The influence of cisplatin on the response of mouse kidneys to multifraction irradiation

Radiotherapy Elsevier

and Oncology,

15 (1989) 93-102

93

RTO 00574

The influence of cisplatin on the response of mouse kidneys to multifraction irradiation F. A. Stewart, Y. Oussoren

and H. Bartelink

Experimental Therapy (H6). The Netherlands Cancer Institute(Antoni van Leeuwenhoekhuis), Amsterdam, The Netherlands (Received

18 April 1988, revision received 14 November

1988, accepted

18 November

1988)

Key wor&: c-DDP; X-rays; Mouse kidney: Fractionation

Summary

The aim of these studies was to measure the extent of renal damage after fractionated irradiation in combination with cis-diamminedichloroplatinum(II) (c-DDP) and to determine whether there was any drug-induced inhibition of repair of sublethal X-ray damage. Fractionated irradiations were given either in a short overall treatment time of 2 days or in a total time of one month, in which irradiations were given in the first and fourth weeks only. A single dose of c-DDP was given before the first X-ray dose for treatments in a total time of 2 days and before the first X-ray dose of each week for the split-course treatments. Renal function was measured at monthly intervals (from 18 to 39 weeks after the start of treatment) by clearance of “Cr-EDTA. The renal damage was always worse after combined treatments (X + c-DDP) than after X-rays alone. A comparison of radiation dose-response curves with and without c-DDP yielded Dose Enhancement Factors (DEFs) of 1.2 to 1.4 for all fractionation schedules, with no trend for an increase in DEF with increasing number of fractions. A Linear-Quadratic (LQ) analysis of the data demonstrated that there was no change in the a//l value for renal radiation damage for the combined treatments compared with X-rays alone (a//t?= 2-3 Gy). These results suggest that the increased renal damage which occurred after combined X + c-DDP was due to independent, additive toxicities and not to radiosensitization or inhibition of X-ray repair by the drug. Introduction

cis-Diamminedichloroplatinum(II) (c-DDP) is now one of the most widely prescribed cancer Address for correspondence: F. A. Stewart, Experimental Therapy (H6), The Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Plesmanlaan 121,1066 CX Amsterdam, The Netherlands 0167-8140/89/$03.50

0 1989 Elsevier Science Publishers

chemotherapeutic agents. In addition to its cytotoxic properties, c-DDP has been shown to be a potent radiosensitizer in several experimental tumour models [2,4,13,18,21] and clinical trials are now in progress to evaluate the potential of this drug as a radiosensitizer [ 1,7,9,24]. In assessing the benefit of c-DDP used in combination with radiotherapy, the increased risk of normal tissue damage must be taken into account. The dose-

B.V. (Biomedical

Division)

94

limiting toxicities in the use of c-DDP as a chemotoxic agent are otoxicity, gastrointestinal and renal toxicity; these tissues may therefore also be at risk when the drug is used as a radiosensitizer. Previous studies have already demonstrated a moderate increase in renal damage (DEF = 1.3) when c-DDP is combined with single dose irradiation [26]. One of the proposed mechanisms whereby c-DDP may act as a radiosensitizer is by inhibition of repair of radiation injury [6,14,31]. If this occurred in normal tissues, as well as in tumours, then a larger modification of X-ray damage would be expected for fractionated treatments than for single doses. The present study was therefore undertaken to investigate the influence of c-DDP on renal damage after fractionated irradiation in order to compare repair of radiation damage after X-rays alone with that occurring after combined treatments of X-rays plus c-DDP. Linear-quadratic (LQ) analysis of the data was used to compare a//I ratios after irradiation alone or in combination with the drug, and hence to determine if there was any modification of the X-rays dose response by c-DDP. The results reported here are an extension of the preliminary results previously published [ 271.

