[Radiobiological aspects of diagnostic X-ray use in dentistry]. / Radiobiologische aspecten van tandheelkundige röntgendiagnostiek.

Soon after the discovery of X-rays, it became clear that their use can cause detrimental effects. The field of radiobiology deals with these detrimental effects. In this article, the theoretical concepts of radiobiology relevant to diagnostic X-ray use are presented. The effects of radiation on living tissues, the relationship between dose and effect, and a translation of these effects into the dental application are discussed. X-rays cannot be considered to be harmless even when used at the relatively low doses as in dentistry. If applied with justification and optimization, the risk to the patient will, however, be small.

Studies of dose-fractionation on early and late responses in pig skin: a reappraisal of the importance of the overall treatment time and its effects on radiosensitization and incomplete repair.

Studies in pig skin have examined the effects of dose fractionation on the acute radiation response. The variation in ED50 values for moist desquamation for doses given as 1-48 fractions over less than or equal to 16 days were best fitted by a log-log plot of iso-effect dose against the number of fractions; the slope of this plot indicated a fraction number exponent (N) of 0.42 +/- 0.007. Based on the assumptions made in applying the linear-quadratic (LQ) model, the alpha/beta ratio was found to decrease with decreasing per fraction: for doses given as 6-27 Gy per fractions the alpha/beta ratio was 8.74 +/- 0.48 Gy, whereas for doses of 2.55-6 Gy per fraction it was only 0.85 +/- 0.29 Gy. A simple approach to a time factor could not be used to calculate iso-effect doses for acute reactions in pig skin when treatment time was increased from less than or equal to 16 days to 28-39 days. This was due to the opposing effects of radiosensitization and repopulation when the cell cycle time of epidermal basal cells was shortened. For late dermal necrosis in pig skin, repair of sublethal damage was not completed in 24 hr. This finding has a significant effect on the interpretation of the results of fractionation studies using this late endpoint. Expressed in terms of a simple power-law function, there was a significant change in the fraction number exponent "N" from 0.43 +/- 0.007 to 0.37 +/- 0.006 for the complete and incomplete repair data, respectively. Many of the fractionation effects reported for acute and late damage to pig skin would appear to be in excellent agreement with those for human skin.

The radiation response of the cervical spinal cord of the pig: effects of changing the irradiated volume.

PURPOSE: An investigation of the field size effect for the cervical spinal cord of the pig after single doses of gamma-rays. In this study, clinically relevant volumes of the spinal cord were irradiated. METHODS AND MATERIALS: The effects of the local irradiation of different lengths of the spinal cord (2.5 cm, 5.0 cm, and 10.0 cm) have been evaluated in mature pigs (37-43 weeks). Single doses of 25-31 Gy were given using a 60Co gamma-source, at a dose rate of 0.21-0.30 Gy/min. The incidence of radiation-induced paralysis was used as the endpoint. The data were analyzed using probit analysis and a normal tissue complication probability (NTCP)-model. RESULTS: Twenty-five animals out of a total of 53 developed paralysis, with histological evidence of parenchymal and vascular changes in their white matter. The slope of the dose-response curves decreased with the decrease in field size; however, there was no significant difference at the radiation dose associated with a 50% incidence of paralysis (ED50) irrespective of the method of analysis. The ED50 values +/- standard errors (+/- SE) were 27.02 +/- 0.36 Gy, 27.68 +/- 0.57 Gy, and 28.28 +/- 0.78 Gy for field lengths of 10, 5, and 2.5 cm, respectively. Analysis of the data with a normal tissue complication probability (NCTP) model gave similar results. The latent period for paralysis was 7.5-16.5 weeks with no significant differences between dose and field size. CONCLUSION: No significant field size-related differences in response were detectable in the cervical spinal cord of mature pigs after single dose irradiations, specifically at a clinically relevant level of effect (< ED10).

Pulsed dose rate and fractionated high dose rate brachytherapy: choice of brachytherapy schedules to replace low dose rate treatments.

