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RADIATION INDUCED GENE MUTATIONS ALTERING ENZYMES AND DOMINANT CATARACT MUTATIONS IN THE MOUSE

Objective

EXTRAPOLATION OF THE EXPECTED FREQUENCY OF RADIATION-INDUCED GENETIC DAMAGE TO HUMANS ASSUMES EQUAL SENSITIVITY OF MAN AND MOUSE TO THE INDUCTION OF MUTATIONS AND A SIMILAR EFFECT OF FACTORS AFFECTING THE INDUCED MUTATION RATE IN MAN AS IS DETERMINED IN THE MOUSE.
THE DOMINANT CATARACT MUTATION TEST PROVIDES FOR THE FIRST TIME, THE POSSIBILITY TO TEST THE BASIC ASSUMPTIONS FOR THE EXTRAPOLATION FROM MICE TO MAN. IF THE ASSUMPTION IS CORRECT THAT MICE AND MAN ARE EQUALLY SENSITIVE, THEN ONE WOULD EXPECT THAT THE INDUCED MUTATION RATE IN MOUSE AND GOLDEN HAMSTERS IS SIMILAR. THEREFORE, THE DOMINANT CATARACT MUTATION TEST WILL BE EMPLOYED TO MAKE THE FIRST SPECIES COMPARISON OF THE RADIATION-INDUCED GENE MUTATION RATE TO DOMINANT ALLELES IN THE LABORATORY MOUSE MUS MUSCULUS AND THE GOLDEN HAMSTER MESOCRICETUS AURATUS.

The objectives of the project are to determine the mutation rates at loci controlling erythrocyte enzyme activity in offspring of irradiated mice, in a combined experiment scoring specific locus mutants as positive control in the same offspring and to characterise genetically and biochemically the recovered enzyme activity mutants.

Results obtained with the recessive specific locus test with mice have been used as a basis for the estimation of the genetic risk of man exposed to radiation. However, the number of loci screened with the specific locus test is a very small fraction of the total genome, the results represent only 1 class of mutations (recessive visibles), and large numbers of animals are required to screen mutations. These disadvantages can be partially overcome by combining the scoring of specific locus mutations with the screening of dominant cataract mutations, protein charge mutations and enzyme activity mutations. The screening for enzyme activity mutations was developed in our laboratory as a new method for the detection of biochemical mutations in germ cells of mice. The principle is based on the determination of specific activity in erythrocyte enzymes of offspring from treated animals. For the specific locus test defined strains of mice have to be used. It is known from other organisms that the mutation frequency is dependent on the genetic background. Therefore, it is an essential advantage of our method that no special strains of mice are required.

It can be concluded that more studies on the genetic effect of radiation in mice are needed in order to gather additional information for an improved comparison between mutation rates obtained with test systems screening different genetic endpoints and to investigate the influence of the genetic background of the experimental animal and expand the database of radiation experiments with biochemical methods.

The objective of the programme was the determination of mutation rates in offspring o f irradiated mice by detecting enzyme activity mutants, in a combined experiment scoring simultaneously specific locus mutants.

The key aims of the project are to provide a better basis for the extrapolation of animal data to man. Experiments were undertaken to study the following assumptions required in procedures to estimate the radiation genetic risk in man from experimental data:
linear relationship of radiation dose and the induction of dominant mutations;
equality of the radiation doubling dose for different genetic mutational endpoints;
equality of the sensitivity to mutation induction of oocytes and spermatogonia;
that results for (102/E1xC3H/E1) F1 hybrid mice are representative in estimating the induced mutation rate in the mouse;
the sensitivity to mutation induction by radiation is similar in the germ cells of different species of mammals.

Dose response analysis in mice:
The frequency of recovered specific locus and dominant cataract mutations indicate that radiation induces per locus, by a factor of 24, more recessive specific locus mutations than dominant cataract mutations. We interpret this to be due to the quality of deoxyribonucleic acid (DNA) damage ultimately resulting in a recessive or a dominant allele. A recessive mutation represents a loss of functional gene product. Radiation, which mainly results in DNA deletions, would be expected to induce a large proportion of loss mutations. Dominant alleles, by comparison, mainly represent alterations of a functional gene product which in the heterozygote interfere with the normal gene product. These mutations would be expected to be more likely due to point mutations. Thus the greater likelihood that radiation induces recessive mutations rather than dominant mutations.