Materials and methods Experimental design

Fractionated renal irradiations were given, with and without c-DDP, in a total treatment time of 1 to 2 days (2,4 and g fractions), or in 4 weeks (2,4,10,20 and 30 fractions). For each irradiation schedule, 5 X-ray dose groups were included and successive irradiations were always separated by a minimum interval of 5 h to allow repair of sublethal radiation injury to occur. If the repair halftime in kidney is longer than 1 h, as recent evidence suggests [23], then repair may not, in fact, be totally completed within 5 h. For the schedules treated in the short overall time, c-DDP was given as a single i.p. injection of 6 mg - kg- ’ 30 min

before the first X-ray dose; no prior hydration was used. For the schedules treated in a total time of one month, irradiations were given in the first and fourth weeks, with a 2-week rest period in the middle of treatment to allow repopulation of the crypt cells of the small bowel and therefore reduce gut toxicity. For these schedules, 4 mg - kg- ’ c-DDP was administered, i.p., without prior hydration, 30 min before the first X-ray dose of each week (i.e. 2 x 4 mg - kg- ‘). The two drug dosing schedules were equitoxic in terms of animal weight loss. Irradiations

Female C3H mice were used aged lo-14 weeks and weighing 22-27 g. The kidneys of unanaesthetized mice were bilaterally irradiated using 250 kV X-rays (15 mA), filtered with 0.5 mm Cu, giving a dose rate of 235 cGy - min - ’ at the position of the kidneys. Details of the irradiation set-up and procedure have been previously published [26]. Measurements of renal function

Kidney function as measured by the clearance of “0-EDTA, using the single sample method previously described [26,361. Repeat measurements were made on individual mice at monthly intervals from 18 to 39 weeks after treatment. In a few cases, some earlier tests of renal function were also carried out. Results are expressed as the residual radioactivity (% of injected dose) remaining in the plasma at 30 min after an i.p. injection of the tracer (approximately 10 @i per mouse).

Results

Functional renal damage after irradiation alone developed progressively, in a dose-related manner, from the first test at 18 weeks. After combined treatment with X-rays + c-DDP there was generally an earlier onset of damage. The time

95 13

10

El

a 6 4

': u

14

zi2

12

#

10 a 6 4 2

OOlO

1 xl

30

L

I

20 41 010 time from reatment (weeks)

30

10

Fig. 1. Time course for the development of renal damage after 2,4, 10, or 20 F of X-rays alone (0) or X + c-DDP) (0). The total treatment time was 4 weeks for all schedules and c-DDP was given before the first X-ray dose in weeks 1 and 4 (2 x 4 mg . kg-‘). The X-ray doses are written beside each response curve. The hatched area shows the response ( + 1 S.E.M.) of untreated controls and the response after drug alone (2 x 4 mg 1kg- ‘) is indicated by the dashed line (- - - - ). Each data point represents the mean + 1 S.E.M. of 4 to 6 mice.

course for development of damage in selected dose groups after irradiations in a total treatment time of one month is shown in Fig. 1. For each fractionation schedule, and at all testing times, there was more damage for a given radiation dose after the combined treatments than after X-rays alone. The late renal damage (30-40 weeks) after X-rays + c-DDP could be matched with damage after a higher dose of X-rays alone (e.g. in the 2fraction schedule 17.5 Gy + c-DDP gave the same amount of late damage as 23 Gy X-rays only). For equal late reactions there was, however, more renal damage in the earlier period (18-30 weeks) after the combined treatments due to the earlier onset of damage. Drug doses of

alone 2 x 4mg.kg-’ (or 1 x 6mgakgg’) caused only a minimal decrease in renal function. The extent to which c-DDP increased late radiation damage for different fractionation regimes was assessed by comparing X-ray doseresponse curves with and without drug at 31 weeks after treatment (Figs. 2 and 3). c-DDP always shifted the X-ray dose-response curves to lower doses giving rise to DEFs* of 1.3 to 1.4. This was true for all schedules tested and the

* DEF (Dose Enhancement Dose of X-ray alone Dose of X + c-DDP

Factor) =

for equal damage.

