Fractionated, Low-Dose-Rate Ionizing Radiation Exposure ... - BioOne

RADIATION RESEARCH

168, 716–724 (2007)

0033-7587/08 $15.00 䉷 2007 by Radiation Research Society. All rights of reproduction in any form reserved.

Fractionated, Low-Dose-Rate Ionizing Radiation Exposure and Chronic Ulcerative Dermatitis in Normal and Trp53 Heterozygous C57BL/6 Mice R. E. J. Mitchel,1 P. Burchart and H. Wyatt Radiation Biology and Health Physics Branch, Atomic Energy of Canada, Chalk River Ontario, K0J 1J0, Canada

culatory, digestive and respiratory systems (1, 2). In contrast to those observations, which resulted from a mixedLET radiation exposure, other epidemiological evidence (3) indicates that exposures to low-LET radiation alone, at very low doses and dose rates, protected workers generally from all diseases of the circulatory system. Understanding the risks of these and other non-cancer diseases associated with low-dose and dose-rate exposures is particularly important for the proper protection of workers who might encounter such exposures in an occupational setting. Animal experiments can help that understanding, but to be useful, they must be conducted under dose and dose-rate conditions that resemble potential occupational exposures. Recent studies useful in this regard have investigated immune activation in mice by continuous, low-dose ␥ irradiation at dose rates of 0.35 mGy/h and 1.2 mGy/h for periods of weeks or continuing up to lifetime exposures (4– 6). Immune activation was observed and in mice with an autoimmune disease (MRL-lpr/lpr), the immune activation was associated with a prolongation of life span and the amelioration of a variety of cancer and non-cancer diseases. Recently, chronic low-dose-rate irradiation has been shown to result in a total-body immunological network activation in a variety of wild-type mouse strains, including C57BL/6, BALB/c, C3H/He, DBA/1, DBA/2 and CBA (7). While in mice exposed at high dose rate, abnormal immune cells increased in a dose-dependent manner, in the mice irradiated continuously at low dose rate, no abnormal immune cells were detected at any radiation dose. Those results suggest that low-dose and dose-rate exposures may initiate a protective effect for any disease in which the immune system may play a role. Heterozygosity for Trp53 is known to make mice prone to spontaneous cancer development and to reduce their average life span by about one-third (11). It is also known to influence the adaptive response in mice, the protective effect of low doses of radiation that reduces the latency of both spontaneous and radiation-induced cancer (12, 13). Heterozygosity for Trp53 additionally influences the sensitivity of fetal mice to teratogenesis induced by high doses of radiation (14) and influences their adaptive response, as measured by the ability of a prior low dose to protect fetal

Mitchel, R. E. J., Burchart, P. and Wyatt, H. Fractionated, Low-Dose-Rate Ionizing Radiation Exposure and Chronic Ulcerative Dermatitis in Normal and Trp53 Heterozygous C57BL/6 Mice. Radiat. Res. 168, 716–724 (2007). The influence of low-dose-rate chronic radiation exposure and adaptive responses on non-cancer diseases is largely unknown. We examined the effect of low-dose/low-dose-rate fractionated or single exposures on spontaneous chronic ulcerative dermatitis in Trp53 normal or heterozygous female C57BL/6 mice. From 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks, and other Trp53ⴙ/⫺ mice were exposed to a single dose of 10 mGy (0.5 mGy/min) at 20 weeks of age. The 90-week exposure produced an adaptive response, decreasing both disease frequency and severity in Trp53ⴙ/ⴙ mice and extending the life span of older animals euthanized due to severe disease. The 30- or 60-week exposures had no significant protective or detrimental effect. In contrast, the chronic, fractionated exposure for 30 or 60 weeks significantly increased the frequency and severity of the disease in older Trp53ⴙ/⫺ mice, significantly decreasing the life span of the animals required to be euthanized for disease. Similarly, the single 10-mGy exposure also increased disease frequency in older animals. However, the chronic, fractionated exposure for 90 weeks prevented these detrimental effects, with disease frequency and severity not different from unexposed controls. We conclude that very low-dose fractionated exposures can induce a protective adaptive response in both Trp53 normal and heterozygous mice, but that a lower threshold level of exposure, similar in both cases, must first be passed. In mice with reduced Trp53 functionality, doses below the threshold can produce detrimental effects. 䉷 2007 by Radiation Research Society

INTRODUCTION

Interest in the effects of radiation on the risk of noncancer diseases has been stimulated by a recent analysis of the data from the Japanese atomic bomb survivors, which reported increasing trends with dose for diseases of the cir1 Address for correspondence: Radiation Biology and Health Physics Branch, Atomic Energy of Canada Limited, Chalk River Laboratories, Chalk River, ON, K0J 1J0, Canada; e-mail: [email protected].

