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Evidence for humoral effects on the radiation response of rat foot skin

Research Institute (University of Oxford), Churchill Hospital, Oxford OX3 7LJ, UK.

	Abstract

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The influence of perturbation of the physiologic state of the whole body on the outcome of radiation exposure has been examined in a rat foot model. Irradiation was carried out using ⁶⁰Co -rays. Moist desquamation was used as an endpoint. Rats were given a priming dose of 2 Gy, 4 Gy or 7 Gy to their whole body except their hind feet (partial body priming dose). After a variable time period both hind feet of these animals were irradiated with graded doses of ⁶⁰Co -rays. The incidence of moist desquamation in the irradiated feet of these animals was compared with the incidence of moist desquamation in animals that had not received the initial partial body priming dose. It was noticed that the incidence of moist desquamation in the rat foot skin ofanimals that received 7 Gy partial body priming dose 4 h prior to irradiation of their hind feet was significantly less than moist desquamation in control animals. The ED₅₀ value of 22.53±0.16 Gy for moist desquamation of the foot skin of control animals was significantly lower (p<0.01) than the value of 25.25±0.29 Gy obtained for animals that received a partial body priming dose of 7 Gy 4 h prior to irradiation of their hind feet. It was concluded that the response of rat foot skin to radiation was not purely the result of epidermal stem cell kill and that it can be modified by alterations in the overall physiological state of the animal's body brought about by a priming dose to the whole of the animal's body except the hind feet.

	Introduction

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According to the accepted concepts of cellular radiobiology, based on the target theory for single cells [1–3] it is assumed that radiation kills cells at random and that the dose of radiation and the radiosensitivity of irradiated cells determine the probability of cell kill. This concept was extended to tissues [4, 5] by viewing normal tissue radiation injury as a result of the sterilization of clonogenic cells within that tissue. According to this concept, tissue-specific function is restricted to functional non-proliferative cells derived from clonogenic cells. Failure of clonogenic stem cells to replace the functional cells, which continue to be lost at anormal rate, results in a gradual depletion of functional cells. When the number of functional cells reaches a critical level, the tissue cannot sustain its function and radiation-induced injury is expressed. A key assumption of this concept is that the cells respond to radiation independently of each other. This might be true of cells in vitro but what about in tissues? Does the response of specific tissue solely depend on the survival of target stem cells and is the response independent of the physiological state of whole system? The present study was designed to answer these questions.

	Material and methods

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Mature (26 weeks old) female Sprague–Dawley rats were used in this study. The animals were housed in groups of three per cage, in conventional housing conditions, 55% humidity, 21–22°C and 12 h:12 h (07:00–19:00) light–dark cycle.They received standard pellet diet food and water ad libitum. Irradiation procedures were carried out under anaesthesia with ⁶⁰Co -rays at a dose rate of 1.5 Gy min^-1. Animals were initially anaesthetized in a Perspex box flushed with oxygen and 2–3% halothane. Pre-anaesthetized rats were then positioned in a Perspex irradiation jig and anaesthesia was maintained by continuous flushing withoxygen and 1–1.5% halothane at a rate of 2 l min^-1. For partial body priming dose irradiations, four rats at a time were placed next to each other in the Perspex jig and their hind feet were stretched outside the irradiation field and shielded by positioning a 5 cm thick lead block on the Perspex lid of the box over the hind foot region. During this procedure the hind feet received only scattered radiation, which was measured to be 7% of the partial body priming dose. For irradiation of the hind feet, the foot to be irradiated was positioned in a slot in a circular Perspex holder (1 cm thick, 11 cm diameter) located at the centre of the irradiation field. Rats, nine at a time, were positioned radially around this central Perspex portion of the jig. A 5 cm lead circular collimator was used to shield the rats' body during foot irradiation. Both hind feet of animals were irradiated.

Following irradiation the irradiated feet were examined daily for the presence of moist desquamation between 10 days and 20 days after irradiation. Quantal data for the incidence of moist desquamation were analysed using probit analysis [6], and the dose at which moist desquamation was observed in 50% of irradiated feet (ED₅₀) (± SE) was obtained for each irradiation schedule. Models involving parallel and non-parallel dose–effect curves were fitted to each set of data. The most appropriate model was selected on the basis of test of parallelity of dose–effect curves using Wald statistics. Mantel–Haenzsel statistics were computed to determine whether there was an association between the response and the dose of radiation and the treatment group. Comparison of quantitative values was carried out by t-test or analysis of variance.

