygous or heterozygous. A mutation was concluded to be homozygous lethal if all 20 offspring genetically tested were shown to be heterozygous (Favor et al., 1989). Litter size at weaning and transmission ratio were determined for the mutants.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Choice of radiation dose
In an earlier study (Favor et al., 1987
; Pretsch et al., 1994) we estimated the radiation-induced intraspecific mutation frequencies to recessive visible, dominant cataract and enzyme activity alleles in germ cells of four different mouse strains. Male mice of the genotypes AKR, BALB/c, (102/ElxC3H/El)F1 and DBA/2 were exposed to 3 + 3 Gy irradiation with a 24 h fractionation interval and mated to test stock females. The observed mutation rates in the offspring per locus per gamete x10–5 for treated spermatogonia were 6.8, 4.9, 2.5 and 1.3 for enzyme activity mutations, 8.6, 24.1, 22.8 and 31.4 for specific locus mutations and 0.7, 0.9, 0.6 and 2.5 for cataract mutations, respectively. Some variability from strain to strain in the frequency of radiation-induced mutations was observed. However, there was no consistent effect of genotype on the frequency of induced mutations and it was concluded that no effect of genetic background exists for the four mouse genotypes tested. Originally it was planned to extend this intraspecific comparison for the same radiation dose to an interspecific one with mice and hamsters. Preliminary studies indicated that a dose of 3 + 3 Gy led to permanent sterility in male hamsters. Therefore, we decided on a treatment of 2 + 2 Gy for this interspecific comparison.
Species sensitivity of X-ray induction of dominant lethals and translocations
Léonard et al. (1972) examined the genetic radiosensitivity of different mouse strains using the rate of induction of dominant lethals in spermatozoa as a criterion. There are striking differences between the mouse strains in the total amount of X-ray-induced dominant lethality (both pre- and post-implantation losses). A comparison of these data with those available for golden hamsters, guinea pigs and rabbits (Lyon, 1970) indicates that the variation between different mouse strains with respect to radiation-induced dominant lethals is as large as that between mouse and the other species.
X-irradiation of female mice causes a reduction in the mean number of implants per female and in the ratio of live embryos to total implants; this reduction is more noticeable in conceptions occurring in the third week than in the first week after irradiation (Searle and Beechey, 1974). In contrast, in golden hamsters and guinea pigs, the yields of dominant lethals for second oestrus matings are lower than those for first oestrus matings (Lyon and Smith, 1971).
For X-ray induction of translocations in post-meiotic male germ cells, the mouse is more sensitive than the rabbit, the latter more sensitive than the guinea pig and the guinea pig more sensitive than the golden hamster (Cox and Lyon, 1975). After spermatogonial irradiation, the rank order of sensitivity is mouse > rabbit > guinea pig 
golden hamster (Lyon and Cox, 1975).
Radiation-induced specific locus mutations in mice
The irradiation-induced specific locus mutation rates of males and females were significantly different from the control rates in the examined germ cell stages (Table II), thus confirming the effectiveness of the radiation treatment. A higher sensitivity to irradiation of post-spermatogonia compared with spermatogonia (P = 0.0079, two-tailed) was observed, whereas no difference in sensitivity was observed between mature and maturing oocytes. In irradiated females the mutants were conceived on day 1 (Tyr), day 2 (Tyrp1 and Ednrb), day 3 (p), day 4 (Ednrb), day 22 (Ednrb) and day 24 (Ednrb) post-irradiation. The distribution of mutations among the loci shows over-representation at the Ednrb locus for oocytes and spermatogonia (Table III).
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Radiation-induced enzyme activity mutations and mutation rates in mice and golden hamsters
Altogether 32 632 mice and 15 662 hamsters were examined for enzyme activity mutations. The results of parental treatment with 2 + 2 Gy
-irradiation are presented in Table IV. However, it must be admitted that the number of tested offspring is small for some germ cell stages (especially for post-spermatogonial stages in hamsters).
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The calculation of enzyme activity per locus mutation frequencies for both tested species has been undertaken based on cited references for the number of loci screened for mice. Mice are genetically well studied and due to their close relationship to golden hamsters we assume that homologous genes are responsible for the enzyme activity alterations in both species. Fourteen loci affect expression of the 10 tested enzymes in erythrocytes (Table I
).
Interestingly, several G6PD mutations were recovered in mutagenicity experiments which express elevated activity and are autosomally transmitted (Charles and Pretsch, 1987; this study). Two loci have been located at central chromosome 1 and proximal chromosome 9 (unpublished data) which may represent regulatory genes for the G6pdx structural locus. Additional experiments have to clarify whether these loci are identical to Gdr1 and Gdr2 (Table I). Concerning mutants with elevated PGAM activity, we have mapped two loci to mouse chromosomes 19 and 4, respectively (Pretsch and Favor, 1996a,b). Three additional loci causing increased PGAM activity have been identified at chromosomes 2, 8 and 12, but mutants were not recovered in radiation mutagenesis experiments (unpublished data). For that reason, the estimation of 14 loci for the 10 enzyme activities screened (Table I) may be a preliminary one and an underestimate.