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RADIATION AND ULCERATIVE DERMATITIS IN MICE

mice from teratogenesis induced by a subsequent high radiation exposure (15). Trp53 can also play a role in non-cancer diseases. In the Apoe⫺/⫺ mouse model, an absence of Trp53 function is known to influence aortic atherosclerosis. At similar serum cholesterol levels, plaque formation was significantly accelerated in Apoe⫺/⫺Trp53⫺/⫺ mice compared to Apoe⫺/⫺Tp53⫹/⫹ mice (16, 17). However, no information is available on the influence of Trp53 function for any radiation-induced effects on heart disease. Similarly, while continuous exposure to low-dose-rate radiation has been shown to activate immune function in autoimmune deficient (lpr/ lpr) mice, prolonging life span and protecting against a variety of non-cancer, autoimmune-related diseases (4, 5), the influence of reduced Trp53 function is unknown. In the work presented here, we examine the effects of fractionated low-dose, low-dose-rate ␥-radiation exposures on chronic ulcerative dermatitis, a spontaneous, autoimmune-type age-related disease in C57BL/6 mice (8, 9) that has been linked to oxidative injury (10). Skin ulcers from this disease can progress to the point where euthanization is required under National guidelines for the care of animals. While previous investigations (4–6) have provided important evidence of the influence of continuous exposure at low dose rate, such an exposure scenario is unlikely in occupationally exposed persons. Workers in the nuclear industry, for example, are more likely to receive a single low external dose only at some time during their working day, and that exposure might then be repeated on each working day for the total period of their employment. To assess the effects of such an occupational exposure pattern, we used a single exposure or fractionated radiation exposures given on a schedule (Monday to Friday) typical of potential human occupational exposures and investigated whether such exposures, which were delivered over increasing portions of the animal’s life span, could induce protective or detrimental effects. Additionally, we compared the influence of these chronic exposures in Trp53 normal mice (Trp53⫹/⫹) and in mice with reduced Trp53 gene function (Trp53⫹/⫺). MATERIALS AND METHODS Breeding Male mice carrying a single defective copy of the Trp53 gene (B6.129S2-Trp53tm1Tyj/⫹) were obtained from the Jackson Laboratory (Bar Harbor, ME) and were crossed with 129X1/SvJ female mice (Trp53⫹/⫹), also obtained from the Jackson Laboratory. The strain of origin of both lines is C57BL/6J. The resulting F1 female progeny were genotyped prior to exposure or assignment to the unexposed control groups. Trp53 heterozygous (⫹/⫺) and normal (⫹/⫹) females were retained for the radiation-exposed groups and unexposed controls. Genotyping A small tail clip was collected from F1 mice for DNA extraction and genotyping. The tail sample was incubated in fresh, sterile DNA extraction mixture: 100 ␮l PBND buffer [50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% (v/v) Nonidet P40 and

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0.45% (v/v) Tween 20] and 2 ␮l of chilled pronase (10 ␮g/ml) (Roche Diagnostics, Laval, QC). Samples were incubated in a PTC-100 thermal cycler (MJ Research, Waltham, MA) at 65⬚C for 1 h, heated to 95⬚C for 10 min, then cooled to 4⬚C. To determine Trp53 genotypes, the DNA extracted from these tail samples was assayed for the wild-type Trp53 gene and the Trp53 knockout construct using PCR. The primer sequences used for amplification of the wild-type Trp53 allele were from exon 6, EX658: 5⬘-CAGCGTGGTGGTACCTTATGA-3⬘, and exon 7, W3⬘: 5⬘-TATACTCAGAGCCGGCCT-3⬘ (12). The PCR product length was ⬃470 base pairs. The deletion allele was identified using the neo generic primers (IMR013: 5⬘-CTTGGGTGGAGAGGCTATTC-3⬘ and IMR014: 5⬘-AGGTGAGATGACAGGAGATC-3⬘). The neo PCR product was ⬃280 base pairs long. PCR was carried out in 25-␮l reaction mixes containing 10 ␮l PCR buffer [25 mM Tris-HCl, pH 8.3; 3.75 mM MgCl2; 125 mM KCl; 0.025% gelatin (Sigma-Aldrich Canada Ltd., Oakville, ON)]; all four deoxynucleotides (Invitrogen Canada Inc., Burlington ON), each at 200 ␮M; 50 pmol of each of the four primers (Invitrogen Canada Inc.); 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems Inc., Foster City CA); and 3 ␮l (⬃15 ng) of template DNA (final MgCl2 concentration 1.8 mM). A 20-␮l mineral oil overlay was used to reduce evaporation during cycling. PCR was carried out in a PTC-100 thermal cycler. The cycling conditions were 95⬚C for 5 min; 40 cycles of 95⬚C for 30 s, 51⬚C for 30 s and 72⬚C for 1 min; and 72⬚C for 5 min. The reaction products were visualized using ethidium bromide fluorescence after electrophoresis in a 1.5% agarose gel. Digital images were captured using a CCD video camera and Image-Pro Plus, version 3 software (Media Cybernetics Inc., Des Moines, IA). Animal Housing Female F1 Trp53 heterozygous (⫹/⫺) or normal (⫹/⫹) mice were randomly assigned to groups, each with about 200–250 animals for the chronically exposed groups. The group of Trp53 heterozygous (⫹/⫺) mice receiving 10 mGy at 20 weeks of age contained 102 mice. All mice were housed in a specific-pathogen-free animal facility using filter-top cages in ventilated cage racks with automatic watering, and their health status was examined daily. Food was supplied ad libitum. Temperature and ventilation were computer-controlled at each rack. Temperature was maintained at 23 ⫾ 2⬚C, and a 12-h light/dark cycle was used. All housing, handling and experimental procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and with the preapproval of the local Animal Care Committee. Mice were kept for the duration of their natural life span or until euthanization was required according to the guidelines of the Canadian Council on Animal Care. Severe ulcerative dermatitis was one condition that required euthanization. Irradiation The chronic radiation exposure was designed to simulate a potential occupational exposure. A fractionated low-dose, low-dose-rate exposure was given once per day, 5 days per week, for increasing fractions of the life span of the mice. The unrestrained mice in their plastic cages were exposed beginning at about 6 weeks of age to 60Co ␥ radiation at low dose rate from an open beam source (GammaBeam 150, Atomic Energy of Canada Limited). Animals were exposed daily to about 0.33 mGy of ␥ radiation delivered at low dose rate (0.7 mGy/h), and the exposure was repeated 5 days/week (Monday to Friday). Trp53⫹/⫹ animals were exposed for about 30 weeks (257 days of age), 60 weeks (467 days of age), or 90 weeks (676 days of age), corresponding to approximately the period prior to the death of any mice, the point midway through the appearance of the first, low rate of appearance of the disease, and the point midway through the overall appearance of the disease, where the rate of appearance of chronic ulcerative dermatitis increases rapidly (Fig. 1). Trp53⫹/⫺ mice were similarly exposed for about 30 weeks (257 days of age), about 60 weeks (467 days of age), or about 90 weeks (676 days of age), cor-