96

observed DEFs appeared to be independent of fractionation or overall treatment time (Table I). In order to examine whether c-DDP influenced the extent of repair of radiation injury, the doseresponse curves for l-30 F X-rays alone and X + c-DDP were analyzed separately at testing times of 27-40 weeks after treatment. There was clearly a large dose-sparing effect with increasing fraction number after both X-rays alone and in combination with drug (Fig. 4). The differences in absolute isoeffect doses for 30 F compared with 1 F (03,, - 0,) were 44 and 32 Gy for X-rays alone or in combination with c-DDP respectively. These dose increments, however, can not be directly compared to assess the relative amounts

lo-I 2Flld 88-

. . 1.4 ii+

4-

: .i *

/

Fig. 2. Dose-response curves for renal damage at 31 weeks atIer 2 or 4 F given in a total treatment time of 1 day. 0 = X only data, 0 = X + c-DDP (6 mg . kg- ’ before first X-ray dose). Each data point represents the mean k 1 S.E.M. of a group of 4 to 6 mice. DEFs for c-DDP enhancement are shown beside the curves.

:&& I

1

L O 0

5

I”

I

I

I

I

15 20 total dose (Gy)

10

25

30

c 0

2F

8

86-

10

20

30

40

total dose (Gy) Fig. 3. Dose-response curves for renal damage at 3 1 weeks after 2,4 or 30 F given in a total treatment time of 4 weeks. 0 = X only, 0 = X + c-DDP (2 x 4 mg . kg- ‘). Each point represents the mean + 1 S.E.M. for groups of 4 to 6 mice and the DEFs for c-DDP are shown beside the curves.

97 TABLE I DEFs and u/j ratios for renal damage after X-rays + c-DDP. Treatment

DEFs from dose-response

curves at:

24-27 wks

28-31 wks

33-35 wks

2 F/l day:

a b

1.37 ?r 0.23 1.53 + 0.29

1.43 + 0.16 1.48 f 0.14

1.27 f 1.27 f

2 F/l month:

a b

1.39 f 0.11 1.41 + 0.08

1.34 + 0.07 1.37 + 0.07

1.31 * 0.05 1.32 + 0.03

4 F/l day:

a b

1.44 * 0.20

1.29 + 0.07 1.31 f 0.08

1.25 f 1.25 f

0.04 0.04

4 F/l month:

a b

(1.24) 1.24 + 0.13

1.27 + 0.10 1.25 + 0.06

1.15 f 1.11 f

0.07 0.05

10 F/l month:

a b

(1.58) 1.66 + 0.08

1.34 f 0.14 1.42 f 0.05

1.39 f 0.10 1.42 + 0.04

20 F/l month:

a b

(1.6) 1.64 + 0.8

(1.5) 1.53 k 0.6

(1.4) 1.43 *

a b

1.28 f 0.27 1.40 f 0.13

1.30 + 0.16 1.34 * 0.10

1.27 + 0.06 1.22 k 0.06

:

2.1 & 0.2 Gy 2.1 f 0.3 Gy

3.2 + 0.2 Gy 3.9 f 0.3 Gy

3.3 & 0.2 Gy 2.9 k 0.4 Gy

30 F/l month:

a/b X only/l month:

0.13 0.05

0.04

or/b X + c-DDP/l

month:

a b

2.1 k 0.6 Gy 2.8 k 1.1 Gy

2.3 k 0.3 Gy 2.5 f 0.5 Gy

2.7 + 0.6 Gy 2.3 f 0.5 Gy

a/j7 X + c-DDP/2

days:

a

2.2 + 0.5 Gy

3.1 f 0.2 Gy

4.3 f 0.4 Gy

(a) DEF calculated at damage levels of 5, 6 and 7% residual [51Cr]EDTA, at the three testing times respectively. Isoeffective doses were read from linear dose-response curves and errors are k 1 SE. computed as BMS values from envelopes through the error bar. Values in brackets were calculated by extrapolation of curves to reach the isoeffect level. (b) DEFs calculated by linear regression of effect versus log dose. Parallel lines could be fitted to the X-ray only and X + drug data, therefore, the DEF was independent of isoeffect level. Errors are f 1 S.E.. a/b (a) calculated using linear regression of reciprocal total isoeffect doses (from best eye fit linear dose-response curves (a) or regression of effect vs. log dose (b) versus dose per fraction. Errors represent f 1 SE. for linear quadratic variance only, with no estimate of uncertainty on determination of isoeffective dose.