Experiment 1
Four groups of 36 animals were used in this study. Each group was divided into two subgroups, "control" and "test". Test groups received a 7 Gy partial body priming dose to the whole body, except the hind feet, which were positioned outside the irradiation field and shielded with 5 cm lead. During this stage of irradiation the shielded feet received 0.5 Gy from scattered radiation (7% of the partial body priming dose). The hind feet in the test groups were then irradiated with graded single doses at 2 h, 4 h, 6 h or 24 h after the initial partial body priming dose. Control groups received no partial body priming dose, only graded single doses to their hind feet.

Experiment 2
Three groups of 36 animals were used in this experiment. Each group was divided into two subgroups, control and test. Test groups received partial body priming doses of 2 Gy, 4 Gy or 7 Gy to the whole body, except to their hind feet, which were positioned outside the irradiation field and shielded with 5 cm lead. The hind feet of test group animals were irradiated with graded single doses at 4 h after the initial partial body priming dose. Control groups received no partial body priming dose, only graded single doses to their hind feet.

Experiment 3
Two groups of 36 rats were used in this experiment. Each group was divided into two subgroups, control and test. Test groups received priming doses of 0.5 Gy or 1 Gy to their hind feet 4 h prior to irradiation of the same feet with graded single doses of -rays. The control groups received no local priming dose, only graded single doses to their hind feet.

	Results

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The dose–effect curves for the incidence of moist desquamation from the control groups used in these studies were not significantly different (p>0.25) from each other, therefore the data were pooled and used as the collective control for all three experiments.

Experiment 1
The dose-related incidence of moist desquamation in the skin of the rat foot in control animals that received single doses of ⁶⁰Co -rays to their hind feet and in test animals that received partial body priming doses of 7 Gy to the whole body except their hind feet 2 h, 4 h, 6 h or 24 h prior tothe irradiation of their hind feet is shown in Figure 1. There was strong evidence (p=0.34) for fitting parallel dose–effect curves, and a p-value of 0.61 for a test of goodness-of-fit indicated that the fitted model was adequate. A clearly significant association (p<0.0001) between the incidence of moist desquamation and the dose of radiation was obtained by computing Mantel–Haenzsel statistics. This implied that as the dose increased, the proportion of irradiated feet showing moist desquamation also increased. Similarly, a significant (p=0.04) association between the incidence of moist desquamation and the treatment group after adjustment for dose was observed. This test, which involved comparison of the entire dose–effect curves, implied that there was a dependency between the incidence of moist desquamation and the length of the time interval between partial body priming dose and subsequent hind foot irradiation.

View larger version (17K): [in this window] [in a new window]   Figure 1. Incidence of moist desquamation in the skin of rat foot in control animals that received single doses of 60Co -rays to their hind feet (•), and in animals that received 7 Gy partial body priming dose to their whole body except their hind feet at 2 h (), 4 h (), 6 h () or 24 h () prior to the irradiation of their hind feet.

Experiment 2
The dose-related incidence of moist desquamation in the skin of rat foot in control animals that received single doses of ⁶⁰Co -rays to their hind feet and in test animals that received partial body doses of 7 Gy 4 h prior to the irradiation of their hind feet is shown in Figure 2.

View larger version (16K): [in this window] [in a new window]   Figure 2. Incidence of moist desquamation in the skin of rat foot in control animals that received single doses of 60Co -rays to their hind feet (•), and in animals that received 2 Gy (), 4 Gy () or 7 Gy () partial body priming dose to their whole body except their hind feet 4 h prior to the irradiation of their hind feet.

Experiment 3
The dose-related incidence of moist desquamation in the skin of rat foot in control animals that received single doses of ⁶⁰Co -rays to their hind feet and in test animals that received 0.5 Gy or 1 Gy to their hind feet 4 h prior to the irradiation of the same feet is shown in Figure 3. There was strong evidence (p=0.53) for fitting parallel dose–effect curves, and a p-value of 0.32 for a test of goodness-of-fit indicated that the fitted model wasadequate. A clearly significant association (p<0.0001) between the incidence of moist desquamation and the dose of radiation was obtained by computing Mantel–Haenzsel statistics. This implied that as the dose increased, the proportion of irradiated feet showing moist desquamation also increased. There was no significant (p=0.08) association between the incidence of moist desquamation and the treatment group after adjustment for dose. It appeared that giving a small priming dose to the foot itself did not alter the sensitivity of foot skin. ED₅₀ values of 23.29±0.40 Gy and 22.97±0.38 Gy obtained for test animals with 0.5 Gy or 1 Gy local priming dose, respectively, were not significantly different (p=0.08) from the value of 22.53±0.16 Gy for control animals.