PURPOSE: Pulsed dose rate (PDR) brachytherapy is a new type of afterloading brachytherapy (BT) in which a continuous low dose rate (LDR) treatment is simulated by a series of "pulses," i.e., fractions of short duration (less than 0.5 h) with intervals between fractions of 1 to a few hours. At the Dr. Daniel den Hoed Cancer Center, the term "PDR brachytherapy" is used for treatment schedules with a large number of fractions (at least four per day), while the term "fractionated high dose rate (HDR) brachytherapy" is used for treatment schedules with just one or two brachytherapy fractions per day. Both treatments can be applied as alternatives for LDR BT. This article deals with the choice between PDR and fractionated HDR schedules and proposes possible fractionation schedules. METHODS AND MATERIALS: To calculate HDR and PDR fractionation schedules with the intention of being equivalent to LDR BT, the linear-quadratic (LQ) model has been used in an incomplete repair formulation as given by Brenner and Hall, and by Thames. In contrast to earlier applications of this model, both the total physical dose and the overall time were not kept identical for LDR and HDR/PDR schedules. A range of possible PDR treatment schedules is presented, both for booster applications (in combination with external radiotherapy (ERT) and for BT applications as a single treatment. Because the knowledge of both alpha/beta values and the half time for repair of sublethal damage (T 1/2), which are required for these calculations, is quite limited, calculations regarding the equivalence of LDR and PDR treatments have been performed for a wide range of values of alpha/beta and T 1/2. The results are presented graphically as PDR/LDR dose ratios and as ratios of the PDR/LDR tumor control probabilities. RESULTS: If the condition that total physical dose and overall time of a PDR treatment must be exactly identical to the values for the corresponding LDR treatment regimen is not applied, there appears to be less need for strong fractionation in PDR schedules. If the overall time is at least as long as that of the LDR schedule and if the total physical dose is (slightly) adapted, PDR schedules can be designed using longer pulse intervals of up to 3 h. Schedules with sufficiently long intervals have significant logistic advantages in terms of patient care and treatment tolerance. However, in general, PDR schedules that apply more fractionation have a lower risk of overdosing normal tissues in comparison to fractionated HDR schedules. Applying probable ranges for the values of alpha/beta and T 1/2, the model calculations indicate that the differences in effects between the proposed fractionated HDR and PDR schedules could be rather small. To detect the magnitude of these differences, (randomized) clinical studies with rather large patient groups might be needed. CONCLUSIONS: Pulsed dose rate treatment schedules with longer intervals of up to 3 h appear adequate to replace LDR treatment schedules. Whether PDR schedules can, indeed, replace LDR treatment schedules and whether they offer detectable advantages over schedules with less fractionation (fractionated HDR) should be tested in clinical studies.

A new model of radiation-induced myelopathy: a comparison of the response of mature and immature pigs.

PURPOSE: The development of an experimental model of radiation-induced myelopathy in the pig which would facilitate the study of the effects of clinically relevant treatment volumes. METHODS AND MATERIALS: The effects of local spinal cord irradiation, to a standard 10 x 5 cm field, have been evaluated in mature (37-42.5 weeks) and immature (15.5-23 weeks) pigs. Irradiation was with single doses of 60Co gamma-rays at a dose-rate of 0.21-0.65 Gy/min. The incidence of paralysis was used as an endpoint. RESULTS: Irradiation of mature animals resulted in the development of frank paralysis with animals showing combined parenchymal and vascular pathologic changes in their white matter. These lesions, in common with those seen in patients, had a clear evidence of an inflammatory component. The latency for paralysis was short, 7.5-16.5 weeks, but within the wide range reported for patients. However, it was shorter than that reported in other large animal models. The ED50 value (+/- SE) for paralysis was 27.02 +/- 0.36 Gy, similar to that in rats taking into account dose-rate factors. The irradiation of immature pigs only resulted in transient neurological changes after doses comparable to those used in the mature animals, ED50 value (+/- SE) 26.09 +/- 0.37 Gy. The reasons for these transient neurological symptoms are uncertain. CONCLUSION: A reliable experimental model of radiation-induced myelopathy has been developed for mature pigs. This model is suitable for the study of clinically relevant volume effects.