of repair in the two treatments, since recovered dose is dependent on X-ray dose and lower total doses were used in all the combined treatment schedules. A better method for assessing whether c-DDP influences repair of X-ray damage is to compare the oc/flvalues obtained by an LQ analysis of data from fractionated treatments with and without c-DDP [3]. LQ analysis of the present data was performed using isoeffective doses derived from dose-response curves (best eye fits through all

data points) for renal damage in the period 27 to 35 weeks after treatment. The result for combined X-rays + drug in overall treatment times of 2 days and one month were analyzed separately, but for X-rays alone given in the short treatment time there were insufficient data to permit an LQ analysis. The a/b values, derived by linear-regression analysis from a plot of reciprocal isoeffect dose versus dose per fraction (Fig. 5, and Table I), varied between 2.1 f 0.2 Gy and 3.3 t 0.2 Gy for X-rays alone given in a treatment

98 12 [X rays only

1

88-

s; 12 2

[X*c-DDPI

I

Oo

Fig. 5. Reciprocal total dose versus dose per fraction for renal damage (6% residual plasma [‘rCr]EDTA level) at 31 weeks after irradiation. 0 = 2 to 30 F X only data from this article, treatment time 4 weeks; A = previously published data for 2-30 F X only, treatment time 4 weeks (ref. [28]); 0 = 2 to 30F X + c-DDP (2 x 4mg*kg-‘) in 4 weeks; A=2to8FX+~-DDP(lx6mg*kg-‘)inlto2days. Errors are k 1 S.E.M. and lines were fitted by linear regression. Back extrapolates on the abscissa ( - - - -) indicate a/b values.

1OF

I

I

I

I

10

20

30

40

1

I

I

50 80 70 total dose (Gy)

Fig. 4. Dose-response curves for renal damage 31 weeks after 1 to 30 F of X-rays alone (top panel) or X + c-DDP (bottom panel). All treatments were in a total time of4 weeks and c-DDP was given before the first X-ray dose in weeks 1 and 4 (2 x 4 mg *kg- I). Each point represents the mean f 1 S.E.M. for groups of 4 to 6 mice. The 1 F data (0) are reanalyzed from a previously published experiment. (ref. [26]).

time of one month, and 2.1 + 0.6 to 2.7 f 0.6 Gy for the combined treatments given in one month. The limited data available for analysis after X + c-DDP in treatment times of 1 to 2 days gave u/Bratios of 2.2 f 0.5 to 4.3 + 0.4 Gy. The addition of c-DDP to X-rays therefore caused no signticant alteration in a//J values for the treatment schedules tested.

Discussion

For combined treatments of X-rays with drugs the total drug dose which can be administered is al-

ways limited by toxicity. This means that for fractionated schedules a choice must be made between giving a high drug dose before a limited number of X-ray doses, or a lower drug dose before each X-ray dose. Clinical trials to assess the potential of cytostatic drugs as radiosensitizers have been initiated using both these approaches. In some of the experiments reported here, we used a single drug dose given before the first of 2, 4 or 8 X-ray fractions. For these schedules the overall treatment time was kept short (2 days), in order to minimize the effects of decreasing drug concentration during irradiation. Although most of the free platinum will have been cleared before the end of a 2-day treatment period [ 5,19,33], c-DDP-DNA adducts have been shown to persist for many weeks. In the rat kidney, for example, approximately 70% of the initial c-DDP-DNA adducts remained after 3 days [30]. An overall treatment time of 2 days restricted the total number of X-ray doses which could be given to 8 (4 doses per day with a minimal interfraction interval of 5 h). In order to increase the number of X-ray fractions we therefore used a second experimental