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Statistics Statistical tests were conducted using the software program SigmaStat 3.01 (Systat Software Inc., Richmond, CA). Mean dermatitis frequencies and mean ages at death were compared using the t test for normally distributed data and the Mann-Whitney Rank Sum test for non-parametric data. Survival probability was analyzed using a Kaplan-Meier analysis, and differences were tested for significance using a Log Rank Analysis. Differences with P ⬍ 0.05 were considered significant.

RESULTS

Ulcerative Dermatitis in Wild-Type (Trp53⫹/⫹) and Heterozygous (Trp53⫹/⫺) Mice

FIG. 1. Comparison of the frequency of acute ulcerative dermatitis in unirradiated Trp53⫹/⫹ and Trp53⫹/⫺ mice. Panel A: All mice with acute ulcerative dermatitis at time of death. Panel B: All mice euthanized due to acute ulcerative dermatitis. Trp53⫹/⫹, solid circles; Trp53⫹/⫺, open squares.

responding to approximately the period prior to the death of any mice, the point midway through the overall appearance of the disease, and the period covering the maximum life span of the animals (Fig. 1). Source decay over this extended period was accommodated by regularly adjusting the distance between the source and the animals to maintain the stated daily dose and dose rate. The total dose received by the animals that lived to the end of each exposure was 48, 97 or 146 mGy. Animals that died prior to the end of the assigned exposure received proportionally less total dose. Control animals that received no exposure were handled in the same way and at the same time as the exposed animals but were held in a shielded portion of the exposure facility during the time the test animals were exposed. Additionally, one group of Trp53⫹/⫺ mice received a single 10-mGy exposure (0.5 mGy/min) at 20 weeks of age to examine the effect of a single low dose.

Chronic ulcerative dermatitis increased as a function of age in the C57BL/6 mice with either full (Trp53⫹/⫹) or reduced (Trp53⫹/⫺) Trp53 gene function (Fig. 1A). The data show that there are two different rates of appearance of this disease, an initial lower rate in mice that died at an age less than about 700 days and a higher rate in mice that lived beyond that age. Since virtually all the cancer-prone Trp53⫹/⫺ mice died prior to 700 days of age, it is unclear whether the second higher rate in mice ⬎700 days of age exists in the Trp53 heterozygous mice. Comparing normal and Trp53 heterozygous mice with an age of death ⬍700 days, there was no significant difference in the mean age of death or in the frequency of animals with dermatitis (P ⬎ 0.05). Restricting the analysis to only those animals euthanized because of the disease can give a measure of severe rather than total disease. As was the case for total disease incidence (Fig. 1A), Fig. 1B shows the same two different rates of appearance of severe disease in the Trp53 normal animals, and again only the initial lower rate in the Trp53 heterozygous animals. In the unexposed animals that were euthanized at an age ⬍700 days, the mean age of death was lower in the Trp53 heterozygous animals (by about 50 days, P ⬍ 0.04), and the mean frequency of euthanization for the disease was also lower (about twofold, P ⬍ 0.02) than in the Trp53 normal animals. Skin samples from 12 unexposed control mice euthanized due to ulcerative dermatitis between 400 and 500 days of age were compared histologically to skin samples from 12 mice euthanized due to ulcerative dermatitis between 750–800 days of age. No significant differences were detected in the extent of epidermal hyperplasia or in the extent of involvement of underlying tissue (data not shown). Radiation and Ulcerative Dermatitis in Chronically Exposed Trp53 Wild-Type (Trp53⫹/⫹) Mice 1. All mice with dermatitis The frequency of dermatitis in control and exposed wildtype mice that died from all causes is shown in Fig. 2A. The figure shows the age of the animals when exposure stopped for each of the chronic exposures: a 30-week exposure, ending when the animals were 257 days of age,