Modification of the radiation response of pig skin by manipulation of tissue oxygen tension using anesthetics and administration of BW12C.

The importance of tissue oxygen tension on radiosensitivity was studied by examining modifications in the incidence of moist desquamation in pig skin after irradiation with strontium-90 plaques. The effects were analyzed using quantal dose-response data and comparisons were made using ED50 values for moist desquamation. Under standard anesthetic conditions of 2% halothane, approximately 70% oxygen, and approximately 30% nitrous oxide, the ED50 value (+/- SE) for moist desquamation was 27.32 +/- 0.52 Gy with no significant variation in radiosensitivity between dorsal, lateral, and ventral skin sites on the flank. Irradiation with 2% halothane and air increased the ED50 to 31.25 +/- 0.94 Gy, primarily due to an increased radioresistance of the dorsal sites. When combined with BW12C, a drug which binds oxygen selectively to hemoglobin and hence reduced the oxygen availability to tissues, a further increase in the ED50 values was observed. This was approximately 39 Gy with BW12C concentrations of 30 mg/kg and 50 mg/kg b.w. of BW12C, indicating a dose modification factor (DMF) of approximately 1.26. However, when animals were breathing the standard gas mixture, this DMF was reduced to 1.15 for 30 mg/kg of BW12C, indicating that a higher level of oxygen partly counteracted the effects of the drug in these studies with BW12C. The greatest variability in radiosensitivity was seen in the dorsal fields. This suggested complex physiological adaptation, a phenomenon that might also explain the absence of any modification of the radiation response when 100 mg/kg of BW12C was used.

Induction of hypoxia in normal and malignant tissues by changing the oxygen affinity of hemoglobin--implications for therapy.

The drug BW12C, which increases the oxygen affinity of hemoglobin, reduces oxygen availability to tissues. This results in protection against radiation damage to the hemopoietic system and epidermal Langerhans cells in CBA mice. The drug also protects against beta-irradiation damage in pig epidermis. BW12C increases the hypoxic cell fraction in tumors and histological examination of an experimental T cell lymphoma shows that the induced hypoxia leads to tumor necrosis.

Amelioration of both early and late radiation-induced damage to pig skin by essential fatty acids.

PURPOSE: To evaluate the possible role of essential fatty acids, specifically gamma-linolenic and eicosapentaenoic acid, in the amelioration of early and late radiation damage to the skin. METHODS AND MATERIALS: Skin sites on the flank of 22-25 kg female large white pigs were irradiated with either single or fractionated doses (20 F/28 days) of beta-rays from 22.5 mm diameter 90Sr/90Y plaques at a dose rate of approximately 3 Gy/min. Essential fatty acids were administered orally in the form of two 'active' oils, So-1100 and So-5407, which contained gamma-linolenic acid and a mixture of that oil with eicosapentaenoic acid, respectively. Oils (1.5-6.0 ml) were given daily for 4 weeks prior, both 4 weeks prior and 10-16 weeks after, or in the case of one single dose study, just for 10 weeks after irradiation. Control animals received a 'placebo' oil, So-1129, containing no gamma linolenic acid or eicosapentaenoic acid over similar time scales before and after irradiation. Acute and late skin reactions were assessed visually and the dose-related incidence of a specific reaction used to compare the effects of different treatment schedules. RESULTS: A reduction in the severity of both the early and late radiation reactions in the skin was only observed when 'active' oils were given over the time course of the expression of radiation damage. Prior treatment with oils did not modify the radiation reaction. A 3.0 ml daily dose of either So-1100 or So-5407 given prior to, but also after irradiation with single and fractionated doses of beta-rays produced the most significant modification to the radiation reactions, effects consistent with dose modification factors between 1.06-1.24 for the acute reactions of bright red erythema and/or moist desquamation, and of 1.14-1.35 for the late reactions of dusky/mauve erythema and dermal necrosis. There was the strong suggestion of an effect produced by the 'placebo' oil, So-1129, after higher daily doses of oil. CONCLUSIONS: Essential fatty acids can modulate normal tissue reactions when given over the time when radiation damage is normally expressed. Dose modification factors suggest that a > or = 10% higher dose is required to produce the same level of normal tissue injury. Clinical application of selected essential fatty acids at appropriate doses may lead to a significant increase in the therapeutic gain in patients treated for cancer by radiotherapy.

