Recessive Specific-locus Mutations Research Articles

Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice.

The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice. Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU). The cumulative dose for each treated animal was 300 mg ENU/kg body weight. Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells. Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus). Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)). Each of the four lacI mutant offspring carried a different mutation. Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition. Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells. These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation.

Mutagenic activity of 2-amino- N6-hydroxyadenine in the mouse spot test

The base analogue 2-amino- N 6-hydroxyadenine (AHA) was mutagenic in the spot test in (T × HT)F 1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 × 10 −3 spots of genetic relevance) than that of mitomycin C (16 × 10 −3), ethylnitrosourea (6.8 × 10 −3) and cyclophosphamide (6.4 × 10 −3) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice.

Ionizing radiation and genetic risks IV. Current methods, estimates of risk of Mendelian disease, human data and lessons from biochemical and molecular studies of mutations

This paper is aimed at a synthesis of conclusions and concepts from the first three papers of this series and an inquiry of their relevance to the estimation of the risk of autosomal dominant and X-linked diseases in man, due to exposure to ionizing radiation. For a population under conditions of continuous irradiation, the doubling-dose method (DD method) enables the prediction of the excess risk of dominant and X-linked diseases at equilibrium. Per unit dose, this quantity is the product of the natural prevalence of these diseases (assumed to be 10,000/10 6 livebirths) and the reciprocal of the DD. The DD currently used is 1 Gy and is based primarily on data on the induction of recessive specific-locus mutations in male mice. The estimate of risk to the first generation is derived from that at equilibrium; the figure is about 15% of the equilibrium value (i.e., 15 cases/10 6 livebirths/cGy). with the direct method, the first-generation risk of dominant disease is estimated using data on the induction of dominant skeletal and cataract mutations in male mice and a number of correction factors. The estimates are about 10–20 cases and 0–9 cases, respectively, for irradiation of males and females, per 10 6 livebirths/cGy. In the Japanese studies, no significant adverse genetic effects, attributable to exposure of the parents to the atomic bombs, could be demonstrated with respect to any of the endpoints used. Most of the latter are clinically and socially relevant but mutationally insensitive. On the basis of these data, Neel and colleagues have estimated that the gametic DD for genetic effects of radiation in man is at least about 4–5 times the 1 Gy value thus far used. The concepts, assumptions, and the data-base used with the DD method have been re-examined. Arguments are advanced to support the thesis that ionizing radiation is probably not very efficient in inducing the very specific molecular changes that are known to underlie spontaneous mutations which cause naturally occurring dominant genetic diseases. It is suggested that (i) the DD estimate of 1 Gy that is used to estimate risk for autosomal dominant and X-linked diseases is conservative and (ii) the 1% prevalence figure for these diseases that is used for this purpose may be too high. If these suggestions are correct, then the estimate of risk for the dominant and X-linked diseases may need to be revised downwards. The question of the total risk of genetic disease (i.e., an estimate which includes the very large class of multifactorial diseases) is not addressed here and is not answerable at present using the DD method, one important reason being that the mutation-responsive component of these diseases cannot be reliably estimated. The choice of an overall DD used for genetic risk estimation depends on what indicators are perceived to be relevant in the human context and is largely judgemental. Since recessive predominate among radiation-induced mutations, adverse genetic effects of radiation exposure are primarily those associated with induced recessive mutations in the heterozygous condition. Some suggestions are made for further work to improve genetic risk estimates.

A dose-response analysis of ethylnitrosourea-induced recessive specific-locus mutations in treated spermatogonia of the mouse

A dose-response analysis was carried out with 2 independent data sets available for ethylnitrosourea-induced specific-locus mutations in spermatogonia of the mouse. It was assumed that the occurence of mutation is binomially distributed and maximum-likelihood procedures were employed to determine the appropriateness of 4 alternative models, Linear, Linear-Quadratic, Power, and Threshold, in describing the dependence of the binomial parameter on dose. For both data sets, the Threshold model yielded a far superior fit and the threshold dose was estimated to be between 34 and 39 mg/kg. These results are supported by the relatively inefficient response of ethylnitrosourea at lower doses in inducing DNA adducts. Relevant specific-locus mutation results in the mouse for low-dose fractionated treatment as well as the recovery of mutation mosaics indicate the threshold model to be an oversimplification. Rather than a threshold dose below which 100% of the induced DNA adducts are repaired, we propose that some DNA adducts which may eventually be fixed as a mutation persist through a number of repair-competent cell divisions and do not interfere with normal cell function nor do they induce a repair response before being eventually fixed as a mutation. We interpret the threshold response for ethylnitrosourea-induced specific-locus mutations to be due to a saturable repair process which at lower doses results in ethylnitrosourea being less efficient in inducing mutation. Once this repair process is saturated, a clear dose-related increase in the mutation rate is observed.

