R ADIOTHERAPY

aONcO~~GY ELSEVIER Radiotherapy and Oncology 34 (1995) 203-209

Renal damage after total body irradiation in a mouse model for bone marrow transplantation: effect of radiation dose rate

Akmal Safwat*a3b, Ole S. Nielsenb, Hoda Abd El-Bakky”, Jens Overgaardb aDepartment of Radiotherapy, National Cancer Institute, Fom El-Khalig, Cairo, Egypt

bDepartment of Oncology and Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus University Hospital, N@rebrogade 44, DK-8000 Aarhus C, Denmark

Received 13 October 1994; revision received 19 December 1994; accepted 1 February 1995

Abstract

Late renal damage after total body irradiation (TBI) and bone marrow transplantation (BMT) is a recently recognised morbidity. We have tested the effect of single fraction TBI given at two different dose rates on late kidney damage in a mouse model. TBI was given at either high dose rate (HDR; 0.71 Gy/min) or low dose rate (LDR; 0.08 Gy/min). Transplantation with syngeneic mar- row cells was done 4-6 h after TBI. Kidney damage was tested using 5’CrEDTA residual activity, blood urea nitrogen (BUN) and percentage haematocrit (Hct). TBI alone given at HDR or LDR caused progressive renal damage with no evidence of recovery or plateau. The time latency before the expression of damage was dependent on both dose and the end point used. It was shorter the higher the dose. 5’CrEDTA detected renal damage at the same doses as BUN but earlier in time, while %Hct showed evidence of renal damage at doses lower than both BUN and “CrEDTA. Using the “CrEDTA the dose-response curves for renal damage were steep and continuously shifting towards lower doses as follow-up time after treatment increased. There was a sparing effect of reducing the dose rate that was more evident at follow-up times of less than a year than at 66 weeks after TBI. Thus, the dose modifying ratio (DMF), defined as the dose needed to cause renal damage in 50% of irradiated animals (ED,,) using LDR divided by the ED, using HDR., was dependent on the time of evaluation. It varied from 1.2 (week 18) to 1 (week 66). Clinically, this study may indicate that patients treated with TBI and BMT should continuously he assessed for late kidney damage. The use of sensitive techniques using radionuclides to measure renal damage is recommended.

Keywords: Total body irradiation; Dose rate; Kidney damage; Bone marrow transplantation

1. Introduction

Until recently, renal complications after total body irradiation (TBI) and bone marrow transplantation (BMT) were considered “uncommon”. However, a few recent retrospective studies have reported relatively high incidence of late renal dysfunction among survivors after TBI and BMT [3,5,6,12,21]. The different clinical syndromes of renal dysfunction after BMT resemble those of radiation-induced renal dysfunction [ 12,191. Also, the histological picture of the reported renal syn-

*Corresponding author, Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus University Hospital, Ngrrebrogade 44, DK-8000 Aarhus C, Denmark.

dromes after BMT are very similar to radiation-induced renal damage [2]. Few experimental studies have ad- dressed the problem of kidney damage after BMT and have indicated an appreciable degree of damage in ani- mals treated with different conditioning TBI regimens followed by BMT [4,8-lo].

Hence, both clinical and experimental studies suggest that irradiation may be a major contributing factor causing this renal damage. However, most of the experi- mental studies on kidney damage after radiation have been done using isolated kidney irradiation and not TBI. It has been suggested that a certain dose of radia- tion given as half or total body irradiation may be more toxic to the kidney than when given as localised irradia- tion [ 11,141. Thus, the available data on kidney damage

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204 A. Safwat et al. /Radiotherapy and Oncology 34 (1995) 203-209

after the complex treatment of BMT are still far from being complete.

Therefore, we initiated a study aiming at analyzing the effect of single fraction TBI given at either low or high dose rate on late renal damage after TBI and BMT in a mouse model.

2. Material and methods

2.1. Animals

Male CsD2FJBOM mice were used in all experiments and treated when 14-16 weeks of age (adults). They were kept eight per cage under normal laboratory condi- tions and given tap water and food ad libitum.