The kinetics of repair for sublethal radiation-induced damage in the pig epidermis: an interpretation based on a fast and a slow component of repair.

The kinetics of repair of sublethal damage were studied for the epidermis of the pig after beta-irradiation. Total doses were given as 3 or 4 fractions with interfraction intervals that ranged from 0.08 h-8.0 h. Three different methods were used to analyse the results: (a) the incomplete repair (IR)-model; (b) theoretical curves showing changes in iso-effect doses, based on the IR-model, were compared with experimentally derived iso-effect doses; and (c) the percentage unrepaired dose was calculated using differences in the iso-effect doses (ED50) for moist desquamation for the various interfraction intervals. Although mono-exponential kinetics are assumed in the IR-model, the half time for repair (T1/2) was not unique, but increased with the increase in interfraction interval. For 3 fractions, with an interfraction interval of 0.25 h, a T1/2 value of 0.27 h (0.19-0.43 h) was obtained, while an interval of 4 h gave a significantly higher T1/2 value of 1.40 h (0.01-2.28 h). Similar results were obtained with the 4-fraction schedule. The experimentally derived iso-effect doses for short interfraction intervals were above the theoretical curve indicating a faster rate of repair than anticipated, based on the IR-model. Iso-effect doses for longer interfraction intervals were below the theoretical curve suggesting a slower rate of repair. For both the 3- and 4-fraction data the percentage unrepaired dose, as a function of the interfraction interval, was significantly better fitted by a bi-exponential equation than a mono-exponential equation (p less than 0.05). Two distinct components of repair were resolved suggesting a fast and a slow component of repair with T1/2 values of approximately 0.14 h and of approximately 2.7 h, respectively. Thus all three methods of analysis suggest that the repair of sublethal radiation-induced damage to pig skin are better explained by bi-exponential rather than mono-exponential kinetics.

Repair and recovery in the epithelial and vascular connective tissues of pig skin after irradiation.

Using split-dose experiments, with varying time intervals between two equal fractions, the total repair capacity and the time of onset of additional recovery was determined for both early and late responses in pig skin. The early epidermal response was studied after beta-irradiation and 250 kV X-rays were used to investigate dermal changes. Based on the results of seven separate single dose studies the ED50 value ( +/- SE) for early moist desquamation was 27.76 +/- 0.91 Gy. With intervals of one and 14 days between two equal fractions similar ED50 values of 35 Gy were obtained. This suggested a recovered dose of approximately 7 Gy for epithelial desquamation, a repair capacity for sublethal injury of 20-25%. Additional recovery possibly due to repopulation, was observed with intervals of greater than or equal to 21 days between fractions. The rate of additional recovery was linearly related to the time interval between doses and was equivalent to 74 cGy/day. Recovery from the first dose was complete within 6 weeks. Evidence for radiation-induced tissue hypoxia was obtained when longer time intervals between doses were used. The more subjective early erythema reaction was also assessed. This reaction produced a similar estimate for the repair capacity and for the time of onset of recovery to that obtained using moist desquamation. This agreement was not maintained with intervals of greater than or equal to 28 days between doses due to an artefact associated with the way erythema reactions were assessed. After irradiation with single doses of X-rays ED50 values of 18.59 +/- 0.49 and 20.53 +/- 0.35 Gy were obtained for the dermal reactions of dusky/mauve erythema and necrosis, respectively. The recovered doses for the dermal responses, with intervals of 1 and 28 days between fractions, were similar, approximately 4.2 Gy, indicating a total repair capacity of 20-25%. Additional dermal recovery was seen only with intervals of greater than 28 days between doses. There was no evidence for "slow repair". Surprisingly complete recovery from the first dose was suggested with an interval of 16 weeks between doses. This finding might be influenced by radiation-induced hypoxia. The time of onset of additional repopulation/recovery and the latency for tissue impairment in epidermal and dermal tissues in pig skin were compared with those for other early and late responding tissues.