99 protocol with a total treatment time of one month. In these schedules, irradiations were given in the first and fourth weeks with c-DDP before the first X-ray dose of each week. There was consequently a maximum interval of 4.5 days between c-DDP administration and the last irradiation of each week. Drug-fading effects may have been more pronounced with these schedules employing longer overall treatment times, although the experiments of Terheggen [ 301 demonstrated that at least 60% of the c-DDP adducts induced remained in rat kidneys at 5 days. The results of the studies reported here demonstrate that c-DDP given in combination with fractionated renal irradiation always led to more damage than X-rays alone. During the late period (30-40 weeks after treatment) the DEF for combined treatment relative to X-rays alone was 1.2-l .4, independent of fractionation schedule or overall treatment time. The renal damage also appeared earlier after combined treatments, however, creating the impression of a greater enhancement of damage in the early testing periods. These results are consistent with either the addition of independent, subthreshold toxicities during the period before expression of radiation injury, or the enhancement of drug damage by radiation. Similar findings were previously reported for single dose studies [26]. Another possible explanation is that the early expression of damage was caused by drug-induced stimulated proliferation in the kidneys with the precipitation of latent radiation injury. This is unlikely, however, since c-DDP causes only a sma.ll increase of renal proliferation [ 161 and other methods for stimulating renal proliferation did not lead to early expression of radiation injury (Lebesque and Stewart, unpublished data). In the present study we were primarily interested in the modification of late radiation damage in the kidneys by c-DDP. It was postulated that any reduction in the extent of repair of radiation damage by the drug would lead to a larger modification of the X-ray response with fractionated treatments than with single doses. The DEFs which were measured in this study did

not increase with increasing fractionation and none of the values were significantly higher than the previously reported value of 1.3 for single doses [ 261. This suggests that there was little or no inhibition of X-ray repair by c-DDP in the treatment schedules employed in this study. Persistent renal damage has been measured for up to one year after c-DDP alone in both rats and mice [ 15,20,26] and the DEFs of 1.2-1.4 measured in the present studies for combined treatment with X-rays + c-DDP will include a component of drug-alone toxicity. It is however, dficult to determine the exact contribution of drug-alone killing from a comparison of dose-response curves for functional damage. Assays for tissue damage, after both X-rays and drugs, usually have large detection thresholds, with very non-linear dose-response curves. Sign&ant cell killing may therefore occur without producing a measurable response; this makes it difficult to distinguish between an additive or interactive response when the two agents are combined [25]. One method of using functional dose-response curves to determine whether there is any modillcation of X-ray response by a drug is to compare a//l ratios obtained from an LQ analysis after treatments with X-rays alone or with drug [ 31. If there is only independent, additive killing from the two agents then the a/j3 ratios for X-rays alone or in combination with drug will be identical. Under these circumstances the extent of drug-alone killing can be estimated by comparing the Y-intercept values on the reciprocal total dose plots from the two treatments. The only other explanation for a lack of change a//l for combined treatments relative to X-rays alone would be a proportional increase in both a and fl. If this occurred, an identical cl/j3 ratio could be obtained for X-rays alone and X + drug despite a change in the shape of the X-ray response curve and this possibility can not be excluded from the present study. If the shape of the radiation-survival curve is modified by drug in any way other than by proportional increases in both a and fi then the a//l ratio for combined treatments will differ from that for X-rays alone [ 31. Any drug-induced increase

