Effect of ionizing radiation on transgenerational appearance of pun reversions in mice

Nicholas Carls and Robert H. Schiestl

Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA


    Abstract
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 Abstract
 Introduction
 References
 
Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally induced cancers show a delayed onset of > 20 years following exposure. In fact, the frequency of genetic changes in cancer cells is higher than can be explained by random mutation. A high level of genetic instability in a subpopulation of cells may be caused by a mutator phenotype transmitted through many cell divisions. We have determined the effects of irradiation of parental male mice on the frequency and characteristics of mitotically occurring DNA deletion events at the pun locus in the offspring. Reversion of the pun marker in mouse embryos is due to deletion of 70 kb of DNA resulting in fur spots in the offspring. We found that irradiation of male mice caused a significantly higher frequency of large spots in the offspring, indicative of the induction of DNA deletions early in embryo development. These deletion events occurred, however, many cell divisions after irradiation. The present data indicate that exposure of the germline to ionizing radiation results in induction of delayed DNA deletions in offspring mice.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally induced cancers show a delayed onset of >20 years following exposure (1). The frequency of genetic changes in cancer cells is higher than can be explained by random mutation (2). It was thus proposed that a subpopulation of cells may develop a mutator phenotype (3), resulting in an elevated level of genetic instability, which may be transmitted through many cell divisions. A common characteristic of cancer cells, widely thought to be a major contributor to the progressive, multistage development of malignant disease, is an unstable `hypermutable' genome. There is a large body of data indicating that irradiation or chemical carcinogens induce a multitude of so-called delayed reproductive effects in a subpopulation of exposed cells in vitro and in vivo (46). These persistent effects are dominant traits (7) that include aneuploidy (8), small colony size (9), low plating efficiency, abortive colonies (10), a high proportion of giant cells (11) and increased mutation, chromosomal abnormalities and recombination frequencies (1216). Delayed reproductive effects persist at least until 95–100 population doublings (see for example ref. 12) and thus cannot be caused by unrepaired lesions of the initial damage. The frequency at which they are seen (up to 50% of the irradiated cells) excludes the possibility of a mutation in any one of the genes required for the maintenance of genomic stability (6).

Delayed reproductive effects can give rise to a transformed phenotype. A series of elegant experiments in Little's laboratory demonstrated that the ultimate appearance of transformed foci in a Petri dish was independent of the number of cells irradiated, as long as these cells eventually grew to confluence on the dish (summarized and cited in ref. 17). Even if only one irradiated cell was seeded on the dish, the descendants of this cell gave rise to transformed foci at the high probability of 80% per dish. Since only a few transformed foci appeared on the dishes the implication was that their appearance was not an immediate effect of the irradiation, but rather a delayed effect occurring at an elevated frequency in the descendants of the irradiated cells.

Genomic instability has also been observed in offspring following irradiation at the zygote stage or following irradiation of the parental animals before mating. In one such study skin fibroblasts from mouse fetuses X-irradiated at the zygote stage showed a significantly higher frequency of chromosome aberrations (18) than cells from control fetuses. Other studies reported transgenerational effects. Mouse minisatellite mutations were found at a higher frequency in offspring after irradiation of one parental mouse and mating with an unirradiated mouse (1921). Interestingly, many offspring showed mosaicism, indicating that the effect was the result of a delayed action of the irradiation in the embryo rather than an acute action on the germline. The inheritance of delayed reproductive effects through the germline may be at the origin of childhood cancers. For instance, a critical review of 32 studies on `parental occupational exposure and childhood cancer' (22) comes to the conclusion that `the preponderance of evidence supports the hypothesis that occupational exposure of parents to chemicals increases the risk of childhood malignancy'.

In the current study we used a model system in the mouse to determine the effects of germline irradiation on frequencies and timing of DNA deletions in the offspring embryo. The mouse pink-eyed unstable (pun) mutation, which leads to a dilution of coat and eye color pigmentation, is due to a 71 kb head-to-tail duplication of the pink-eyed dilute locus (23). A deletion event of one copy of the duplicated sequence that happens in a melanocyte precursor cell of the embryo can be detected after this cell has multiplied and developed into a black spot on the dilute coat of the offspring (Figure 1Go; 24,25). pun reversion events are inducible in the offspring after they have been exposed in utero to X-ray and chemical carcinogens (24,2628). The nature of the carcinogen-induced spots was identified as bona fide DNA deletion events using RT–PCR (24). Deletion events occurring early in embryogenesis should yield larger spots than events occurring later. Here we irradiated male mice and 4 weeks or more after irradiation crossed them with unirradiated dams. The frequency and size of spots were determined and compared with control matings. Most interestingly, irradiation of the male mice before mating caused a significant increase in the frequency of large spots, the result of deletion events occurring early in embryogenesis, but after many cell divisions of the zygote.



