1978, British Journal of Radiology, 51, 923-924
NOVEMBER
1978
Short communications
Radiation response of mice maintained on a defined diet By Shirley Lehnert, Ph.D.* University Surgical Clinic, Montreal General Hospital, Montreal, Quebec, Canada {Received April, 1978) The use of a defined diet consisting of readily absorbable nutrients ("elemental diet") has been reported to protect the intestinal epithelium of rats against lesions associated with 5-fluorouracil (Bounous et al., 1971a) and to increase 30-day survival in mice following 900 rad of X rays (Hugon and Bounous 1972). A diet of this type has been used in the management of patients with intestinal lesions induced by 5-fluorouracil (Bounous et al., 19716) and other antitumour agents (Cousineau et al., 1973) and has been administered to patients undergoing a course of radiotherapy for tumours of the abdominal region (Bounous et al., 1973). The effect of ingestion of a defined diet on the response of mice to whole body irradiation has been investigated in this •Present address: Department of Therapeutic Radiology, Royal Victoria Hospital, 687 Pine Avenue W., Montreal H3A 1A1, Quebec, Canada.
laboratory. Mice maintained on the defined diet (diet-fed) were allowed unlimited access to Meade Johnson Product 3200-A.S. (Table I) in powdered form from seven days prior to irradiation until the end of the experimental period. They were observed to gain weight at the same rate as their control counterparts who were fed Purina mouse chow ad libitum (chow-fed). Table II shows the mean lethal dose (MLD) of X rays at seven days and 30 days post-irradiation for chow-fed and diet-fed mice. Clearly post-irradiation survival is unchanged in mice maintained on the defined diet. This observation is at variance with the findings of Hugon and Bounous (1972) who showed enhanced survival for diet-fed mice at 30 days post-irradiation. Survival curves for clonogenic cells of the intestinal crypts are shown in Fig. 1. The exponential slopes of the
TABLE I COMPOSITION OF THE DEFINED DIET
Components
g.%
Mead Johnson casein hydrolysate* Sucrose Mead Johnson medium-chain triglycerides* Corn oil Olive oil Cod liver oil Vitamin mixturef Salt mixture (Hegsted)
24.9 44.7
100-
6.9 4.3
10.4 1.3 2.5 5.0
101-
*Mead Johnson Company of Canada Ltd., Belleville, Ontario. fVitamin diet fortification mixture, Nutritional Biochemical Corporation, Cleveland, Ohio. TABLE II MEAN LETHAL DOSE (RAD) AT 7 AND 30 DAYS
Mean Lethal Dose ± S.E. 7 Days
30 Days
Control
1186±13.0 (1160-1212)*
575±8.0 (559-591)
Fed defined diet 7 days prior to irradiation
1180±11.0 (1158-1202)
560±9.0 (542-578)
1.0
1200
1300 1400 Dose, Rad
1500
1600
FIG.1.
*95% Confidence limits of Mean Lethal Dose. Swiss ICR/HAM male mice (weight 25.0±2.0 gm.) were irradiated with 225 kV, 15 mA X rays (FSD 37.5 cm, dose rate 54 rad/minute). Approximately 120 mice were irradiated in the appropriate dose range, and MLD was calculated from the survival curve by the probit method.
Survival curves for crypt stem cells from chow-fed (o—o) or diet-fed mice (•—#) mice. Animals were killed approximately three and a half days after irradiation and the number of viable crypts per circumference was counted on cross-sections of the jejunum. The number of cells surviving a given dose or irradiation was calculated by correcting for crypts regenerating from more than one surviving cell according to Poisson statistics (Withers and Elkind, 1970).
923
VOL.
51, No. 611 Short communications
2 3 Days after irradiation
mice, but in diet-fed animals the initiation of the increase in incorporation of tritiated thymidine following irradiation was delayed by approximately 16 hours and the compensatory overshoot in proliferative activity observed in control was not seen. The results of the two experiments just described suggest that feeding of the defined diet to mice prior to irradiation has produced changes in the growth kinetics of the intestinal epithelium with the result that the number of proliferative cells in the crypt is reduced at the time of irradiation and post-irradiation proliferation is delayed and reduced in extent. In summary, the results of the investigations described here indicate that feeding of the defined diet to mice before irradiation has no radioprotective effect in terms of mortality and that the ability of the animal to regenerate intestinal epithelium rapidly following irradiation is slightly impaired, possibly as a result of changes in intestinal cell kinetics induced during the pre-irradiation feeding period.