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frequency in the group receiving the longest (to 676 days of age) exposure (disease frequency, P ⫽ 0.06, survival probability P ⫽ 0.08 compared to the unexposed animals). While the data in Fig. 2A show disease frequency in both younger and older animals, the data can be arbitrarily divided into those two age categories. Figure 2B considers only those control and exposed animals alive at 676 days of age (the end of the 90-week exposure). There was again no significant change in the mean frequency of disease, but the mean age at death was significantly increased for the group receiving the longest exposure (90 weeks, 146 mGy total dose) compared to the unexposed group (P ⬍ 0.01). Similarly, a survival probability analysis indicated increased survival time in that exposed group (median survival 833 days) compared to the unexposed controls (median survival 784 days, P ⬍ 0.04). 2. Mice euthanized due to dermatitis

FIG. 2. The influence of chronic radiation exposure on the frequency of acute ulcerative dermatitis at time of death in Trp53⫹/⫹ mice. Panel A: Dermatitis frequency in all Trp53⫹/⫹ mice. Panel B: Dermatitis frequency in all exposed and unexposed Trp53⫹/⫹ mice alive at 676 days of age (the end of the 90-week, 146-mGy chronic exposure). Unexposed mice, solid circles; mice exposed for 30 weeks (48 mGy); open triangles, mice exposed for 60 weeks (97 mGy); open circles, mice exposed for 90 weeks (146 mGy), open squares. In panel A, the vertical dotted line represents the end of the 30-week exposure, the vertical dashed line, the end of the 60-week exposure, and the vertical solid line the end of the 90-week exposure.

with a maximum total dose of 48 mGy; a 60-week exposure, ending when the animals were 467 days of age, with a maximum total dose of 97 mGy; or a 90-week exposure, ending when the animals were 676 days of age, with a maximum total dose of 146 mGy. While there was no apparent influence of any radiation exposure on the appearance of the disease in the younger animals (mice that died at ⬍700 days of age), there was a trend toward a reduced

An analysis of only those Trp53 wild-type animals euthanized because of the disease was used as a measure of the impact of the radiation exposure on the severity of the disease. Figure 3A shows the frequency of dermatitis in only those control or exposed animals that were euthanized due to the disease. While there was no significant influence of the exposure on the mean age of death, a survival probability analysis showed, compared to unexposed control animals, an overall increased survival probability in the animals that were exposed to radiation for 90 weeks (median survival 862 days compared to 777 days, P ⬍ 0.02) but not for animals whose exposure stopped after 30 or 60 weeks. Likewise, a similar comparison of the frequency of disease also showed a reduced frequency in the 90-weekexposed group (P ⬍ 0.01). As was the case when disease was considered in all the animals (Fig. 2), a chronic radiation exposure appeared to influence only the severity of the disease in older animals (as measured by a required euthanization). Again, Fig. 3B considers only those animals alive at 676 days of age (the time of completion of the 90-week radiation exposure). Compared to the unexposed mice, both the mean age of death (P ⬍ 0.02) and the mean frequency of disease (P ⬍ 0.02) were reduced in the animals receiving 146 mGy (90 weeks of exposure). There was no significant change in either parameter for the animals receiving 48 mGy (30 weeks of exposure) or 97 mGy (60 weeks of exposure). A survival probability analysis confirmed an increased survival in the group euthanized for the disease but receiving the 90-week exposure (median survival 864 days compared to 822 days, P ⬍ 0.004). Radiation and Ulcerative Dermatitis in Chronically Exposed Trp53 Heterozygous (Trp53⫹/⫺) Mice 1. All mice with dermatitis The frequency of dermatitis in control and exposed Trp53 heterozygous mice that died from all causes is shown

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FIG. 3. The influence of chronic radiation exposure on the severity of acute ulcerative dermatitis in Trp53⫹/⫹ mice. Panel A: Dermatitis frequency in all mice euthanized due to severe acute ulcerative dermatitis. Panel B: Dermatitis frequency in all exposed and unexposed Trp53⫹/⫹ mice alive at 676 days of age (the end of the 90-week, 146-mGy chronic exposure) and subsequently euthanized for severe acute ulcerative dermatitis. Unexposed mice, solid circles; mice exposed for 30 weeks (48 mGy), open triangles; mice exposed for 90 weeks (146 mGy), open squares. In panel A, the vertical dotted line represents the end of the 30week exposure, the vertical dashed line, the end of the 60-week exposure, and the vertical solid line, the end of the 90-week exposure.