2.2. Treatment

Unanaesthetized mice were restrained in acrylic jigs placed in a specially constructed acrylic box as previous- ly described in detail [22]. Whole body irradiation was delivered with a 250 kV Phillips X-ray unit (10 mA, 2.2 mm Cu HVL). For the groups given high dose rate sin- gle fraction, the dose rate was 0.71 Gy/min. The dose rate for the groups receiving single fraction at low dose rate was 0.08 Gy/min. Changing the dose rate was done by increasing the distance of the acrylic mouse box to the source. An integrating dosimeter (dosimentor Sn4, PTW Freiburg) was used for dosimetry.

Intravenous injection (into the tail vein) of -2 x lo6 syngeneic bone marrow cells 4-6 h after the last treatment was done in all animals. The marrow was obtained from both femurs of normal untreated mice and diluted appropriately in Hank’s balanced salt solution.

2.3. Assessment of kidney damage

“CrEDTA clearance. The single sample method described by Stewart et al. [16] was used with minor modifications. Sterile aqueous solution of chromium EDTA containing 1% benzyl alcohol and the salt of EDTA were used (Amersham; pack size 37 MBq, 10 mCi vials). The residual activity of the material was calculated as percentage of injected material in 20 ~1 of plasma, 20 min after i.p. injection of 10 j&i (in 0.1 ml) per mouse. Counting the samples was done using a gamma counter (Cobra Auto-Gamma, Packard A Canberra Co.).

The haematocrit (Hct). The %Hct values were measured after centrifugation of the blood samples taken during the chromium test. The values were deter- mined by the use of a Hawksley micro-haematocrit reader.

Blood urea nitrogen (BUN). Blood samples for the urea nitrogen were taken from the retro-orbital sinus in

a 125 ~1 non-heparinized capillary tube. The blood was centrifuged for 5 min at a 64 000 cycle/min, and 10 ~1 of plasma was examined in a Kodak Ektachrome DT60 analyzer. Blood samples were withdrawn 5 days before the “CrEDTA test to avoid using radioactive samples in the analyzer.

2.4. Experimental design and data analysis

The animals receiving a single fraction at high dose rate (HDR) were distributed between the following dose levels: 4 Gy, 6 Gy, 8 Gy, 9 Gy, 10 Gy, 10.5 Gy, 12 Gy, 13.5 Gy and 14 Gy. The animals receiving a single frac- tion at low dose rate (LDR) were distributed between the following dose levels: 6 Gy, 9 Gy, 11 Gy, 13 Gy, 14 Gy, 17 Gy and 22 Gy. In addition, there was a control group to which only BMT was performed. There were 24 animals in the control group and from 8-16 animals in each of the others. The animals were tested on week 12 after treatment and every 6 weeks thereafter for the first year. Starting from the second year, testing was done every 12 weeks. For clarity of graphical representa- tion only five dose levels from each of the TBI regimens were shown in Figs. l-3.

For each functional test a mean value of each group was calculated at each point of follow-up and plotted against time. For numerical analysis, the data were con- verted to quanta1 response (all or nothing) data. Each experimental group was divided into responders and non-responders. Responders were defined as those ani- mals in which the values for a specific functional param- eter exceeded a predetermined level. In all the functional tests, the iso-effect level was chosen to be values ex- ceeding three times the standard deviation of the control group mean. All the animals that reached this level showed persistent and progressive damage until death or the end of the follow-up period.

Based on the quanta1 response data both time- response and dose-response data were generated. The time-response data were gathered by using a Kaplan Meier estimate for responders. The dose-response curves were computed using a logit programme. The dose-response data were used to calculate the iso- effective doses for different times after treatment. The error bars in the curves represent one standard error (S.E). The dose modifying factor (DMF) is defined as the ratio between the dose needed to cause response in 50% of the animals (EDSo) using the LDR protocol and the EDSo using single fraction at HDR.

3. Results

Fig. 1 shows the residual activity of “CrEDTA in plasma plotted against time after treatment. Each data point represent the mean value of the residual activity measurements for the group of mice receiving the same

A. Safwai et al. /Radiotherapy and Oncology 34 (1995) 203-209 205

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Fig. I. The change in the average residual activity of S’CrEDTA after a single fraction TBI at HDR (A) or LDR (B). All the animals in the group receiving 12 Gy at HDR and 17 Gy at LDR died before week 36. Error bars represent I SE.

dose. A single fraction of 8 Gy at high dose rate (HDR) was needed to cause detectable kidney damage. The level of damage increased gradually with higher doses. Also the rate of progression was dose dependent, i.e. it was faster the higher the dose. The data did not fit a straight line. Instead, there was some evidence of an ini- tial high level of residual activity that was partially resolved around week 36 after treatment and followed later on by an accelerated rate of progression of damage that showed no plateau or evidence of recovery. The early damage and its partial resolution was clearer when we traced individual animals, i.e. it was not an artifact caused by death of animals having the highest degree of damage (data not shown). The accelerated progression of damage after the low doses (e.g. 8 Gy HDR and 11 Gy LDR) started around week 54 after treatment.