The data may be analysed to estimate the radiation doubling dose for the induction of recessive specific locus or dominant cataract mutations. The doubling dose is defined as that dose which results in an induced mutation rate equal to the spontaneous mutation rate. It is calculated as the ratio of the fitted regression parameters, a/b. For the speci fic locus mutation results the doubling dose is estimated to be 0.7 Gy whereas for the dominant cataract mutation results the doubling dose is estimated to be 2.4 Gy. This observation is most important for 2 reasons. First, the doubling dose method of genetic risk estimation has assumed the doubling doses for different genetic endpoints to be the same and can be based upon the doubling dose estimated for the specific locus alleles. Our present results suggest that the different genetic endpoints may have different doubling doses, which violates the basic assumption of the doubling dose method as presently employed. Should the doubling dose method be used to estimate the genetic risk to man, an estimate of the doubling dose for each genetic endpoint would be required. Second, the radiation doubling dose has been estimated for induced mutations recovered in the offspring of the survivors of the Hiroshima and Nagasaki atomic bombings and it has been suggested that the doubling dose in humans is higher than that in the mouse. The relevance of the mouse experimental data for extrapolation to humans has been questioned. However, the human doubling dose for dominant genetic traits has been compared with the mouse doubling dose for recessive specific locus mutations. Our present results suggest that the comparison is confounded by a difference in the genetic endpoints screened. Thus, differences observed can not be concluded to be species differences. In fact, the doubling dose which we estimate for dominant cataract mutations in the mouse is similar to the doubling dose for dominant genetic traits in humans and supports the assumption that mouse germ cell mutagenicity data are the most relevant experimental data for extrapolation to humans.

Oocyte experiments in mice:
Results showed that radiation was effective in inducing dominant cataract as well as recessive specific locus mutations. Comparative results at the same doses for spermatogenic stages of the mouse are inc luded and indicate the induced mutation rate in oocytes to be similar to the radiation induced mutation rate in spermatogonia. These results support an assumption now required in the estimation of the human radiation genetic risk, ie the sensitivity of oocytes to the inductionof dominant mutations by radiation is similar to the sensitivity of spermatogonia.

Mouse strain comparisons:
Results indicate that the mutation rate to specific locus alleles following radiation is dependent upon the genotype of male treated. A similar observation was not observed for dominant cataract alleles.

The sensitivity to mutation induction presently observed does not comply with our experimental hypothesis based on radiation toxicity, dominant lethals and DNA repair. Until these discrepancies can be clarified we maintain the cautious interpretation that no clear cut demonstration of an effect of genotype on the sensitivity to mutation induction in germ cells of the mouse was observed and assume results from the hybrid mice are representative.

Mouse hamster species comparison:
The critical assumption in an extrapolation from experimental results in the mouse to a human genetic risk is a similarity of the sensitivity to mutation induction in germ cells of mammals. Since comparable experimental results for mouse and man are not possible, this assumption can only be tested in an array of mammalian laboratory species. Results for radiation induced dominant cataract alleles in the golden hamster and the mouse are compared. The first results for the induction of comparable mutations in the germ cells of different mammalian species indicate no observable difference in the radiation induced mutation rate which supports the basic assumption critical for an extrapolation of experimental results to man.

Our most important observation is that the critical assumption in the doubling dose method of genetic risk estimation as presently employed, ie the radiation doubling dose is similar for all genetic endpoints, may not be correct. We suggest that extrapolation procedures to estimate dominant deleterious mutations in humans should be based on experimental results for similar genetic endpoints in animals.

The Cat-2no (formerly Nop) cataract of the mouse is a nuclear opacity which is inherited as an autosomal dominant gene. Among the proteins of the lens, gamma crystallins were diminished as compared to the wild type. The reason for the decreased amount of these proteins in the Cat-2no lenses was investigated using biochemical analysis including protein analysis, determination of metabolite concentrations and enzyme activities. Furthermore, sequencing analysis and regulation studies of gamma crystallin genes were undertaken.

A set of spontaneous (Cat-2no, formerly Nop: nuclear opacity; Cat-2sc, formerly Scat: suture cataract) and radiation induced cataract mutations in mice (Asc-1, Asc-2, Cat-2t, Cat-3vao, Coc, Pcs-2 and Tcm) and rat (cat) were investigated for biochemical alterations in the cataractous lenses. Mutants from the Cat-2 allelic group were selected for further analysis of their protein composition and also for histological examination.

Experiments found that for wild type lenses, an enhanced oxidised glutathione (GSSG) content could be observed in females as compared to males. Such a sex effect could not be detected for the superoxide dismutase (SOD) activity.

7 cataract mutations were investigated for effects on osmotic alterations in the lenses. The water content is enhanced only in lenses of homozygous mutants from the Cat-2no and Cat-2sc line. Additionally, it is enhanced both in the heterozygous and homozygous Cat-2t and Cat-3vao lenses.

In no mutant line investigated could a decreased sodium potassium adenosine triphosphatase (ATPase) activity be found similar to the causative factor in the Nakano mutant line leading to an increase of lenticular water content and swelling of lens fibre cells.

The mutants Cat-2no, Cat-2sc and Cat-2t were found to exhibit alterations in the lenticular protein composition.