View larger version (15K): [in this window] [in a new window]   Figure 3. Incidence of moist desquamation in the skin of rat foot in control animals that received single doses of 60Co -rays to their hind feet (•), and in animals that received 0.5 Gy () or 1 Gy () local priming dose to their hind feet 4 h prior to the irradiation of the same feet.

	Discussion

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The results involving partial body irradiation prior to total foot irradiation suggest that the response of the skin of the rat foot was not determined solely by the radiation dose to the foot but was also dependent on the change of physiological state of the whole organism brought about by partial body irradiation. When the animals received a priming dose to their whole body except the hind feet, ED₅₀ values for moist desquamation of the foot varied with the quantity of priming dose and the time interval between the priming dose and subsequent foot irradiation.

The maximum variation was obtained for feet irradiated 4 h after delivery of a partial body priming dose of 7 Gy, with an ED₅₀ value of 25.25±0.29 Gy. This resulted in a small but significant DMF of 1.12±0.01, which indicated an increase in resistance of rat foot skin to irradiation as a consequence of alterations in the other parts of rat's body. This modification in response was still present at relatively lower levelsafter smaller priming doses and after longer time courses. However, the biggest effect was observed after 7 Gy, the largest partial body priming dose studied, delivered 4 h prior to hind foot irradiation.

According to cellular radiobiology, which has evolved around the concept of survival of target cells, moist desquamation of the skin of rat foot, as observed in these experiments, could be viewed as a direct consequence of reduction of the clonogenic cells of the epidermis to a critical level. A few surviving stem cells could not produce sufficient number of cells to replace those lost at the end of their useful life. If this was the only mechanism, and the cells in the irradiated foot responded to radiation independently of the othercells and of the physiological state of whole organism, the survival of the epidermal cells should have been determined by the dose ofradiation received by the epidermal cells of thefoot only. In such a case, the chance of development of moist desquamation should not be influenced by alterations in the physiological state of the animal's body, nor should it be modified by post-irradiation treatment.

Interest in the effects of irradiation that cannot be attributed to direct DNA damage [7] has recently been renewed by the description of a bystander effect induced by both high- and low-linear energy transfer radiation [8]. These involve mainly in vitro studies and point to unexpectedly high radiosensitivity at low doses leading to a significantly higher frequency of damage than would have been predicted simply by the number of radiation particles traversing the nucleus [9, 10]. Bystander effect also refers to radiation effects in cells that were not directly hit by the radiation [11]. Mutations induced by irradiation of cytoplasm by the microbeam have been reported [12]. The phenomenon of bystander effect predominates at low doses, reaches a plateau and appears to have higher expression after lower doses [13]. This is contrary to the present findings where the largest effect was observed after the higher priming dose. However, the present findings, which give support to the view of indirect effects influencing radiosensitivity, are supported by the results of a study by Mothersill and Seymour [13]. In a variety of different experiments, they showed that medium taken from irradiated human epithelial cells altered the clonogenic survival of unirradiated cells. Although their findings pointed to an increased radiosensitivity, not reduced sensitivity as in the present study, both results indicate the possibility of factors other than DNA damage caused directly by radiation having an influence on outcome. Mothersill and Seymour [13] concluded that a factor was secreted by the irradiated cells into the medium in the post-irradiation period that influenced the survival of cells that had not sustained any direct radiation damage. It was also reported that the sensitivity of endothelial cells in vitro was significantly affected by themicroenvironmental conditions [14]. These authors demonstrated that growing bovine aortic endothelial cells in dishes pre-coated with autologus natural basement membrane-like extracellular matrix increased their capacity to repair radiation lesions. Jolles and Harrison [15] reported that 4 Gy partial or total body dose prior to skin irradiation reduced radiation-induced leakage of dermal vasculature in rabbits. This reduction in response of the vasculature was found to be temporary, as the restoration of response occurred within 6 days and it was proportional to the extent of the pre-treated volume of tissue [16, 17]. This implies that some distant factors are involved in determining the tissue response to radiation. These authors suggested protease and its inhibitor to be an important factor in their observation.