With low dose rate (LDR), the animals tolerated higher doses of radiation than with HDR. The lowest dose needed to cause kidney damage was 11 Gy and the differences in the degree of kidney damage caused by the

different dose levels given at LDR was less clear than with HDR. None of the animals receiving doses higher than 10.5 Gy at HDR or 14 Gy at LDR survived beyond week 30 after treatment.

In contrast to the “CrEDTA, the percentage haematocrit could detect kidney damage at doses as low as 4 Gy at HDR and 9 Gy at LDR. Fig. 2 shows the average reduction in percentage haematocrit for dif- ferent dose levels against time after treatment. The evi- dence of early initial damage that was resolved around week 36 after treatment with the “CrEDTA end-point (vide supra) was only seen with 4 Gy at HDR using the %Hct end-point. Otherwise, the reduction in the %Hct was noticeable 18 weeks after treatment and progressed steadily as the time after treatment increased. The degree of reduction was proportional to the dose. Again, higher doses were needed to give detectable reduction in %Hct when LDR was used, but the dose dependence was less clear.

For about a year after treatment, the serum level of

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Fig. 2. The change in the average %Hct after a single fraction TBI at HDR (A) or LDR (B). All the animals in the group receiving 12 Gy at HDR and 17 Gy at LDR died before week 36. Error bars represent I S.E.

206 A. Safwat et al. /Radiotherapy and Oncology 34 (1995) 203-209

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Fig. 3. The change in the average BUN after a single fraction TBI at HDR (A) or LDR (B). All the animals in the group receiving 12 Gy at HDR and 17 Gy at LDR died before week 36. Error bars represent 1 SE.

the BUN could not detect any shift from normal levels in the groups that showed progressive renal damage as evaluated by the “CrEDTA residual activity and the %Hct. Fig. 3 shows the average serum BUN plotted against time after treatment. In contrast to the “CrEDTA and the %Hct there was an evident latent period before the expression of renal damage. This latency was around 42 weeks with HDR and 60 weeks with LDR. Thereafter, the BUN values increased pro- gressively with time. The threshold for renal damage is difficult to judge since the data at longer observation times are missing because of a technical error. Yet, it seemed similar to that detected by the ‘ICrEDTA, i.e. 8 Gy single fraction HDR and 11 Gy single fraction LDR. If this is true, then the residual activity of “CrEDTA may not be more sensitive than BUN, but rather it detects renal damage earlier.

The Kaplan Meier estimate of the data from the “CrEDTA and %Hct end-points showed that the per- centage of responders increased with time after treat-

ment and reached 100% in all the groups that expressed kidney damage. The higher the dose the higher the per- centage of responding animals at any given time. Also, the latent period needed to give a certain percentage’ of responders (e.g. 50% response) decreased as the dose in- creased. On the other hand, serum BUN detected renal damage in a smaller percentage of animals and at a later time than the “CrEDTA residual activity and the %Hct. Moreover, the dependence of the latency on the total dose was less clear.

Dose-response curves were generated from the “CrEDTA data at weeks 18, 30 and 66 after treatment and the results are shown in Fig. 4. There was clear evi- dence that the dose-response curves shifted towards lower doses as the time after treatment increased. The steepness of the dose-response curves did not show a consistent change between the tested weeks after treat- ment. A single fraction of TBI at LDR was less nephrotoxic than a single fraction at HDR at observa- tion times of less than a year. Later, this sparing effect

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Fig. 4. “CrEDTA dose-response curves at week 18 (O), week 30 (0) and week 66 (v) after a single fraction TBI at HDR (A) or LDR (B). The graphs were analyzed by the use of a logit programme. Error bars represent 1 S.E.