Preclinical studies with the Faure high energy neutron facility: response of pig skin to fractionated doses of fast neutrons (66 MeVp----Be).

The early and late responses of pig skin to fractionated doses of both unfiltered and filtered (i.e. hardened) neutrons using the Faure neutron therapy facility (66 MeVp----Be) were determined and compared with those following fractionated doses with 60Co gamma-rays. Dose-effect curves for the quantal responses of moist desquamation (early epithelial response) and dermal necrosis (late response) were fitted by probit analysis and ED50 values obtained. For a neutron fractionation scheme comprised of 12 fractions in 26 days, and using an unfiltered beam, the ED50 values for moist desquamation and dermal necrosis were 18.67 +/- 2.22 and 22.25 +/- 0.48 Gy, respectively, whereas in the case of the filtered beam, the corresponding ED50 values were 24.78 +/- 1.44 and 23.30 +/- 0.47 Gy. In order to provide a comparison, the values for 24 fractions of 60Co gamma-rays given in 39 days (a clinical protocol used in the Groote Schuur Hospital) were 74.02 +/- 2.92 and 66.72 +/- 1.93 Gy for moist desquamation and dermal necrosis, respectively. For the unfiltered beam, values for the comparative biological effectiveness (CBE) were 3.96 and 3.00 for the early and late skin response, respectively. The corresponding CBE values were for the filtered beam 2.99 and 2.86. These results for the Faure neutron therapy facility can be extrapolated to the human situation with a high degree of confidence, so that the neutron dose which would yield acceptable skin damage in patients may be determined using the data presented here.

Effect of age on radiation-induced early changes of rat rectum. A histological time sequence.

BACKGROUND AND PURPOSE: Radiation treatment of the elderly (> 75 years) is often modified due to an assumed decrease in normal tissue tolerance in this age group. Since more radiobiological data concerning normal tissue toxicity as a function of age are needed, a histological study of age-related radiation changes of the rectum was performed. MATERIALS AND METHODS: The rectum of young and old female Wistar rats (12 and 78 weeks, respectively) was irradiated with single doses of 22 and 39 Gy. The field size was 1.5 x 2.0 cm. The animals were sacrificed at 1, 2, 4 and 10 weeks after treatment. To evaluate radiation damage, 12 histological parameters were scored in four areas of the rectum. A total radiation injury score was calculated. The number of proliferative epithelial cells was evaluated by 5-bromo-2'-deoxyuridine labeling. RESULTS: Some age-related histological differences were observed; especially, the incidence of ulceration and vascular occlusion was higher in the older group. In the low dose group of the older animals, 60% showed ulceration, which was 0% for the young low dose animals. Severe vascular changes occurred early and were more extensive in older animals (4 weeks) than in the younger group (10 weeks). In the area adjacent to the treatment field, cell proliferation increased significantly in older rats at 1 week after 22 Gy, which did not occur in the young group. CONCLUSIONS: Discrete radiation-induced histological differences were observed between the rectum of young and old Wistar rats, especially in the development of ulceration and vascular changes. Although the survival of these Wistar rats in earlier studies was not affected by age, the impact of the observed histological differences for their importance in the long-term is currently being investigated.

A comparison of the radiation response of the epidermis in two strains of pig.