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics in F 1 mice derived from post-spermatogonial treatment with ethylnitrosourea

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics was determined in F 1 mice derived from post-spermatogonial germ-cell stage treatment with 2 × 80, 160 or 250 mg/kg ethylnitrosourea. A total of 5 dominant cataract mutations, 3 dominant cataract mutation mosaics, 1 specific-locus mutation and 9 specific-locus mutation mosaics were recovered in 15,542 screened F 1 offspring. Results indicate that ethylnitrosourea treatment increases the mutation rate of dominant cataract and recessive specific-locus alleles in post-spermatogonial germ-cell stages of the mouse and that the mutations occur mainly as mosaics. Genetic confirmation of newly induced mutations occurring as mosaics is more problemtical for induced recessive alleles than for induced dominant alleles and should be considered when evaluating such mutagenicity results.

The effect of dose fractionation on the frequency of ethylnitrosourea-induced dominant cataract and recessive specific locus mutations in germ cells of the mouse

A combined dominant cataract-recessive specific locus mutation experiment for fractionated exposure to ethylnitrosourea (2 × 80 mg/kg, 24-h fractionation interval) was designed to determine if lower doses of ethylnitrosourea are more effective in inducing dominant cataract mutations as suggested by previous results. This observation was not confirmed by the present experiment. The extensive, statistically more reliable specific locus results indicate an additive effect of fractionated ethylnitrosourea treatment. A saturable repair system for ethylnitrosourea-induced DNA damage has been previously documented (Karran et al., 1979; Sega et al., 1986; Van Zeeland et al., 1985). Two parameters inherent to a saturable system, the minimal time required for the saturated system to recover and the minimal dose to saturate the system are important, and results of experiments employing a fractionation exposure protocol must be interpreted relative to these two parameters. Longer fractionation intervals or smaller doses result in a reduced mutagenic effect. Due to the inherently lower experimental variability of the specific locus mutation assay as compared to the dominant cataract assay, the specific locus assay is the test of choice to determine factors affecting the mammalian germ cell mutation rate. The dominant cataract test requires a larger investment of experimental resources to achieve a comparable degree of accuracy. The dominant cataract mutation test is important in assessing the mutation rate to dominant alleles in germ cells of mammals. Due to the immediate expression of the mutant phenotype in newly occurring dominant mutations, a dominant mutation assay screens a genetically relevant endpoint in an assessment of the mutagenic hazard for man in mouse experiments. A multi-endpoint design screening specific locus, dominant cataract, and biochemical mutational endpoints (Ehling et al., 1985) allows a systematic comparison of mutagenic results for different classes of mutations in the same animals.

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 × 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with γ-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.

Linear dose-response relationship of erythrocyte enzyme-activity mutations in offspring of ethylnitrosourea-treated mice

The specific activity of 10 erythrocyte enzymes was measured to detect gene mutations in F 1 offspring of male mice treated with 3 different doses of ethylnitrosourea (ENU). After administration of ENU or of the solvent (controls), the (101/El × C3H/El)F 1 hybrid males were mated to untreated T-stock females. No enzyme-activity mutant was found in 3610 F 1 offspring of the control group. After treatment of postspermatogonial germ-cell stages, 1 mutant in 1125 F 1 offspring of males treated with 160 mg ENU/kg body weight, and 2 mutants in 1319 F 1 offspring of a 250-mg/kg group were observed. After treatment of spermatogonia, 9 enzyme-activity mutants in 4247 F 1 offspring of males treated with 80 mg ENU/kg body weight, 15 mutants in 3396 F 1 offspring of a 160-mg/kg group, and 9 mutants in 1402 F 1 offspring of a 250-mg/kg group were detected. The mutation frequencies in spermatogonia were significantly different from that of the controls ( P < 0.01). The dose-response curve was found to be linear. The frequencies of enzyme-activity mutations are comparable to those of recessive specific-locus mutations determined in the same experiments. Enzyme-activity mutants with reduced activity as well as mutants with enhanced activity were found. Genetic and biochemical characterization of enzyme-activity mutants was routinely performed. In inter se crossings of heterozygotes, no offspring expressing a third phenotype other than the wild type and the heterozygote were found in approximately half of the mutation studies. The recovered mouse mutants might be used as animal models to study corresponding genetic diseases in humans.

The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia

A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22 594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227 805, 20/13 274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overlay represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed to phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events. First, variants which are sterile can be identified, and the penetrance and fertility of confirmed mutations can be estimated. Second, a group of recovered mutations controlling a phenotype system is required to analyse genetically the loci controlling the system.

Enzyme-activity mutations detected in mice after paternal fractionated irradiation

(101/E1 × C3H/E1)F 1-hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F 1-offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F 1-offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F 1 offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations.

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 × 10 −5 in post-spermatogonial stages and 3.7 × 10 −5 in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 × 10 −6 in the post-spermatogonial stages and 1.1 × 10 −6 in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 × 10 −5 and for dominant cataract mutations 0.9 × 10 −6. In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 × 10 −5. In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 158 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.