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Fig. 1. Schematic representation of the pun locus and the recombination product adapted from Schiestl et al. (24). The pun mutation is due to an internal duplication of ~71 kb of DNA (3738) spanning exons 6–18 of the p gene (25). Deletion of one copy of exons 6–18 results in reversion to p+ (2425). Mechanisms responsible for reversion include intrachromatid crossing over, single-strand annealing, unequal sister chromatid exchange and sister chromatid conversion, as discussed in detail previously (3940).

 
Mice homozygous for pink-eyed dilution unstable (C57BL/6J pun/pun) were obtained from the Jackson laboratory (Bar Harbor, ME). Mice were bred in the institutional animal facility under SPF conditions with a 12 h light/dark cycle and were fed standard diet and water ad libitum.

Male C57BL/6J pun/pun mice received an acute dose of 1 Gy X-rays 28 days before being paired with unirradiated female C57BL/6J pun/pun mice. Female mice were examined every day for the presence of a vaginal plug (evidence of successful mating). The females were separated after a plug was found and another unirradiated female was paired with the irradiated male. Females were exchanged after 2–3 days if no matings were discovered. This process was repeated for up to 1 week. Four additional weekly rounds of such crossings were performed. Offspring were examined for fur spots at 10 days of age and the size of the spots was measured with a ruler.

A Westinghouse 150 Industrial X-ray Machine produced 130 kVp X-rays, delivered by a self-rectifying tube with an inherent filtration of 1.65 mm aluminum. Using a current of 8 mA, the intensity obtained at 40 cm distance was 0.24 ± 0.02 Gy/min. Mice were exposed in individual sterile polypropylene containers resting on a 24 cm diameter steel turntable and were rotated to ensure a more accurate average value of the irradiated field. The dose delivered was measured for each irradiation with a Victoreen C-r 570 meter.

We were interested in the effects of irradiation of male mice before mating on recombination events in the offspring by monitoring the frequency and size of spots. We postulated that the DNA repair and recombination machinery may be most active during prophase of meiosis (in which meiotic recombination occurs), i.e. in male mice ~28 days before the mature sperm enters the cauda epididymis (29,30). Both a meiotic recombination event leading to p+ as well as a very early event in the embryo would lead to offspring with an entirely black coat. The spontaneous frequency of entirely black offspring in our breeding colony was ~1 in 2000 mice. We did not find any black mice among 180 live offspring of irradiated males. Thus, any induction of germline pun reversion events by X-rays would be of a magnitude <10-fold. The average litter size was comparable (7.3 versus 7.6 in Table IGo) but the pregnancy efficiency per detected plug was only ~25% (Table IGo) in the male irradiated group compared with an average of 75% (not shown) for control matings. Of the 180 offspring, 31 (17%) had fur spots (Table IGo). This frequency was slightly, but not significantly, elevated compared with the control frequency of 14%. A classification of the numbers of spots per offspring also did not yield any significant difference between irradiated and control offspring (data not shown).


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Table I. Effect of treatment of male mice with ionizing radiation on intrachromosomal recombination in offspring mice
 
While the frequency of spots did not significantly differ between control and male irradiated groups, there were significant differences in the spot sizes. Early pun reversion events in the premelanocytes of the developing embryo should result in larger spots than later reversions. The spots were classified by size in 5 mm2 intervals (Table IIGo and Figure 2Go). We added the size of different spots from offspring with multiple spots since multiple spots could occur from one initial event after mixing of premelanocytes in the embryo. A shift in the size of offspring spots from small spots towards larger spots compared with the control was apparent. Fourteen of the 31 spots (45.2%) were larger than 21 mm2 compared with 42 out of 203 (20.7%) in the control (Table IIGo and Figure 2Go). This difference is statistically highly significant (P << 0.005) based on {chi}2 distribution values. Of the offspring of irradiated male mice 9.7% were >30% black (mosaic mice in Table IIGo) compared with 12.0% in the control, again a significant difference at P < 0.05. Furthermore, a logistic regression model was fitted to evaluate whether the offspring of irradiated males were more likely to have larger spots than the control mice (31). The median size of the spots was larger for the offspring from irradiated males (12 mm2) compared with control mice (8 mm2). This difference is significant, based on the Wilcoxon ranked sum test (P = 0.01). These results indicate that after irradiation of male mice the number of early arising deletion events in the developing offspring embryo is significantly elevated. This increase, however, was not enough to significantly raise the overall frequency of spots in the current sample. The expected increase in the spotting frequency based on the increase in the number of large spots would be ~25%, which would raise the frequency from 14 to 17.5%, in agreement with the data we obtained (Table IGo). Finally, there is an apparent decrease in the frequency of small spots among the offspring of irradiated males (Table IIGo and Figure 2Go). This decreased frequency is mainly the artificial result of an added portion of large spots, which as a result decreases the portion of small spots. Thus, if we deduct 25% (the added portion of large spots) from the sum of irradiated male spotted mice and divide the frequency of small spots (<20 mm2) by that new number (23.3) there is actually an increase in the frequency in most categories of small spots (1–5, 11–15 and 16–20 mm2).