FIG. 2. Incorporation of tritiated thymidine into the jejunum of mice at various times after 1100R whole body irradiation. ACKNOWLEDGMENTS Six mice were sacrificed in each group. Vertical bars These investigations were supported by the John A. represent standard error.3 (O—O) chow-fed mice (•—•) Hartford Foundation. I am indebted to Claudette Labelle diet-fed mice. 50 ju,Ci H-Thymidine (specific activity 2.0 Ci/mM, New England Nuclear) was injected intra- and Kathy Key for technical assistance. peritoneally 60 minutes prior to sacrifice. Radioactivity was REFERENCES assayed in a weighed solubilized portion of jejunum by BOUNOUS, G., HUGON, J. and GENTILE, J. M., 1971a. liquid scintillation counting. Elemental diet in the management of lesions produced by 5-fluorouracil in rats. Canadian Journal of Surgery, 14, 298-311. curves for control or diet-fed mice are the same at all dose BOUNOUS, G., GENTILE, J. M., and HUGON, J., 1971b. Elemental diet in the management of intestinal lesions levels studied, although the survival of crypt stem cells in produced by 5-fluorouracil in man. Canadian Journal of chow-fed mice was approximately twice that found in Surgery, 74,312-324. diet-fed mice. The fact that if extrapolated back to zero dose the curve for diet-fed mice would cut the abcissa at a BOUNOUS, G., TAHAN, W., SHUSTER, J., GOLD, P., CouSINEAU, L., ROCHAN, M and LEBEL E. 1973. The use of an lower point than that for control mice suggests that the elemental diet during abdominal radiation. Clinical number of clonogenic cells in the non-irradiated crypt is Research, 21,1066. lower in diet-fed mice than in chow-fed mice. In another experiment, the post-irradiation proliferative COUSINEAU L., BOUNOUS, G., ROCHON, M., SHUSTER J., GOLD P. and TAHAN W., 1973. The use of an elemental capacity of the gut was studied in chow-fed and diet-fed diet during treatment with anticancer agents. Clinical mice. Following a dose of 1100R whole body radiation, Research, 27,1066. incorporation of tritiated thymidine into the jejunum fell to a minimum within one day of irradiation, remained low for HUGON, J. S. and BOUNOUS G., 1972. Elemental diet in the management of the intestinal lesions produced by the following day and then increased reaching a maximum radiation in the mouse. Canadian Journal of Surgery, 15, at four days after irradiation (Fig. 2). At this time, the rate of 18-25. incorporation exceeded that seen prior to radiation and this overshoot reflects the occurrence of the high level of com- WITHERS, H. R. and ELKIND, M. M., 1970. Microcolony survival assay for cells of mouse intestinal mucosa expensatory proliferation which repopulates the intestinal posed to radiation. International Journal of Radiation epithelium following sub-lethal doses of radiation. The Biology, 77,261-267. same pattern was observed in both diet-fed and chow-fed
924
NOVEMBER
1978
Short communications
Radiation response of mice maintained on a defined diet By Shirley Lehnert, Ph.D.* University Surgical Clinic, Montreal General Hospital, Montreal, Quebec, Canada {Received April, 1978) The use of a defined diet consisting of readily absorbable nutrients ("elemental diet") has been reported to protect the intestinal epithelium of rats against lesions associated with 5-fluorouracil (Bounous et al., 1971a) and to increase 30-day survival in mice following 900 rad of X rays (Hugon and Bounous 1972). A diet of this type has been used in the management of patients with intestinal lesions induced by 5-fluorouracil (Bounous et al., 19716) and other antitumour agents (Cousineau et al., 1973) and has been administered to patients undergoing a course of radiotherapy for tumours of the abdominal region (Bounous et al., 1973). The effect of ingestion of a defined diet on the response of mice to whole body irradiation has been investigated in this •Present address: Department of Therapeutic Radiology, Royal Victoria Hospital, 687 Pine Avenue W., Montreal H3A 1A1, Quebec, Canada.
laboratory. Mice maintained on the defined diet (diet-fed) were allowed unlimited access to Meade Johnson Product 3200-A.S. (Table I) in powdered form from seven days prior to irradiation until the end of the experimental period. They were observed to gain weight at the same rate as their control counterparts who were fed Purina mouse chow ad libitum (chow-fed). Table II shows the mean lethal dose (MLD) of X rays at seven days and 30 days post-irradiation for chow-fed and diet-fed mice. Clearly post-irradiation survival is unchanged in mice maintained on the defined diet. This observation is at variance with the findings of Hugon and Bounous (1972) who showed enhanced survival for diet-fed mice at 30 days post-irradiation. Survival curves for clonogenic cells of the intestinal crypts are shown in Fig. 1. The exponential slopes of the
TABLE I COMPOSITION OF THE DEFINED DIET
Components
g.%
Mead Johnson casein hydrolysate* Sucrose Mead Johnson medium-chain triglycerides* Corn oil Olive oil Cod liver oil Vitamin mixturef Salt mixture (Hegsted)
24.9 44.7
100-
6.9 4.3
10.4 1.3 2.5 5.0
101-
*Mead Johnson Company of Canada Ltd., Belleville, Ontario. fVitamin diet fortification mixture, Nutritional Biochemical Corporation, Cleveland, Ohio. TABLE II MEAN LETHAL DOSE (RAD) AT 7 AND 30 DAYS
Mean Lethal Dose ± S.E. 7 Days
30 Days
Control
1186±13.0 (1160-1212)*
575±8.0 (559-591)
Fed defined diet 7 days prior to irradiation
1180±11.0 (1158-1202)
560±9.0 (542-578)
1.0
1200
1300 1400 Dose, Rad
1500
1600
FIG.1.