Table I

A. Safwai et al. /Radiotherapy and Oncology 34 (1995) 203-209 201

The dose-response data for renal damage evaluated by “CrEDTA residual activity in mice at various weeks after total body irradiation (TBI) and bone marrow transplantation. TBI was given at high dose rate (HDR) or low dose rate (LDR)

Week HDR WY) LDR (GY) DMF* zt 1 S.E.

18 30 66

No. of mice EDso + I S.E. No. of mice ED,, f I S.E.

57 11.0 l 0.4 46 13.1 f 0.9 1.2 l 0.1 45 10.1 f 0.4 40 11.3 l 1.0 1.1 l 0.1 41 7.5 f 0.5 26 7.5 f 0.8 1.0 * 0.1

*DMF, dose modifying factor.

seemed to become less evident. The dose modifying fac- tor (DMF) using LDR decreased from 1.2 to 1.1 as the time after treatment increased from 18 to 30 weeks. At 66 weeks after treatment the DMF value became 1 (Table 1).

4. Discussion

For all the functional tests used to assess kidney damage, mean values of the groups of animals that received the same dose were presented. This approach may have a few disadvantages. It assumes that the data are normally distributed, which is difficult to prove be- cause of the few animals in each data point. It also assumes that there is no individual difference between animals, which is true only within certain biological limits. Moreover, it has the potential risk of under- estimating both the degree of damage and progression rate, i.e. the animals dying during the follow-up period are most likely those with the highest degree of damage. Therefore, the use of mean values are insufficient for estimating iso-effective doses or latent times of different radiation schedules. Instead, the data were converted into quanta1 response (responders and non-responders) for estimating the iso-effective doses and DMF. How- ever, despite these limitations, presenting the results as average values of the functional tests against time was preferred for illustrating the progressive and increasing ‘degree’ of damage, a criterion which the quanta1 analy- sis of data fails to demonstrate. The estimation of laten- cy is important in late reacting tissues and in the kidneys in particular. This kind of analysis is currently being done and will be presented together with data using frac- tionated TBI in a separate publication.

Using the average residual activity of “CrEDTA in plasma, kidney damage was detectable 54 weeks after a single fraction of 8 Gy at HDR. This is lower than the 12 Gy reported by Williams and Denekamp [23] in mice using the same end-point, but with local&d, bilateral kidney irradiation. However, Williams and Denekamp didn’t follow their animals beyond 52 weeks. In a recent report by Stewart et al. [17], renal damage was seen in mice 60 weeks after a single fraction of 8 Gy was given as localised kidney irradiation at HDR. These results

emphasise the importance of latency in radiation- induced renal damage.

The animals in the irradiated groups were in general of lower average body weight, probably because of late effect on the gastrointestinal tract, than the unirradiated age-matching control group. Therefore, we examined the dependence of the ‘ICrEDTA residual activity on body weight in our animals. There was no correlation between the residual activity of %rEDTA and body weight (data not shown). It has been demonstrated previously that no simple factor could be applied to cor- rect for differences in body weight between groups of treated animals [23]. It is quite possible that any increase in the residual activity of “CrEDTA due to reduction in the body weight was compensated for by the increase in plasmacrit induced by renal damage.

Radiation-induced kidney damage is known to be in- creasing progressively with time. Our data also indicated the possibility of early damage that was partially resolv- ed and followed by late progressive damage. Robbins et al. [13] reported that a dose of 7.8 Gy (60Co) in pigs caused early damage that was resolved by week 24 after treatment, while at higher doses the damage progressed continuously. Unfortunately, Robbins et al. did not follow their pigs beyond week 42 after treatment. In our study, resolution of damage was temporary. In agree- ment with the literature the rate of progression of kidney damage was clearly dose-dependent [7]. Also, the depen- dence of the latent period on both end-point and radia- tion dose (the higher the dose, the shorter the latency) is similar to that reported by Stevens et al. [15].

The dose-response curves for kidney damage were very steep. However, one should be very cautious not to compare different TBI schedule on the basis of steepness of dose-response curves because of the few data points on the curves.