FIG. 4. The influence of chronic radiation exposure on the frequency of acute ulcerative dermatitis at the time of death in Trp53⫹/⫺ mice. Panel A: Dermatitis frequency in all Trp53⫹/⫺ mice. Panel B: Dermatitis frequency in all exposed and unexposed Trp53⫹/⫺ mice alive at 467 days of age (the end of the 60 week, 97 mGy, chronic exposure). Unexposed mice, solid circles; mice exposed for 30 weeks (48 mGy), open triangles; mice exposed for 60 weeks (97 mGy), solid squares; mice exposed for 90 weeks (146 mGy), open squares. Vertical dotted line, end of 30-week exposure; vertical dashed line, end of 60-week exposure; vertical solid line, end of 90-week exposure.

in Fig. 4A. The figure shows the age of the animals when exposure stopped for one of three chronic exposures: a 30week exposure, ending when the animals were 257 days of age, with a maximum total dose of 48 mGy; a 60-week exposure, ending when the mice were 467 days of age, with a maximum total dose of 97 mGy; and a 90-week exposure, ending when the animals were 676 days of age, with a maximum total dose of 146 mGy. The figure shows that, compared to unexposed control animals, a 30-week chronic

exposure that ended prior to the death of any animals with dermatitis strongly amplified the appearance of this disease in the Trp53⫹/⫺ mice, with the mean disease frequency significantly increased (P ⫽ 0.001). Continuing exposure for 60 weeks partly reversed this effect, and continuing exposure for 90 weeks, essentially the whole life span of the Trp53 ⫹/⫺ mice, completely reversed the effect, with neither result being significantly different from the lower frequency seen in the unexposed control animals. However, the mean


frequency of disease in the animals receiving up to 60 weeks of exposure was significantly higher than the frequency in the animals exposed for up to 90 weeks (P ⫽ 0.01). Survival probability analysis showed a reduced survival probability (P ⬍ 0.006) in the animals exposed for 30 weeks (median survival 625 days) compared to unexposed controls (median survival 659 days) or to the animals exposed for up to 90 weeks (median survival 669 days, P ⬍ 0.002). The same analysis showed that when the exposure was continued for 90 weeks, survival probability was not different from that of the unexposed controls. An examination of the data in Fig. 4A suggested that much of the influence of radiation on dermatitis in the heterozygous mice occurred in the older mice, as was the case for the Trp53 normal mice (but with an opposite effect). The analysis of the Trp53 heterozygous mice was therefore repeated, restricting the data to mice that were alive at an age of 467 days, the time that the 60-week exposure ended. Figure 4B shows dermatitis frequency in those mice. It is clear that in Trp53⫹/⫺ mice 467 days of age or older, chronic radiation exposures that stopped at that time or prior to that time (at 257 days of age) markedly increased the appearance of the disease. Chronic radiation exposures that ended when the mice were 257 days of age (where all mice had received 48 mGy) or 467 days of age (where all mice had received 97 mGy) increased dermatitis frequency (P ⬍ 0.01 and P ⬍ 0.02, respectively) compared to the unexposed controls. By comparison, when the chronic exposure continued to near the end of the maximum life span of all the mice (676 days of age, where the mice received doses more than 97 mGy and up to 146 mGy depending on the age at death), there was no significant difference in disease frequency between the exposed mice and the unexposed animals. Similarly, compared to the unexposed controls (median survival 659 days), survival probability analysis showed a reduced survival probability for the mice exposed for 30 weeks (median survival 641 days, P ⫽ 0.004) or 60 weeks (median survival 645 days, P ⬍ 0.03), but there was no significant difference between the unexposed mice and mice exposed for up to 90 weeks (median survival 702 days). Survival probability was also significantly reduced when the 30- or 60-week-exposed groups were compared to the group in which exposure was continued for 90 weeks (P ⬍ 0.02) (Fig. 4B). 2. Mice euthanized due to dermatitis Like the Trp53⫹/⫹ mice, the severity of the disease was estimated by analysis of the Trp53⫹/⫺ animals euthanized due to dermatitis. Figure 5A shows that, compared to unexposed controls, the frequency of animals euthanized due to dermatitis was significantly increased in mice whose exposure ended after 30 weeks (P ⫽ 0.001). The increased frequency in these 30-week-exposed Trp53⫹/⫺ mice resembled the spontaneously higher frequency observed in the

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FIG. 5. The influence of chronic radiation exposure on the severity of acute ulcerative dermatitis in Trp53⫹/⫺ mice. Panel A: Dermatitis frequency in all Trp53⫹/⫺ mice euthanized due to severe acute ulcerative dermatitis. Panel B: Dermatitis frequency in all exposed and unexposed Trp53⫹/⫺ mice alive at 467 days of age (the end of the 60-week, 97-mGy chronic exposure) and subsequently euthanized for severe acute ulcerative dermatitis. Unexposed mice, solid circles; mice exposed for 30 weeks (48 mGy), open triangles; mice exposed for 60 weeks (97 mGy), solid squares; mice exposed for 90 weeks (146 mGy), open squares. Vertical dotted line, end of 30-week exposure; vertical dashed line, end of 60week exposure; vertical solid line, end of 90-week exposure.