It might be argued that the radioresistance observed after a partial body priming dose might have been due to stimulatory effects of low doses of ionizing radiation (radiation hormesis/adaptive response) induced by scattered irradiation received by shielded feet during partial body priming dose irradiation procedure. Scattered dose received by the feet during this procedure was measured to be 7% of the priming dose (50 cGy). This is therefore an unlikely explanation because radiation hormesis has been reported only after lower radiation doses of 1–15 cGy [18–20] and usually develops after relatively longer periods of a few days after low dose irradiation. In vitro, an increased X-ray sensitivity was observed after small doses of less than 0.3 Gy [21]. This was followed by an increased radioresistance for doses0.3–1 Gy. Radioresistance induced by prior treatment with ionizing radiation in human lymphocytes has also been reported [22] and reviewed [23]. Increased radioresistance after small doses ofradiation was attributed to the induction of new proteins involved in DNA repair [24]. The scattered dose of 50 cGy received by the rat feet during the partial body priming procedure was within this limit, therefore this was specifically tested in Experiment 3, results of which indicated that a small priming dose to the foot itself did not alter the sensitivity of foot skin. ED₅₀ values of 23.29±0.40 Gy and 22.97±0.38 Gy obtained for 0.5 Gy or 1 Gy priming dose, respectively, were not significantly different (p=0.08) from the value of 22.53±0.16 Gy for control animals. This indicated that the present findings were not due to an adaptive response.

Shirazi et al [25] reported an increase in the tolerance of mouse skin to irradiation by pre-exposure to ultraviolet B (UVB) light. UVB-induced increase in the tolerance of skin to X-irradiation was dependent on the time interval between the UVB exposure and X-irradiation. The effect was maximum for 14 days time interval and non-existent for a 6 h time interval. These observations were explained by an increase in thenumber of target epidermal cells induced by UVB. In the present studies, a maximum effect was observed around 4 h after the partial body priming dose. It is highly unlikely that an increase in the number of target epidermal cells could have occurred during this time.

The partial body priming dose irradiation procedure, which involved the whole body except the hind feet of the animals, resembled total body irradiation, which is an established radiotherapy procedure used as an immunosuppressive regimen in renal or bone marrow transplantation. The partial body priming dose of 7 Gy will be sufficient to cause a significant reduction in lymphocyte numbers in the test animals [26]. This may have suppressed the inflammatory response that is undoubtedly a contributing factor in the development of radiation injury.

Decreased oxygenation of epidermal target cells might be another explanation, but is not likely here because animals were flushed with a mixture of oxygen and 1–1.5% halothane and wereeffectively breathing pure oxygen during irradiations.

Total body irradiation alters the biochemistry of the body by overexpression or underexpression of biologically active substances [27–29]. It can be speculated that certain circulating biochemical substances, enzymes, cytokines or growth factors induced or inhibited after the partial body priming dose might have been involved in the induction of radioresistance in the animals that received a 7 Gy priming dose.

It can be concluded that the physiological state of the whole system is a factor in defining the response of tissues to irradiation. It is likely that the priming dose initiated the release of one or many biologically active factors that influenced the radiosensitivity of cells that had not received any direct irradiation. The present study was only an observational one and the cause of this induced radioresistance was not studied. Further work is needed to determine the nature of these biologically active agents and the cascade of events by which their perturbation by the partial body priming dose might have resulted in the modification of the response of the rat foot skin to irradiation. Furthermore, the findings support the view that the development of radiation lesions is not simply determined by the reduction of target cells in the skin.

Received for publication February 26, 2001. Revision received June 29, 2001. Accepted for publication August 13, 2001.

	References

Top Abstract Introduction Material and methods Results Discussion References

 