The response of the epidermis was compared in two strains of pig, the English Large White and the Göttinger Miniature, after irradiation with 90Sr beta rays. The effects of two types of anesthesia were also tested in pigs of each strain, a volatile gas mixture of approximately 70% oxygen, approximately 30% nitrous oxide, and 2% halothane, and an intravenously administered narcotic azaperon/etimodat with the animals breathing air. Strain- and anesthetic-related changes were compared on the basis of dose-effect curves for the incidence of moist desquamation from which ED50 values (+/- SE) were determined, i.e., the dose required to produce this effect in 50% of the fields irradiated. For English Large White pigs anesthetized with the volatile gas mixture, an ED50 of 27.32 +/- 0.52 Gy was obtained for moist desquamation. Irradiation with the azaperon/etomidat anesthesia in this strain of pig produced a significantly higher ED50 of 33.36 +/- 0.76 Gy (P less than 0.001). This appeared to be related to the fact that the animals were breathing air, i.e., a lower oxygen concentration (approximately 21%), at the time of irradiation. For the Göttinger Miniature pig the ED50 values for moist desquamation were 38.93 +/- 3.12 Gy and 43.36 +/- 1.34 Gy while using the gaseous anesthetic mixture and the azaperon/etomidat anesthesia with the animals breathing air, respectively. These ED50 values are 10-11 Gy higher than those obtained for the English Large White pig under identical conditions of anesthesia, which resulted in a strain difference ratio of approximately 1.35. Radiation under the volatile gas mixture anesthesia resulted in a uniform irradiation response over the skin of the flank in both strains of pig. Radiation under azaperon/etomidat anesthesia resulted in a nonuniform skin response over the flank. The ED50 for moist desquamation was significantly higher in dorsal sites on the flank compared with the ventral area of English Large White pigs; a similar trend was seen in Göttinger Miniature pigs. This difference in the radiosensitivity over the flank skin while the animals are under azaperon/etomidat anesthesia at the time of irradiation was associated with the animals breathing air and is in agreement with findings published previously for animals under halothane anesthesia and breathing air.

The kinetics of repair of sublethal radiation-induced damage in pig skin: studies with multiple interfraction intervals.

The kinetics of the repair of radiation-induced sublethal damage (SLD) was studied for the epidermis of the pig. A total of either 7 or 14 interfraction intervals with incomplete repair was achieved by giving 28 fractions either as 7 x 2 fractions/day plus a top-up dose of 17 Gy (half tolerance) or as 14 x 2 fractions/day. The dose per fraction ranged from 1.96-4.82 Gy. A total of 9 intervals ranging from 0.17 h up to 8 h between fractions was used. The incidence of moist desquamation, as an estimate of acute epidermal response, was used as an end point to establish dose-effect relationships. The data were analyzed using either the incomplete repair model of Thames, assuming mono-exponential repair kinetics, or a modified version of the incomplete repair model, assuming bi-exponential repair of sublethal damage. Both methods of analysis allowed for the longer overnight interval between fractions. Analysis assuming mono-exponential repair gave a T1/2 of 0.74 h for the combined data, although there was a trend toward a longer half-time when only the longer interfraction intervals ( > 1.0 h) were used in the analysis. A further analysis using the modified version of an incomplete repair model gave a fast and a slow component of repair with significantly different half-times of 0.09 and 4.5 h, respectively. Varying the number of incomplete repair intervals by replacing half the number of fractions with a single half-tolerance top-up dose did not modify the kinetics of repair significantly, in terms of either the repair half-times or the proportion of repair associated with a fast and slow component. Reanalysis of data published previously for 3 and 4 fractions using the modified incomplete repair model again resulted in two components of repair, represented by the significantly different half-times of 0.17 and 3.0 h. These values were similar to those obtained from the multiple-fraction experiment. These data clearly demonstrate that an acutely responding tissue is associated with a long T1/2 for the repair of SLD which is independent of the dose per fraction. For accelerated fractionation schedules in the clinic, using multiple fractions per day, these results suggest a need to control the intervals between fractions carefully and when appropriate to reduce the total dose to avoid serious normal-tissue complications.

Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons.