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Table II. Effect of irradiation of male mice on size distribution of spots in the offspring
 


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Fig. 2. Representation of the spot size distribution of control matings (open bars) and matings after irradiation of the males (filled bars). The percentage of offspring showing spots of the respective sizes are shown.

 
Melanocytes develop as a cell lineage from the neural crest, which in turn develops from the ectoderm. Since there is considerable mixing of cells apparent in the embryo and the melanocytes are not monoclonal (32), it is difficult to narrow down the exact timing of the reversion events. Reversion events in the germline or in the first few cell divisions will result in black mice or mice with very large spots, such as mottled mice. Unrepaired lesions may last for those first few cell divisions, but will be repaired and/or diluted out in further cell divisions. An analysis of the size distribution of the spots >21 mm2 (subclass 21–100 mm2) indicated no significant difference in any class compared with the control (data not shown). If the reversion events were due to unrepaired damage still present after irradiation one would expect the majority of the induced events to result in very large spots, mottled mice or black mice, contrasting with the control. Since this was not the case, it appears that the radiation-induced effect was inherited for many cell divisions before giving rise to a recombination event.

A correlation between clones showing persistent genetic instability and low plating efficiency has been shown (14). Thus, cells transmitting genetic instability may be less fit and slowly losing out in competition against cells not showing increased frequencies of genetic instability. This may be a possible reason for an increase in the number of large (early events) but less of an increase in the number of small spots (later events). It is also possible that some cells are eventually healed from the phenotype of persistent genetic instability.

We used offspring from matings that occurred 4–9 weeks after irradiation. At weeks 6 and 7 type B spermatogonia are hit and at weeks 8 and 9 stem cells, all of which proliferate before developing into spermatids. Thus, after irradiation these cells proliferated before forming spermatids, which is another indication that these effects are delayed and not directly induced by the DNA damage caused by irradiation.

As mentioned above, many reports have demonstrated delayed genetic effects of radiation. Transgenerational effects of radiation include competitive cell proliferation disadvantage of embryo cells from matings in which one parent has been irradiated versus cells from control matings (33), inherited over at least two generations (34). These effects occur when the male mice are mated at weeks 6 and 7 but not at week 5 post-irradiation (33). In our experiment we pooled offspring from matings 4–9 weeks post-irradiation to obtain sufficient data. When we analyzed the fractions of large spots separately for each week of mating, there was no difference between weeks 5–8 post-irradiation. This indicates that the differences regarding the male germ cell stages cited above for competitive cell proliferation disadvantage do not exist for the effects on deletion frequency.

Finally, it was found that some irradiated males transmitted a higher frequency of competitive cell proliferation disadvantage than others (35). We used a total of 14 males in our experiment. We determined whether there was any clustering in some litters or in the offspring from certain males. Generalized estimating equations (36) were used to adjust for correlation between litter mates. Models were run assuming that a correlation existed among the offspring from the same male and assuming that a correlation existed among the offspring of the same litter. The estimated correlation between the offspring from a male and from a litter was low (0.03 and –0.001, respectively). The modeling results indicated no difference in the probability of large spots arising among the offspring from different males or from different litters.

Transgenerational genotoxic effects include a higher frequency of microsatellite instability in offspring of irradiated animals than can be accounted for by the numbers of lesions produced by the radiation (1921). The present data indicate that ionizing radiation exposure of the germline can induce delayed DNA deletions in offspring mice. DNA deletion events have been implicated in the onset of carcinogenesis and a similar phenomenon in humans may account for a portion of childhood cancers.


    Acknowledgments
 
Lynn Wiley and the members of the Schiestl laboratory are thanked for comments on the manuscript. We also thank Edie Weller for statistical analysis. This research was supported by grant no. CN-142 from the American Cancer Society and Research Career Award no. ES00299 from the National Institutes of Environmental Health Sciences, NIH to R.H.S., as well as Center Grant no. ES0002 from the NIEHS.


    Notes
 
1 To whom correspondence should be addressed Email: schiestl{at}hsph.harvard.edu Back


    References
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Received May 21, 1999; revised August 11, 1999; accepted August 19, 1999.