Assuming 14 loci responsible for the screened enzyme activity alterations, the observed mutation rates per locus per gamete x10–5 for treated post-spermatogonial stages, spermatogonia and 1–7 and >7 days post-treatment oocytes were 6.5, 1.5, 8.8 and 7.0, respectively, for mice and 16.7, 0, 7.6 and 0, respectively, for golden hamsters. Although mutation rates vary, mouse to golden hamster comparisons for the four tested germ cell stages indicate no significant differences. With the exception of mouse oocytes 1–7 days post-treatment, there are no significant differences for mutation rates in all germ cell stages tested compared with the control value. In contrast to the specific locus results we suggest that the experimental sizes are too small for enzyme activity mutations and the results are inconclusive rather than negative.
Description of enzyme activity mutations
Mutations causing a decrease as well as mutations causing an increase in enzyme activity were found in offspring of mice and golden hamsters (Tables V and VI). Recovered enzyme activity mutants were back-crossed to wild-type animals and litter sizes and transmission ratios determined from the results of these crosses. Genetic analyses showed most of the mutations to be homozygous lethal (mice, seven of 12; golden hamsters, three of three). This is consistent with the observation that radiation-induced mutations are more likely deletions (Rinchik et al., 1994; Johnson et al., 1995).
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Both mouse and golden hamster LDH mutations (LDH 144 and LDH 2128, respectively) present similar phenotypic properties: blood LDH activity in heterozygotes is reduced to 55–60% of that of wild-type, whereas almost no activity decrease can be observed in the heart. Within the scope of characterizing all our mouse LDH mutations, the homozygous viable mutant LDH 144 (Ldh1a12Neu) has been studied in more detail (Pretsch et al., 1998
). In inter se crosses there is a significant reduction in the number of homozygotes observed compared with the number expected from the Mendelian 1:2:1 ratio for wild-type and heterozygous and homozygous mutants, respectively, suggesting a reduced viability of homozygotes. Homozygous animals displayed a strong increase in spleen volume and alterations in blood parameters, which are typical characteristics of haemolytic anaemia. Sequencing of the coding region of the Ldh1 gene revealed a T/A
C/G transition at codon 241 (ATCACC) in exon 6. The three GR mutant lines, two in mice (GR 4241 and GR 4413) and one in golden hamsters (GR 3664), exhibit a reduction of ~50% wild-type GR activity in the heterozygous condition. Linkage studies with MIT markers demonstrated a mutation of the Gr1 structural gene at chromosome 8 in mice.
In three mutant lines (G6PD/GR 1101, GAPDH/TPI 4437 and GAPDH/TPI 4300), the activities of two enzymes are altered, from which we deduce that the affected enzymes are metabolically closely connected. In the line G6PD/GR 1101 both G6PD and GR are elevated to ~150% of wild-type activity in heterozygotes. Litter size in back-crosses as well as transmission of heterozygotes is strongly reduced, thus we conclude that the mutation is homozygous lethal. Heterozygotes are smaller in size than wild-type siblings, show a belly spot, small eyes and sometimes a curly tail, a forehead spot, paralysed hind legs or spina bifida. Mapping with microsatellites demonstrated a deletion at chromosome 1 in region C4–C5.
Two mutations cause a combined activity alteration of TPI and GAPDH: in the murine line GAPDH/TPI 4437 activities are decreased to ~50% of wild-type, connected with reduced litter size in back-crosses and sterility of males. In contrast, the golden hamster line GAPDH/TPI 4300 exhibits an activity increase to ~150% of wild-type. As a homology to mice, we could demonstrate a linkage of the responsible gene with microphthalmia in golden hamsters (unpublished data).
Conclusion
From our results we could not demonstrate a difference in enzyme activity mutation rates in mouse and golden hamster following irradiation exposure, suggesting no species differences in the sensitivity to radiation mutation induction. In summary, these results give some degree of confidence for the extrapolation from laboratory mammal experimental results to an estimation of human radiation genetic risk, i.e. no species differences in the sensitivity to mutation induction by radiation. The recovered mutations from such mutagenesis studies provide genetic variation with which to characterize the affected genes.
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Acknowledgments |
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The competent technical assistance of Margit Ellendorff, Sybille Frischholz, Brigitta May, Ursula Schaefer and Sylvia Wolf is appreciated. The studies were supported in part by contract no. BI6-156 from the Commission of the European Communities.
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Notes |
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1 To whom correspondence should be addressed. Tel: +49 89 3187 2642; Fax: +49 89 3187 3099; Email: pretsch{at}gsf.de
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References |
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Received on April 29, 1999; accepted on September 6, 1999.
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