unexposed Trp53 normal mice euthanized due to dermatitis (Fig 1B). This increased frequency of severe disease in the 30-week-exposed Trp53⫹/⫺ mice was no longer statistically significant when the exposure was continued for 60 or 90 weeks. Comparing the exposed animals, the frequency of animals euthanized for dermatitis when the exposure ended after 30 weeks was significantly higher than that in the

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animals exposed for 60 or 90 weeks (P ⫽ 0.01 and P ⬍ 0.001, respectively). As was done to estimate disease severity in the older Trp53⫹/⫹ mice, the analysis of the Trp53 heterozygous mice was repeated, restricting the analysis to only the older animals (alive at 467 days, the time at which the 60-week exposure ended) that were subsequently euthanized because of the disease. Figure 5B shows that chronic radiation exposures that stopped when these mice were either 257 or 467 days of age significantly increased the frequency at which the older mice were euthanized because of the disease compared to controls (P ⬍ 0.01 and P ⬍ 0.02, respectively) or compared to mice exposed continuously for essentially their entire life span (up to 676 days, P ⬍ 0.02 and P ⬍ 0.04, respectively). Survival probability analysis of these older mice that were euthanized due to the disease showed that exposures that stopped at 257 days of age significantly decreased survival probability compared to unexposed controls (P ⬍ 0.02) or to exposures that continued for essentially their entire life span (up to 676 days of age, P ⬍ 0.02). Exposures that stopped at 467 days of age conferred a nonsignificant (P ⫽ 0.06) trend toward decreased survival probability compared to unexposed controls, but survival probability was significantly decreased compared to mice exposed for up to 676 days (P ⬍ 0.03). Exposure that continued for 676 days conferred no significant survival advantage or disadvantage when compared to Trp53 heterozygous animals receiving no radiation exposure. Radiation and Ulcerative Dermatitis in Singly Exposed Trp53 Heterozygous (Trp53⫹/⫺) Mice In older Trp53⫹/⫺ animals (alive at 467 days), a single 10-mGy exposure at 20 weeks of age significantly increased the frequency of dermatitis in animals that died from all causes (Fig. 6, P ⬍ 0.02), a result that paralleled the effect of the chronic multiple exposures ending after 30 or 60 weeks (Fig. 4B). However, this single exposure had no significant effect on the frequency at which the older mice were euthanized for the disease or on their survival probability, suggesting that, unlike chronic exposures that stopped after 30 or 60 weeks, the severity of the disease was not influenced by a single exposure given at 20 weeks of age. DISCUSSION

Chronic ulcerative dermatitis is a severe, spontaneous, autoimmune-related skin disease that appears in aging C57BL/6 mice (8–10). In the work presented here, we examined the frequency and severity of this disease in Trp53 normal and heterozygous mice and tested the influence of fractionated low-dose-rate ␥-radiation exposure delivered for various fractions of the animal’s life span. The exposure dose and delivery were designed to represent a potential human occupational exposure, typically occurring during each normal working day. In unexposed Trp53 normal

FIG. 6. The influence of a single 10-mGy low-dose-rate exposure on the frequency of acute ulcerative dermatitis in Trp53⫹/⫺ mice. The figure shows dermatitis frequency in all exposed and unexposed Trp53⫹/⫺ mice alive at 467 days of age. Unexposed mice, solid circles; mice exposed to 10 mGy at 140 days of age, open circles.

mice, the disease first appeared at about 400 days of age. There were two distinct rates at which the frequency of disease increased, with the second much higher rate beginning around 700 days of age (Fig. 1A). The rate of appearance of severe disease, the point at which mice must be euthanized, appeared to follow the same two distinctly different rates of increase (Fig. 1B). No histological differences were detected in ulcerative skin samples taken from unexposed control mice euthanized for dermatitis in these two different periods. In cancer-prone Trp53⫹/⫺ mice, the maximum life span is near 700 days, and only one rate of increase in ulcerative dermatitis disease frequency was observed, which was not different from the first lower rate in the Trp53⫹/⫹ mice. In this period, however, the Trp53⫹/⫺ mice had a significantly lower frequency of severe disease, and it appeared significantly earlier than in the Trp53⫹/⫹ mice (Fig. 1B), indicating a link between the appearance of this disease and the level of Trp53 function. Chronic exposure of the Trp53⫹/⫹ mice to fractionated doses for 30, 60 or 90 weeks had no significant effect on the disease frequency or rate of appearance of the disease in younger animals, i.e., during the initial slower rate of increase in disease frequency (⬍700 days of age, Fig. 2A). However, when older animals were specifically examined (⬎657 days of age, during the second, faster rate of increase of disease, Fig. 2B), the data showed that the 90week exposure induced an adaptive response, significantly slowing the appearance and reducing the severity of the disease. The 90-week exposure also significantly increased the life span of animals that ultimately required euthanization as a result of severe skin disease (Fig. 3B). This protective response is very similar to that reported previ-