*95% Confidence limits of Mean Lethal Dose. Swiss ICR/HAM male mice (weight 25.0±2.0 gm.) were irradiated with 225 kV, 15 mA X rays (FSD 37.5 cm, dose rate 54 rad/minute). Approximately 120 mice were irradiated in the appropriate dose range, and MLD was calculated from the survival curve by the probit method.
Survival curves for crypt stem cells from chow-fed (o—o) or diet-fed mice (•—#) mice. Animals were killed approximately three and a half days after irradiation and the number of viable crypts per circumference was counted on cross-sections of the jejunum. The number of cells surviving a given dose or irradiation was calculated by correcting for crypts regenerating from more than one surviving cell according to Poisson statistics (Withers and Elkind, 1970).
923
VOL.
51, No. 611 Short communications
2 3 Days after irradiation
mice, but in diet-fed animals the initiation of the increase in incorporation of tritiated thymidine following irradiation was delayed by approximately 16 hours and the compensatory overshoot in proliferative activity observed in control was not seen. The results of the two experiments just described suggest that feeding of the defined diet to mice prior to irradiation has produced changes in the growth kinetics of the intestinal epithelium with the result that the number of proliferative cells in the crypt is reduced at the time of irradiation and post-irradiation proliferation is delayed and reduced in extent. In summary, the results of the investigations described here indicate that feeding of the defined diet to mice before irradiation has no radioprotective effect in terms of mortality and that the ability of the animal to regenerate intestinal epithelium rapidly following irradiation is slightly impaired, possibly as a result of changes in intestinal cell kinetics induced during the pre-irradiation feeding period.
FIG. 2. Incorporation of tritiated thymidine into the jejunum of mice at various times after 1100R whole body irradiation. ACKNOWLEDGMENTS Six mice were sacrificed in each group. Vertical bars These investigations were supported by the John A. represent standard error.3 (O—O) chow-fed mice (•—•) Hartford Foundation. I am indebted to Claudette Labelle diet-fed mice. 50 ju,Ci H-Thymidine (specific activity 2.0 Ci/mM, New England Nuclear) was injected intra- and Kathy Key for technical assistance. peritoneally 60 minutes prior to sacrifice. Radioactivity was REFERENCES assayed in a weighed solubilized portion of jejunum by BOUNOUS, G., HUGON, J. and GENTILE, J. M., 1971a. liquid scintillation counting. Elemental diet in the management of lesions produced by 5-fluorouracil in rats. Canadian Journal of Surgery, 14, 298-311. curves for control or diet-fed mice are the same at all dose BOUNOUS, G., GENTILE, J. M., and HUGON, J., 1971b. Elemental diet in the management of intestinal lesions levels studied, although the survival of crypt stem cells in produced by 5-fluorouracil in man. Canadian Journal of chow-fed mice was approximately twice that found in Surgery, 74,312-324. diet-fed mice. The fact that if extrapolated back to zero dose the curve for diet-fed mice would cut the abcissa at a BOUNOUS, G., TAHAN, W., SHUSTER, J., GOLD, P., CouSINEAU, L., ROCHAN, M and LEBEL E. 1973. The use of an lower point than that for control mice suggests that the elemental diet during abdominal radiation. Clinical number of clonogenic cells in the non-irradiated crypt is Research, 21,1066. lower in diet-fed mice than in chow-fed mice. In another experiment, the post-irradiation proliferative COUSINEAU L., BOUNOUS, G., ROCHON, M., SHUSTER J., GOLD P. and TAHAN W., 1973. The use of an elemental capacity of the gut was studied in chow-fed and diet-fed diet during treatment with anticancer agents. Clinical mice. Following a dose of 1100R whole body radiation, Research, 27,1066. incorporation of tritiated thymidine into the jejunum fell to a minimum within one day of irradiation, remained low for HUGON, J. S. and BOUNOUS G., 1972. Elemental diet in the management of the intestinal lesions produced by the following day and then increased reaching a maximum radiation in the mouse. Canadian Journal of Surgery, 15, at four days after irradiation (Fig. 2). At this time, the rate of 18-25. incorporation exceeded that seen prior to radiation and this overshoot reflects the occurrence of the high level of com- WITHERS, H. R. and ELKIND, M. M., 1970. Microcolony survival assay for cells of mouse intestinal mucosa expensatory proliferation which repopulates the intestinal posed to radiation. International Journal of Radiation epithelium following sub-lethal doses of radiation. The Biology, 77,261-267. same pattern was observed in both diet-fed and chow-fed
924