100 in radiosensitivity (either by repair inhibition or a change in D,) would be reflected as a change in the a//I ratio. An LQ analysis of the present results using reciprocal total dose plots [ 1l] demonstrated that the a/b ratios for X-rays alone or in combination with drug varied between 2 and 3 Gy (see Table I). There was no increase in the ratio u/b after X-rays + c-DDP compared with X-rays alone. Another method for obtaining values for a/b has recently been developed by De Boer [8]. For this analysis, total isoeffect dose (0) is plotted against the product of total dose and dose per fraction (Dd). The a/p ratio can then be obtained from the inverse slope of the linear-regression line through these points. This method has been shown to give values for alpalmost identical to the statistically correct, but more complex, method described by Tucker [32]. The De Boer method was also used to analyze the present data and u//l values of 3.4 + 0.7 Gy for X-rays alone and 2.6 & 0.9 Gy for X + c-DDP were obtained for tests at 31 weeks. This confirms the conclusions from the reciprocal total dose analysis; i.e. c-DDP did not influence the u//l ratio for renal radiation injury. These data imply that c-DDP did not modify the renal radiation sensitivity and that enhanced damage after the combined treatments was probably the result of independent additive toxicities. The results for the kidney contrast, to some extent, with those obtained for mouse intestine [lo], in which there was a slight (but not significant) increase in a//3 after combined treatments with X-rays + c-DDP compared with irradiation alone (a//% of 20.0 _+4 Gy cf 13.0 f 1.7 Gy). Some inhibition of repair of X-ray damage by c-DDP in the intestine is therefore possible, although the contribution of this effect was estimated to be small in comparison with the effects of independent killing [lo]. Repair inhibition by c-DDP has also been implicated in the lung where DEFs of up to 1.4 were measured for fractionated irradiation schedules [29], whereas separate single dose studies did not demonstrate any enhancement [22,35]. There is a single report

suggesting inhibition of repair of X-ray damage by c-DDP in mouse skin [ 121, but the majority of investigators have found little or no influence of c-DDP on skin damage after single or fractionated irradiation [2,18,34]. There was also no c-DDP enhancement of radiation mucositis in mice after either single or fractionated irradiations [ 171. Most of the normal tissues studied have therefore demonstrated only modest increases of radiation damage by c-DDP and these effects can largely be explained by additive toxicities. The possible exceptions are intestine and lung, where some repair inhibition and sensitization may occur. In conclusion, c-DDP given in combination with fractionated renal irradiation led to an increase in the severity of late kidney damage which was mainly due to additive, independent toxicities. There is little evidence from these studies for significant radiosensitization of the kidneys by c-DDP or for any inhibition of repair of radiation injury.

Acknowledgements

We are grateful to Drs. Adrian Begg, Joos Lebesque, Ben Mijnheer and Roe1 de Boer for many helpful discussions during the planning of these experiments and in preparation of the manuscript. We would also like to thank Mr. Ton Luts for help with irradiation and testing of the mice, Guus Hart for statistical analysis of the data and Miss Thea Eggenhuizen for typing the manuscript. This work was fully supported by The Netherlands Cancer Foundation (KWF), project NKI 85-2.

References Al-Saraff, M., Pajak, T. F., Marcial, V. A., Mowry, P., Cooper, J. S., Stetz, J., Ensley, J. F. and Velez-Garcia, E. Concurrent radiotherapy and chemotherapy with cisplatin in inoperable squamous cell carcinoma of the head and neck. An RTOG study. Cancer 59: 259-265, 1987.