The longitudinal outgrowth of spermatogenetic colonies arising from stem cells that survived neutron doses of 150, 300, and 350 rad was studied up to 30 weeks in histological sections of CBA mouse testes. Two methods were used: (1) the repopulation index (RI) as a measure of the length of total colonies per testis and (2) measurement of the individual length of all colonies in serially sectioned testes 4 and 15 weeks after 300 rad and 15 weeks after 350 rad. The mean initial growth of the colonies is linear up to 8, 15, and 20 weeks after 150, 300, and 350 rad, respectively. Although after these doses the mean initial colony growth rate did not differ significantly (about 27 microns/day), both methods showed that the colonies grow about 20% slower after 350 rad. Screening of individual colonies revealed a great variation in colony length per testis and a higher frequency of short colonies with higher neutron doses. Counting of colonies after 300 rad showed that all surviving stem cells had started to form a colony within 4 weeks after irradiation. The development of spermatogenetic cells to mature spermatozoa was studied after 100, 150, 300, and 350 rad in sections of repopulating tubules used for RI determination as well as in serial sections of individual colonies. Although after 300 and 350 rad spermatogenetic cell types beyond the stage of young spermatocytes reappeared 1 week late, we found no great disturbances in the regular reappearance of the successive spermatogenetic cell types after irradiation. However, from the study of individual colonies it appeared that colonies differ widely in their development even within one testis. Moreover, the frequency of less developed colonies was higher after 350 rad than after 300 rad. Our data suggest that this retardation in the reappearance of further developed cells is caused by a delay in the production of developed cells in spermatogonia in an increasing fraction of the colonies after higher neutron doses. Even in fully developed colonies the production of differentiating spermatogenetic cell types was subnormal after 300 and 350 rad. This was caused by an extensive cell degeneration in the colonies as well as by a tendency of the undifferentiated and/or A1-spermatogonial population to increase its own number at the cost of the production of further developed cells.

Manipulation of the radiosensitivity of pig epidermis by changing the concentration of oxygen and halothane in the anaesthetic gas mixture.

A gas mixture of halothane, oxygen and nitrous oxide has been used to anesthetize pigs for irradiation. The effects of various concentrations of halothane and oxygen on the radiosensitivity of the epidermis were examined after irradiation with single doses of beta-rays from strontium-90 plaques. The incidence of moist desquamation was used as an endpoint, and experiments were compared on the basis of the dose associated with a 50 per cent incidence of moist desquamation (ED50 +/- SE). For pigs inspiring an anaesthetic gas mixture of 2 per cent halothane, approximately 70 per cent oxygen and approximately 30 per cent nitrous oxide the ED50 for moist desquamation was 27.32 +/- 0.52 Gy. A similar ED50 value of 27.39 +/- 1.20 Gy was obtained when 4 per cent halothane was used in place of 2 per cent. When the pigs were breathing air (approximately 21 per cent oxygen) in place of oxygen and nitrous oxide the ED50 values were increased significantly to 31.25 +/- 0.94 Gy and 33.72 +/- 1.08 Gy for 2, and 4 per cent halothane, respectively. This change in the radiosensitivity of the epidermis was represented by dose modification factors of approximately 1.13 and approximately 1.23 for 2 and 4 per cent halothane, respectively. Irradiation with a high oxygen concentration in the inspired gas mixture did not result in any significant variation of the dose required to produce moist desquamation in 50 per cent of the fields irradiated for dorsal, lateral and ventral positioned skin fields on the flank. However, pigs breathing air and halothane during irradiation showed marked differences in the radiosensitivity of the various sites on the flank, with ED50 values for moist desquamation of approximately 37 Gy and 26-30 Gy for dorsal and ventral positioned fields, respectively. This marked difference in radiosensitivity suggests variations in the physiological compensation over the flank when pigs are breathing oxygen at low concentrations under anaesthesia.

Repair kinetics in pig epidermis: an analysis based on two separate rates of repair.