The mutants derived from the different alleles of the Cat-2 locus were analysed histologically. In sections of the Cat-2sc mutants , a hydropic swelling of lens epithelium was observed in the heterozygotes, whereas in homozygous mutants interruptions and degeneration of lens fibres as well as clefts and folds of the capsule were observed.

The most intensive studies among our different cataract mutant lines have been performed concerning the Cat-2no mutants. At the protein level, the cataractous Cat-2no lenses exhibit a reduced content of gamma crystallins as demonstrated by polyacrylamide gel isoelectric focussing (PAGIF). Northern blots probed with complementary deoxyribonucleic acid (cDNA) specific for alpha crystallin, beta crystallin and gamma crystallin genes suggested a reduced transcription of the gamma crystallins genes. In contrast, the transcription of alpha crystallins and beta crystallins appeared to be similar in wild type and the mutants.

The murine gamma E-crystallin gene was isolated from a genomic deoxyribonucleic acid (DNA) library and sequenced from 650 basic pairs (bp) in the 5' region down to the second exon.

Factors from mouse and rat lenses were identified binding to a 208 bp AluI/NcoI fragment of the 5' region from the murine gamma E-crystallin gene which includes some regulatory elements (2 thymine adenine thymine adenine (TATA) boxes, a rudimentary cytosine adenine adenine thymine (CAAT) box) and the putative transcriptional and translational start site.

Experiments have been initiated to extend the control sample sizes for mutation rate studies in the mouse and hamster as well as to determine the mutation rate following 2 plus 2 Gy (0.75 Gy/min; 24 h fractionation interval) irradiation in male and female mice and hamsters. However sample sizes are still too small to make meaningful comparisons.

The dominent cataract test has been employed in one mouse in multiple endpoint test procedures to systematically compare the induced mutation rate to visible, dominent visible, electrophoretic and enzyme activity alleles. Over 85 independant dominent cataract mutations have been recovered. A dominant cataract mutation recovered in a 3 plus 3 Gy spematogonial irradiation experiment, shown to be X-linked in genetic confirmation crosses, has been mapped to the distal region of the X-chromosome between jimpy and hypophosphatemia.
2 independant mutations have been recovered in irradiation experiments and shown to be alleles. Linkage studies have located the dominant cataract gene on mouse chromosome 10, 3 to 4 cM from one marker S1.

Selected fitness paramters (homozygous viability, segregation ratio and relative fitness) were characterized for a total of 19 radiation induced enzyme activity mutations and compared with results for 33 ethylnitrosourea (ENU) induced enzyme activity mutations. The ratio of homozygous lethal mutations was higher following radiation mutagenic treatment than in ENU experiments. Segregation ratio was reduced in 12 out of the 19 radiation induced mutations studied while the segregation ratio of ENU induced mutations at the corresponding loci was normal. The mean relative fitness for radiation induced and ENU induced enzyme activity mutations was distributed close to the expected value of 1.
Two methods are available to estimate the human radiation genetic risk. The direct approach estimates the frequency of induced dominant deleterious mutations in man based on experimental results in the mouse for induced dominant mutations. An estimation of the frequency of induced chromosomal aberrations is extrapolated from a combination of mouse and primate data. The second approach, the indirect method, estimates the frequency of dominant deleterious mutations in man based on the estimated doubling dose determined in the mouse for recessive specific locus mutations or on a combination of mouse data for the most relevant genetic endpoints.

Both extrapolation procedures require the assumption that there are no species differences in the sensitivity to radiation mutation induction. The indirect approach requires the additional assumption that the doubling dose estimated in the mouse for induced specific locus mutations is representative for other genetic endpoints.

In humans, epidemiological results to estimate the genetic risk due to radiation exposure are available for the offspring of exposed survivors of the atomic bombings at Hiroshima and Nagasaki. Traits scored were embryonic mortality, juvenile mortality, juvenile cancer, reciprocal translocation, enzyme charge or enzyme activity alteration, and sex chromosome aneuploidy. The probability of observing an increased mutation rate in the exposed group was low since the human database was meagre by animal experimentation standards due to a low parental generation exposure and a small population size. Despite these limitations Neel and coworkers have estimated the radiation doubling dose in man, defined as that dose of radiation which induces as many mutations as occur spontaneously per generation.

For the contract period 1990 to 1991, the mouse and hamster dominant cataract and enzyme activity mutation rate group is to be extended until mutations are recovered for both genetic endpoints in both species to provide a more accurate estimation of the spontaneous mutation rate. These data are critical in estimating the radiation doubling dose for these endpoints in germ cells of mammals. The mutation rate in spermatogonia following two exposures of 2 Gy will be determined for dominant cataract and enzyme activity alleles in mouse and hamster, as well as experiments to measure the radiation induced mutation rate in oocytes for both genetic endpoints in both species.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

GSF - FORSCHUNGSZENTRUM FUER UMWELT UND GESUNDHEIT GMBH
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