101 2 Bartelink, H., Kallman, R. F., Rapacchietta, D. and Hart, A. A. M. Therapeutic enhancement in mice by clinically relevant dose and fractionation schedules of cisdiamminedichloroplatinum(I1) and irradiation. Radiother. Oncol. 6: 61-74, 1986. 3 Begg, A. C. Additivity versus repair inhibition in fractionated treatments combining drugs and X rays: a theoretical analysis. Int. J. Radiat. Oncol. Biol. Phys. 13: 921-927, 1987. 4 Begg, A. C., Van der Kolk, P. J., Dewit, L. and Bartelink, H. Radiosensitization by cisplatin of RIFl tumour cells in vitro. Int. J. Radiat. Biol. 50: 871-874, 1986. 5 Belt, R. J., Himmelstein, K. J., Patton, T. F., Bannister, S. J., Sternson, L. A. and Repta, A. J. Pharmacokinetics of non-protein-bound platinum species following administration of cis-dichloroplatinum(I1). Cancer Treat. Rep. 63: 1515-1521, 1979. 6 Carde, P. and Laval, F. Effect of cis-dichlorodiammine platinum II and X rays on mammalian cell survival. Int. J. Radiat. Oncol. Biol. Phys. 7: 923-933, 1981. 7 Crissman, J. D., Pajak, T. F., Zarbo, R. J., Martial, V. A. and Al-Saraf, M. Improved response and survival to combined cisplatin and radiation in non-keratizing squamous cell carcinomas of the head and neck. Cancer 59: 1391-1397, 1987. 8 De Boer, R. W. The use of the D versus dD plot to estimate the a//3 ratio from iso-effect radiation damage data. Radiother. Oncol. 11: 361-367, 1988. 9 Dewit, L. Combined treatment of radiation and cisdiamminedichloroplatinum(I1): a review of experimental and clinical data. Int. J. Radiat. Oncol. Biol. Phys. 13: 403-426, 1987. 10 Dewit, L., Begg, A. C., Kohler, Y., Stewart, F. A. and Bartelink, H. Inlluence of cis-diamminedichloroplatinum(I1) on mouse duodenal crypt stem cell survival after multifraction X ray treatment. Int. J. Radiat. Oncol. Biol. Phys. 11: 1809-1816, 1985. 11 Douglas, B. G. and Fowler, J. F. The effect of multiple small doses of X rays on skin reaction in the mouse and a basic interpretation. Radiat. Res. 66: 401-426, 1976. 12 Douple, E. B., Eaton, W. and Tulloh, M. E. Skin radiosensitization studies using combined cis-dichlorodiammineplatinum(I1) and radiation. Int. J. Radiat. Oncol. Biol. Phys. 5: 1883-1885, 1979. 13 Douple, E. B. and Richmond, R. C. Enhancement of the potentiation of radiotherapy by Platinum drugs in a mouse tumor. Int. J. Radiat. Oncol. Biol. Phys. 8: 501-503, 1982. 14 Dritschillo, A., Piro, A. and Kelman, A. D. The effect of cis-platinum on the repair of radiation damage in plateau Chinese hamster (V-79) cells. Int. J. Radiat. Oncol. Biol. Phys. 5: 1345-1349, 1979. 15 Jongejan, H. T. M., Van der Kogel, A. J., Provoost, A. P. and Molenaar, J. C. Interaction of cis-diamminedichloroplatinum and renal irradiation on renal function in