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ously for chronic low-dose-rate irradiation in immune-compromised MRL-lpr/lpr mice (4, 5), but it suggests that in Trp53 normal mice a lower dose or exposure time threshold must be passed before a protective adaptive response is initiated in vivo. In comparison, chronic exposure of mice with reduced Trp53 function (heterozygotes) produced different results, again depending on the duration of the exposure (and hence the dose). Since the maximum life span of these animals was less than about 700 days, radiation effects could be observed only during the initial, slower rate of increase in disease (Fig. 1). In these Trp53 heterozygous mice, chronic exposures that stopped prior to (at 30 weeks of age) or midway through disease development (at 60 weeks of age) significantly increased the frequency (Fig 4B) and severity of the disease in older (⬎457 days of age) animals (Fig. 5B). Similarly, a single lower exposure at 20 weeks of age also increased disease frequency in the older mice but, in contrast, had no influence on severity. These results indicate that for these doses, detrimental effects outweighed any constitutive or induced protective mechanisms. These increases in frequency or severity from single or multiple radiation exposures of the Trp53 heterozygous mice raised the rates closer to but did not exceed the normally higher rates observed in the unexposed Trp53 normal mice. Interestingly, if the chronic, fractionated exposure was not stopped but was continued for essentially the whole life span of the Trp53 heterozygous animals, the frequency and severity of the disease were not different from these observed in the unexposed control heterozygotes. Apparently, with the larger total dose, adaptive responses were sufficiently induced to at least balance the otherwise detrimental effects of the exposure. As with the Trp53 normal mice, the results from the heterozygotes suggest that protective adaptive responses are activated only when a lower dose threshold is surpassed. A single 10-mGy dose at 20 weeks of age also increased disease frequency in the older mice, suggesting that increasing the dose rate, at least up to a dose of 10 mGy, does not influence the detrimental outcome. Since in both Trp53 heterozygous and normal mice doses between 97 and 146 mGy (up to 90 weeks of exposure time) were required to either protect against (in Trp53 normal mice) or balance (in Trp53 heterozygous mice) detrimental effects, Trp53 functionality may not greatly influence this lower dose threshold for adaption. The observation of a lower threshold for the dose that is required to induce protective effects has been reported previously for adaptive responses measured by chromosomal break repair in human cells (18), for cell killing in human cells in tissue culture (19), and for chromosomal inversions in mice in vivo (20, 21). Trp53 is a radiation-inducible regulatory gene involved in DNA repair, apoptosis and cell cycle control. Any specific role in ulcerative dermatitis and its response to radiation were unknown, but the data presented here suggest a large influence. The mechanism of action is unknown.

However, the link between chronic ulcerative dermatitis and oxidative stress (10) suggests that in animals with fully or partially functional Trp53, radiation may act on ulcerative dermatitis in a manner similar to its action in radiation carcinogenesis, where a low dose induced an adaptive response that protected against the carcinogenic effects of an oxidative stress from a subsequent radiation exposure (13, 22). For cancer, however, heterozygosity for Trp53 reduced the magnitude and upper dose threshold of the protective effect of radiation (13), and radiation exposure increased disease risk only when the dose exceeded an upper threshold value (12, 13). Those results, however, were unlike the results presented here for ulcerative dermatitis in the Trp53 heterozygous mice, where single or multiple low doses given earlier in life increased disease risk, while continuing multiple doses that extended later into life (and resulted in higher cumulative doses) did not. These observations on dermatitis risk in the Trp53 heterozygous mice and the protective effects for dermatitis seen only at higher cumulative doses in the Trp53 normal mice, coupled with previous observations for lower dose thresholds of adaption (18–21), suggest that these are general phenomena. The data suggest that radiation risk is not linear with dose for many end points, including cancer and dermatitis, and just as there is in general an upper dose threshold where protective adaptive responses give way to detrimental effects, there is also in general a lower dose threshold for adaptive responses below which detrimental effects may or may not occur depending, at least in the case of dermatitis, on the level of Trp53 function. While the upper dose threshold for adaption appears to depend strongly on Trp53 functional status (13), the lower dose threshold may be relatively unaffected by Trp53 functionality. It is clear, however, that in all cases dose is not a good predictor of radiation risk. The observations presented here for this autoimmuneand age-related non-cancer disease in mice should be considered in the assessment of low-dose radiation risk for similar diseases in occupationally or repetitively exposed persons. The data indicate that low doses may generally produce either no effect or protective effects. However, if risk was observed to increase, that increase may be closely linked to the genetic status of a subpopulation and may not reflect the risk in the majority of persons. ACKNOWLEDGMENT This work was supported by Atomic Energy of Canada Limited and by Health Canada. Received: June 12, 2007; accepted: August 28, 2007

REFERENCES 1. Y. Shimizu, D. A. Pierce, D. L. Preston and K. Mabuchi, Studies of the mortality of atomic bomb survivors. Report 12, part II. Noncancer mortality: 1950–1990. Radiat. Res. 152, 374–389 (1999). 2. M. Yamada, F. L. Wong, S. Fujiwara, M. Akahoshi and G. Suzuki,

724

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.