Pig skin was irradiated using 90Sr/90Y plaques and the dose-related incidence of induced moist desquamation was determined. The repair of radiation-induced sublethal damage (SLD) was studied by fitting these response data to the generalized LQ equation for incomplete repair using quasilikelihood methods with binomial statistics, and either a Poisson or logistic link to relate the probability of response to the covariates. A Poisson response analysis based on the assumption that SLD was governed by two repair processes gave estimated repair half-times of 0.20 [(95% confidence limits) 0.12, 0.34] and 6.6 [4.3, 10.0] h. The estimates of the short and long repair half-times were significantly different, although there was no significant difference between the results using the Poisson and logistic modes of analyses. The partition coefficient for the longer repair process was 0.5 [0.34, 0.71] indicating that about 33% of SLD-derived lethal damage is associated with the longer repair process in the case of 'complete repair' protocols. However, this proportionation is, in general, protocol dependent for incomplete repair protocols. A chi2 test on the residual deviance showed that the assumption of two repair processes for SLD gave a superior fit to the data than a single repair process at a significance level >99%. The radiation dose to the assumed target cell population depends upon their depth from the skin surface, due to the relatively short range of the electron emission from the 90Sr/90Y plaques. However, further modelling analyses have shown that the estimated repair half-times were independent of the assumed target cell distribution in the skin. This is in contrast with the alpha/beta ratio, where different (clinically significant) estimates can be obtained depending upon the assumed target cell distribution. If the target cells were at 16 micrometer depth from the surface of the skin, the estimated value for the alpha/beta ratio using the biphasic repair model would be 4.6[3.6, 5.6] Gy(Poisson analysis). However, the estimates decrease with the assumed depth (distribution) of the target cells.

Protection of pig epidermis against radiation-induced damage by the infusion of BW12C.

BW12C, which was developed as an agent for the treatment of sickle cell anaemia, increases the binding of oxygen to haemoglobin and hence reduces the availability of oxygen to tissues. Due to these changes in oxygen availability BW12C could act as a protector against radiation-induced injury to normal tissues. In this study the potential value of BW12C, as a radioprotector, was studied in the irradiated epidermis of the pig. The infusion of BW12C caused an instant left shift of the oxygen dissociation curve, an effect that lasted for approximately 1.5 h. This left shift in the oxygen dissociation curves increased with increasing dose of the drug. There appeared to be no long-term systemic effects produced by doses of 20-100 mg/kg of BW12C. In the first 90 min after the infusion of BW12C skin fields were irradiated with single doses of beta-rays from strontium-90 plaques. The incidence of moist desquamation was used as an endpoint for assessing the severity of the radiation response. With animals breathing approximately 70% oxygen in the anaesthetic gas mixture, the ED50 values for moist desquamation were 30-31 Gy after a dose of 30 and 50 mg/kg, and 37-38 Gy for 75 and 100 mg/kg doses of BW12C. These ED50 values were significantly higher than the value of 27.3 Gy for radiation alone. This indicated dose modification factors (DMF) with mean values of approximately 1.13 and approximately 1.40 for irradiation following the infusion of low (30-50 mg/kg) and high (75-100 mg/kg) doses of the drug, respectively. With the animals breathing air (approximately 21% of oxygen) in the 2% halothane anaesthesia gas mixture, irradiation in the presence of 30 and 50 mg/kg of BW12C resulted in ED50 values of approximately 39 Gy for moist desquamation, which was significantly higher than the value of 31.2 Gy for radiation alone. Surprisingly, a higher dose of 75 mg/kg of BW12C resulted in a lower ED50 value for moist desquamation of 34.38 Gy. Irradiation in the presence of a dose of 100 mg/kg of BW12C produced an ED50 value which was not significantly different from that for radiation alone. In the situation where animals were breathing air (approximately 21% oxygen) during irradiation a DMF of 1.14 was obtained for irradiation alone, when the results were compared with those for irradiation alone with approximately 70% oxygen in the anaesthetic gas mixture.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling.

A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing > or = 16 cells were counted as colonies. These colonies were first seen on day 14/15 after irradiation. The number of colonies per cm2, as a function of skin surface dose, yielded a cell survival curve with a D0 (+/- SE) of 3.87 +/- 0.57 Gy. The ED50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm2. After higher doses, abundant smaller colonies of 4-8 BrdU-positive cells were seen and these were more radioresistant, as represented by higher D0 values.

Changes in epidermal radiosensitivity with time associated with increased colony numbers.

Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of these small colonies were abortive and did not progress to larger colonies. There was no statistical evidence for significant variations in the interanimal responses.