16

17

18

19

20

21

22

23

24

25

26

27

28

the young and adult rat. Radiother. Oncol. 10: 49-57, 1987. Kovacs, C. J., Braunschweiger, P. G., Schenken, L. L. and Burholt, D. R. Proliferative defects in renal and intestinal epithelium after cis-dichlorodiammineplatinum(I1). Br. J. Cancer 45: 286-294, 1982. Landuyt, W., Ang, K. K. and van der Schueren, E. Combinations of single doses and fractionated treatments of cis-dichloro-diammineplatinum(II) and irradiation. Effect on mouse lip mucosa. Br. J. Cancer 54: 579-586, 1987. Lelieveld, P., Stoles, M. A., Brown, J. M. and Kallman, R. F. The effect of treatment in fractionated schedules with the combination of X-irradiation and six cytostatic drugs on the RIF-1 tumor and normal mouse skin. Int. J. Radiat. Oncol. Biol. Phys. 11: 111-121, 1985. LeRoy, A. F., Lutz, R. J., Dedrick, R. L., Litterst, C. L. and Guarino, A.M. Pharmacokinetic study of cis-dichlorodiammineplatinum(II) (DDP) in the beagle dog: thermodynamic and kinetic behaviour of DDP in a biologic milieu. Cancer Treat. Rep. 63: 59-71, 1979. Moulder, J. E. and Fish, B. L. Effect of sequencing on the combined toxicity of renal radiation and cis-platinum. J.N.C.I. Monographs 6: 35-39, 1988. Overgaard, J. and Kahn, A. R. Selective enhancement of radiation response in a C3H mammary carcinoma by cisplatin. Cancer Treat. Rep. 65: 501-503, 1981. Peckham, M. J. and Collis, C. H. Clinical objective and normal tissue response in combined chemotherapy and radiotherapy. Bull. Cancer. (Paris) 68: 132-141, 1981. Rojas, A. and Joiner, M. C. The influence of dose per fraction on repair kinetics. Radiother. Oncol. 14: 329-336, 1989. Schaake-Koning, C., Bartelink, H., Hora Adema, B., Schuster-Uitterhoeve, L. and Van Zandwijk, N. Radiotherapy and cis-diamminedichloroplatinum(I1) as a combined treatment modality for inoperable non-small cell lung cancer: a dose finding study. Int. J. Radiat. Oncol. Biol. Phys. 12: 379-383, 1986. Steel, G. G. and Peckham, M. J. Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int. J. Radiat. Oncol. Biol. Phys. 5: 85-91, 1979. Stewart, F. A., Bohlken, S., Begg, A. C. and Bartelink, H. Renal damage in mice after treatment with cisplatinum alone or in combination with X-irradiation. Int. J. Radiat. Oncol. Biol. Phys. 12: 927-933, 1986. Stewart, F. A.,Luts,A., Oussoren,Y., Begg,A. C.,Dewit, L. and Bartelink, H. Renal damage in mice after combined treatment with cisplatin and X-rays: a comparison of fractionated and single dose studies. N.C.I. Monographs, 6: 23-27, 1988. Stewart, F. A., Oussouren, Y., Luts, A., Begg, A. C., Dewit, L., Lebesque, J. and Bartelink, H. Repair of sublethal radiation injury after multiple small doses in mouse

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kidney an estimate of flexure dose. Int. J. Radiat. Oncol. Biol. Phys. 13: 765-772, 1987. Tanabe, M., Godat, D. and Kallman, R. F. Effects of fractionated schedules of irradiation combined with cisdiamminedichloroplatinum(I1) on the SCCVII tumor and normal tissues of the C3H/Km mouse. Int. J. Radiat. Oncol. Biol. Phys. 13: 1523-1532, 1987. Terheggen, P.M. A. B., Floot, B., Scherer, E., Begg, A. C., Fichtinger-Schepman, M. J. and Den Engelse, L. Immunocytochemical detection of interaction products of cis-diamminedichloroplatinum(I1) (cis-DDP) and cis-diammine (l,l-cyclobutanedicarboxylato)-platinum(I1) (CBDCA) with DNA in rodent tissue sections. Cancer Res. 47: 6719-6725, 1987. Towle, L. R., Hall, E. J. and Capuono, L. Studies with cisplatinum diamminedichloride at the cellular level. Radiat. Res. 83: 37-38, 1980. Tucker, S. L. Tests for the fit of the linear-quadratic

33

34

35

36

model to radiation isoeffect data. Int. J. Radiat. Oncol. Biol. Phys. 10: 1933-1939, 1984. Vermorken, J. B., Ten Bokkel Huinink, W. W., McVie, J G., Van der Vijgh, W. J. F. and Pinedo, H. M. Clinical pharmacology of cisplatin and some new platinum analogs. Proceedings of 2nd World Conference on Clinical Pharmacology and Therapeutics, Washington, 1983. Am. Sot. Pharmacol. Exp. Ther. pp. 967-983. Editors: L. Lemberger and M. Reidenberg, 1984. Von der Maase, M. H. Effect of cancer chemotherapeutic drugs on radiation-induced skin damage in mouse feet. Br. J. Radiol. 57: 697-707, 1984. Von der Maase, M., Overgaard, J. and Vaeth, M. Effect of cancer chemotherapeutic drugs on radiation-induced lung damage in mice. Radiother. Oncol. 5: 245-257,1986. Williams, M. V. and Denekamp, J. Sequential functional testing of radiation induced renal damage in the mouse. Radiat. Res. 94: 305-317, 1983.