Noncancer disease incidence in atomic bomb survivors, 1958–1998. Radiat. Res. 161, 622–632 (2004). R. Sponsler and J. R. Cameron, Nuclear shipyard worker study (1980–1988): a large cohort exposed to low-dose-rate gamma radiation. Int. J. Low Radiat. 1, 463–478 (2005). Y. Ina and K. Sakai, Prolongation of life span associated with immunological modification by chronic low-dose-rate irradiation in MRL-lpr/lpr mice. Radiat. Res. 161, 168–173 (2004). Y. Ina and K. Sakai, Further study of prolongation of life span associated with immunological modification by chronic low-dose-rate irradiation in MRL-lpr/lpr mice: Effects of whole-life irradiation. Radiat. Res. 163, 418–423 (2005). Y. Ina, H. Tanooka, T. Yamada and K. Sakai, Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice. Radiat. Res. 163, 153–158 (2005). Y. Ina and K. Sakai, Activation of immunological network by chronic low-dose-rate irradiation in wild-type mouse strains: Analysis of immune cell populations and surface molecules. Int. J. Radiat. Biol. 81, 721–729 (2005). A. G. Andrews, R. C. Dysko, S. C. Spilman, R. G. Kunkel, D. Brammer and K. J. Johnson, Immune complex vasculitis with secondary ulcerative dermatitis in aged C57BL/6Nnia mice. Vet. Pathol. 31, 293–300 (1994). Ace Animals Inc., http://www.aceanimals.com/C57BL6.htm. G. W. Lawson, A. Sato, L. A. Fairbanks and P. T. Lawson, Vitamin E as a treatment for ulcerative dermatitis in C57BL/6 mice and strains with a C57BL/6 background. Contemp. Top. Lab. Anim. Sci. 44, 18– 21 (2005). M. Harvey, M. J. McArthur, C. A. Montgomery, Jr., J. S. Butel, A. Bradley and L. A. Donehower, Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat. Genet. 5, 225–229 (1993). R. E. J. Mitchel, J. S. Jackson, D. P. Morrison and S. M. Carlisle, Low doses of radiation increase the latency of spontaneous lymphomas and spinal osteosarcomas in cancer-prone, radiation-sensitive Trp53 heterozygous mice. Radiat. Res. 159, 320–327 (2003).

13. R. E. J. Mitchel, J. S. Jackson and S. M. Carlisle, Upper dose thresholds for radiation-induced adaptive response against cancer in highdose-exposed, cancer-prone, radiation-sensitive Trp53 heterozygous mice. Radiat. Res. 162, 20–30 (2004). 14. D. R. Boreham, J-A. Dolling, J. Misonoh and R. E. J. Mitchel, Radiation-induced teratogenic effects in fetal mice with varying Trp53 function: Influence of prior heat stress. Radiat. Res. 158, 449–457 (2002). 15. R. E. J. Mitchel, J-A. Dolling, J. Misonoh and D. R. Boreham, Influence of prior exposure to low-dose adapting radiation on radiationinduced teratogenic effects in fetal mice with varying Trp53 function. Radiat. Res. 158, 458–463 (2002). 16. N. V. Guevara, H. S. Kim, E. I. Antonova and L. Chan, The absence of p53 accelerates atherosclerosis by increasing cell proliferation in vivo. Nat. Med. 5, 335–339 (1999). 17. B. J. van Vlijmen, G. Gerritsen, A. L. Franken, L. S. Boesten, M. M. Kockx, M. J. Gijbels, M. P. Vierboom, M. van Eck, B. van De Water and L. M. Havekes, Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3-Leiden transgenic mice. Circ. Res. 88, 780–786 (2001). 18. E. J. Broome, D. L. Brown and R. E. J. Mitchel, Dose responses for adaption to low doses of 60Co ␥ rays and 3H ␤-particle radiation in normal human fibroblasts. Radiat. Res. 158, 181–186 (2002). 19. B. Marples, P. Lambin, K. A. Skov and M. C. Joiner, Low dose hyper-radiosensitivity and increased radioresistance in mammalian cells. Int. J. Radiat. Biol. 71, 721–735 (1997). 20. A. M. Hooker, M. Bhat, T. K. Day, J. M. Lane, S. J. Swinburne, A. A. Morley and P. J. Sykes, The linear no-threshold model does not hold for low-dose ionizing radiation. Radiat. Res. 162, 447–452 (2004). 21. G. Zeng, T. K. Day, A. M. Hooker, B. J. Blyth, M. Bhat, W. D. Tilley and P. J. Sykes, Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure. Mutat. Res. 602, 65–73 (2006). 22. R. E. J. Mitchel, J. S. Jackson, R. A. McCann and D. R. Boreham, Adaptive response modification of latency for radiation-induced myeloid leukemia in CBA/H mice. Radiat. Res. 152, 273–279 (1999).