Studies of the effect of LDR on the kidneys are very few. Travis et al. [20] studied the effect of TBI given as a single fraction at various dose rates. Their lethality dose-response curves are not exclusive for renal damage, but rather for ‘late non-haematopoietic syndromes’. Using histopathology changes at 1 year as the end-point, their results showed marked sparing of renal damage after LDR TBI versus HDR. Since a wide range of dose

208 A. Safwat et al. /Radiotherapy and Oncology 34 (199.5) 203-209

rates was tested they could demonstrate the importance of reducing the dose rate below 0.05 Gy/min to achieve maximum sparing. The low dose rate in our model was only 0.08 Gy/min. Further reduction in dose rate is ex- pected to yield more sparing of kidney damage, but it would be impractical from the clinical point of view. Moulder and Fish [8] have also studied the effect of LDR on TBI-induced renal damage in rats. They reported that the late toxicities seen after LDR (0.0325 Gy/min) were qualitatively similar to those after HDR (0.64 Gy/min), but at twice the radiation dose (DMF = 1.86). Our dose modifying values are lower than those reported by Moulder and Fish [8]. This could be explained by the very low dose rate in their study (0.0325 Gy/min) compared to ours (0.08 Gy/min). To our knowledge, the reduction in the sparing effect achieved by using LDR with the passage of time has not been described before in the literature. Given the few data points and the small number of animals it would be difficult to comment on how solid this observation might be. It is important, however, to notice that in these literature reports the sparing effect of LDR was not tested beyond 1 year of follow up.

For both the HDR and LDR schedules, the EDSo values were continuously shifting toward lower doses as the time after treatment increased. This reflects an increase in the number of ‘responders’ for a given dose as the latency after treatment increases, which is in agreement with the kidney being a late reacting tissue. Comparison with other published ED+ is difficult be- cause of differences in end-points, time of evaluation, animal strain and dose rate. However, the published data suggest that the late EDSo values in our study, e.g. 7.52 Gy at week 66 for single fraction at HDR, are lower than what would be expected from bilateral kidney irra- diation in mice. This could be explained by the longer observation time in our study or, on the basis of the re- sults of Moulder et al. [ 111, by the fact that a single frac- tion of 9 Gy caused a higher degree of kidney damage when given as TBI than when given as localised kidney irradiation.

Our animals were followed for most of their life span. In order to account for any possible effect of ageing on renal function, an age-matched control group was used for determining the base line in the estimation of ‘responders’. In our study the untreated animals did not show any measurable change in the level of the “CrEDTA and BUN tests, whereas the %Hct showed a consistent reduction. This reduction is probably a nor- mal physiological phenomenon in this animal strain and not due to multiple blood sampling, since only a very small amount of blood was withdrawn from the retro- orbital sinus every 6 weeks. Our results confirmed the well-known notion that the reduction in haematocrit is a very sensitive estimate of renal damage, detecting the injury at radiation doses below that detected by

“CrEDTA [ 1,181. However, the dose levels at which Hct started to decline were lower than that reported with localised kidney irradiation [ 181. Hct is also known to yield a well-defined dose-effect curve which can be used to estimate the extent of radiation damage and re- pair [18]. However, using TBI, other factors such as ra- diation damage to the liver, gastrointestinal tract and chronic infections should also be considered as con- tributing to the development of anaemia.

The experimental set-up was meant to mimic the clini- cal situation, thus lungs were not shielded in these animals. Interstitial pneumonitis (IP) was carefully monitored and the details of these findings, as well as the relative radiation sensitivities of the lungs and kid- neys, have been discussed in separate publications. However, we would like to emphasize here that although both kidney and lung damage did develop in some animals, especially those in the higher dose levels, there was a certain dose range at which only kidney damage occurred.

Clinically, renal damage after BMT may turn out to be an important late complication. In the present study we have demonstrated that radiation-induced kidney damage in mice after TBI and BMT is a potentially lethal dose-limiting complication. It occurs at radiation doses lower than that causing interstitial pneumonitis, regardless of dose rate. It could be spared by using low dose rate. Renal damage after TBI is most likely similar to that induced by localised kidney irradiation. The use of sensitive techniques using radionuclides to measure renal damage and monitoring the reduction in %Hct should be the preferred tests for assessing renal damage. Although extrapolating these results to the clinic is dif- ficult, the data indicate that it would be prudent to carefully assess the patients treated with TBI and BMT for late kidney damage. A long observation time is cer- tainly needed before declaring any specific TBI schedule safe.

Acknowledgment

The authors would like to thank P. Schjerbeck for en- thusiastic and skillful technical help. The study was sup- ported by a grant from the Danish Cancer Society.

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