A comparison of the dominant cataract and recessive specific-locus mutation rates induced by treatment of male mice with ethylnitrosourea

Mice were derived from parental males treated with 250 mg ethylnitrosourea per kg body weight. The mice were screened simultaneously for induced dominant cataract and recessive specific-locus mutations. In the spermatogonial treatment group, 16 dominant cataract, 1 dominant corneal opacity and 60 recessive specific-locus mutations were recovered and genetically confirmed in 9352 offspring observed. This lower yield of dominant cataract mutations, when compared with the yield of recessive specific-locus mutations, is similar to results observed by Kratochvilova in a series of experiments on dominant cataract mutations induced by radiation treatment. These results taken with reported results from other dominant mutation test systems, suggest a lower per-locus mutation rate to dominant than to recessive alleles. A corollary to the hypothesis that most dominantly expressed alleles code for an alteration in the function of the normal gene product is that a limited subset of mutations could normally lead to a dominantly expressed mutation. This may explain the lower per-locus mutation rate to dominant than to recessive alleles. Genetic confirmation tests of recovered presumed dominant cataract mutations indicate that a certain category of phenotypic variants (bilateral, severe or unique lens opacity) is likely to be a true mutation but only represents 7 of the 19 mutations recovered. A second category of phenotypic variants (unilateral, neither severe nor unique lens opacity) has an extremely low probability of being a true mutation. Only 1 confirmed mutation in 181 phenotypic variants was obtained. The remaining category of phenotypic variants (either unilateral severe or unique, or bilateral neither severe nor unique lens opacity) represented the majority, 11, of the confirmed mutations obtained. However, 266 presumed mutations in this category were recovered. If a sub-class of phenotypic variants within this category could be identified that could be ignored owing to a very low probability of being a true mutation, the efficiency of recovery of confirmed dominant cataract mutations would be greatly increased with no sacrifice in the accuracy of the observed mutation rate. Finally, the 17 confirmed dominant cataract mutations obtained included a class of 7 that produced significantly fewer than the Mendelian expectation of offspring exhibiting the mutant phenotype. This class probably represents both mutations with penetrance effects and mutations with viability effects. The present experiments represent the first systematic comparison of induced genetically confirmed dominant and recessive mutations for a chemical mutagen in mice. Such results contribute to our limited understanding of the mutation process to dominant alleles.

Thresholds?

Research ArticleOpen AccessThresholds? D P Rall D P Rall Search for more papers by this author Published:1 February 1978https://doi.org/10.1289/ehp.7822163Cited by:9AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit FiguresReferencesRelatedDetailsCited by Woodruff T (2011) Bridging epidemiology and model organisms to increase understanding of endocrine disrupting chemicals and human health effects, The Journal of Steroid Biochemistry and Molecular Biology, 10.1016/j.jsbmb.2010.11.007, 127:1-2, (108-117), Online publication date: 1-Oct-2011. Huff J (2018) Chemicals and cancer in humans: first evidence in experimental animals., Environmental Health Perspectives, 100, (201-210), Online publication date: 1-Apr-1993. Favor J, Sund M, Neuhäuser-Klaus A and Ehling U (1990) A dose-response analysis of ethylnitrosourea-induced recessive specific-locus mutations in treated spermatogonia of the mouse, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 10.1016/0027-5107(90)90175-4, 231:1, (47-54), Online publication date: 1-Jul-1990. EHRENBERG L (1989) Risk Assessment of Genotoxic Agents: Problems and Possible Solutions New Trends in Genetic Risk Assessment, 10.1016/B978-0-12-388176-2.50035-7, (433-448), . Menzer R (1987) Selection of Animal Models for Data Interpretation Toxic Substances and Human Risk, 10.1007/978-1-4684-5290-7_8, (133-152), . Brown C (1987) Approaches to Intraspecies Dose Extrapolation Toxic Substances and Human Risk, 10.1007/978-1-4684-5290-7_10, (237-268), . Bella D and Tabesh T Cancer, Carcinogens and Dispersal: A Disciplinary Dysfunction, Journal of Environmental Systems, 10.2190/KDRP-D40E-FLP9-X59L, 15:3, (211-218) Jukes T (2016) Chasing A Receding Zero: Impact of the Zero Threshold Concept on Actions of Regulatory Officials, Journal of the American College of Toxicology, 10.3109/10915818309140698, 2:3, (147-160), Online publication date: 1-May-1983. Fishbein L (2009) Overview of some aspects of quantitative risk assessment, Journal of Toxicology and Environmental Health, 10.1080/15287398009529947, 6:5-6, (1275-1296), Online publication date: 1-Sep-1980. Vol. 22February 1978Metrics About Article Metrics Publication History Originally published1 February 1978Published in print1 February 1978 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact ehponli[email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.