1. Puck TT, Marcus PI. Action of X-rays on mammalian cells. J Exp Med 1956;103:653–66.[Abstract]
2. Lea DE. Actions of radiations on living cells (2nd edn). Cambridge, UK: Cambridge University Press, 1962.
3. Chadwick KH, Leenhouts HP. A molecular theory of cell survival. Phys Med Biol 1973;18:78–87.[Medline]
4. Withers HR, Peter LJ, Kogelnik HD. The pathobiology of late effects of irradiation. In: Meyn RE, Withers HR, editors. Radiation biology in cancer research. New York: Raven Press, 1980:439–48.
5. Wheldon TE, Michalowski AS. Alternative models for the proliferative structure of normal tissues and their response to irradiation. Br J Cancer 1986; 53(Suppl. vii):382–5.
6. Finney DJ. Probit analysis (2nd edn). London: Cambridge University Press, 1964.
7. Michalowski A. Radiopathology of normal tissues. Are clonogenic survival and cell kinetics all that matter? In: Stigbrand T, et al, editors. Somatic and genetic effects of ionising radiation. Proceedings of the XVth Berzelius Symposium, 1988 November 10–11; Umea. Umea, Sweden: Nyheternas Tryckeri AB, 1988.
8. Seymour CB, Mothersill C. Relative contribution of bystander and targeted cell killing to the low-dose region of the radiation dose-response curve. Radiat Res 2000;153:508–11.[Medline]
9. Nagasawa H, Little JB. Induction of sister chromatid exchanges by extremely low doses of alpha-particles [see comments]. Cancer Res 1992;52:6394–6.[Abstract/Free Full Text]
10. Nagasawa H, Little JB. Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect. Radiat Res 1999;152:552–7.[Medline]
11. Belyakov OV, Malcolmson AM, Folkard M, Prise KM, Michael BD. Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts. Br J Cancer 2001;84:674–9.[Medline]
12. Wu LJ, Randers Pehrson G, Xu A, et al. Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells. Proc Natl Acad Sci USA 1999;96:4959–64.[Abstract/Free Full Text]
13. Mothersill C, Seymour C. Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells. Int J Radiat Biol 1997;71:421–7.[Medline]
14. Fuks Z, Vlodavsky I, Andreeff M, McLoughlin M, Haimovitz-Friedman A. Effects of extracellular matrix on the response of endothelial cells to radiation in vitro. Eur J Cancer 1992;28A:723–4.
15. Jolles B, Harrison RG. Proteases and the depletion and restoration of skin responsiveness to radiation. Nature 1965;205:920–1.
16. Jolles B, Harrison RG. Abolition and restoration of tissue responsiveness to radiation. Br J Radiol 1964;37:803.
17. Jolles B, Harrison RG. Enzymatic processes and vascular changes in the skin radiation reaction. Br J Radiol 1966;39:12–8.[Medline]
18. Feinendegen LE, Bond VP, Booz J, Muehlensiepen H. Biochemical and cellular mechanisms of low-dose effects. Int J Radiat Biol 1988;53:23–37.
19. Yonezawa M, Misonoh J, Hosokawa Y. Two types of X-ray-induced radioresistance in mice: presence of 4 dose ranges with distinct biological effects. Mutat Res 1996;358:237–43.[Medline]
20. Miyachi Y. Analgesia induced by repeated exposure to low dose X-rays in mice, and involvement of the accessory olfactory system in modulation of the radiation effects. Brain Res Bull 1997;44:177–82.[Medline]
21. Marples B, Joiner MC. The elimination of low dose hypersensitivity in Chinese hamster V79-379A cells by pretreatment with X rays or hydrogen peroxide. Radiat Res 1993;141:160–9.
22. Olivieri G, Bodycote J, Wolff S. Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. Science 1984;223:594–7.[Abstract/Free Full Text]
23. Shadley JD. Chromosomal adaptive response in human lymphocytes. Radiat Res 1994;138:S9–S12.[Medline]
24. Marples B, Lambin P, Skov KA, Joiner MC. Low dose hyper-radiosensitivity and increased radioresistance in mammalian cells. Int J Radiat Biol 1997;71:721–35.[Medline]
25. Shirazi A, Liu K, Trott K-R. Exposure to ultraviolet B radiation increases the tolerance of mouse skin to daily X irradiation. Radiat Res 1996;145:768–75.[Medline]
26. Muller IW. Cytokinetics of the megakaryocyte system in irradiated and non-irradiated rats and human. Inaugural Dissertation, Albert-Ludwigs University, Freiburg, 1967.
27. Abdul-Hameed JMA, Haley TJ. Plasma and adrenal gland corticosterone levels after X-ray exposure in rats. Radiat Res 1964;23:620–9.
28. Steel LA, Hughes HN, Walden TL. Quantitative, functional and biochemical alterations in the peritoneal cells of mice exposed to whole-body gamma-irradiation. I. Changes in cellular protein, adherence properties and enzymatic activities associated with platelet-activating factor formation and inactivation and arachidonate metabolism. Int J Radiat Biol 1988;53:943–64.
29. Navratil L, Pospisil J, Blehova Z. Changes of 6-keto PGF₁ concentration in plasma and vessel wall abd TxB₂ in plasma of whole-body irradiated rats in the early stages of irradiation. Prostaglandins Leukot Essent Fatty Acids 1990;41:39–43.[Medline]

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