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Radiosensitization of EL4 lymphoma cells by a commonly used food additive-butylated hydroxytoluene (BHT)
Mutation Research, 21 (I973) 293-295 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
293
Radiosensitization of EL4 lymphoma cells by a commonly used food additive-butylated hydroxytoluene (BET) R e c e n t experiments have suggested t h a t a phenolic a n t i o x i d a n t , B H T , is a p o t e n t radiosensitizer for the sex-linked recessive lethal m u t a t i o n s in the m a t u r e sperm of Drosophila melanogaster although at the c o n c e n t r a t i o n used (o.ooi g %) it appeared to be n o n - m u t a g e n i c a n d non-lethal~, 6. Since phenolic a n t i o x i d a n t s ( B H T along with BHA, a n d PG) are widely used to stabilize fats a n d oils c o n t a i n e d in food 7 a n d are considered harmless 9 when used in low c o n c e n t r a t i o n s it was decided to test the p o t e n t i a l radiosensitizing ability of B H T on mouse E L 4 l y m p h o m a cells. The results presented here are in complete agreement with the earlier Drosophila work~, 6 a n d indicate t h a t B H T is a very p o t e n t radiosensitizer for the E L 4 l y m p h o m a cells. E L 4 l y m p h o m a was originally o b t a i n e d from the Chester B e a t t y Research I n s t i t u t e , London, a n d m a i n t a i n e d in our l a b o r a t o r y b y serial i n t r a p e r i t o n e a l passage in the C57BL mice. Freshly collected E L 4 l y m p h o m a cells were i n c u b a t e d for 30 rain in B H T dissolved in alcohol a n d resuspended in PBS to a final c o n c e n t r a t i o n of o.ooi g°/o or o.oooi g % B H T in 0.2% ethyl alcohol. Controls w i t h o u t B H T were suspended in PBS c o n t a i n i n g 0.2 % ethyl alcohol for the same duration. The cell suspension was exposed to o, 400, 600, 800, iooo, 125o, 15oo, 175o a n d 2000 rad of v-rays from a 137Cs 7 - b e a m (Atomic E n e r g y of C a n a d a Ltd.) at a dose rate of 4.1 rad per second. Groups of five mice were injected i n t r a p e r i t o n e a l l y with l y m p h o m a cells t h a t had been i n c u b a t e d in PBS or PBS + B H T for all the different radiation doses. The viabilit y of the cells in all t r e a t m e n t s was assessed b y t r y p a n blue exclusion test a n d lO 6 viable cells were inoculated per mouse. Some of the t r e a t m e n t s were r e p e a t e d a n d "FABLE I SURVIVALOF C57BL MICE Mean survival time (days 4- S.D.) of groups of mice (5-1o per treatment) inoculated with lO6 EL 4 lymphoma cells pretreated with:
Radiation dose (rad)
PBS
PBS+BHT (o.oor g%)
o 400 600 800 iooo I250 I5oo
I3.8 ± 1.33 I4. 4 :/~ 1,5I 16.8 ± 2.04 I8.6 :t: 1.26 2o. 3 -4- x.7r 24.0 4- 1.65 6 died, mean survival 25.I ± 3.3 days 4 surviving All surviving All surviving 16.8 Jr 2.04
I2. 4:4- 0.47 18.6 :-r 1,5I 25.0 2~ I.O All surviving All surviving All surviving
i75o 20oo 600 800
All surviving All surviving All surviving I6.8 a 4- 1.64 I2.6b ~: 0. 9
a O.OOOI g~0 BHT used. There is no radiosensitization effect at this concentration. b Unirradiated lymphoma cells were exposed to the radiolytic products of o.ooi g% BHT in PBS at 8oo rad. No deleterious effects were observed. A b b r e v i a t i o n s : BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; PBS, phos-
phate-buffered saline; PG, propyl gallate.
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consistently reproducible results on the mean survival time of tumor-inoculated mice were obtained. Table I summarises the details of survival 125 days after tumor cell inoculation. The inoculation of the lO 8 viable E L 4 cells into the peritoneal cavity of C57BL mice resulted in the development of ascites tumor and lethality in about 13.8 -~ 1. 3 days. B H T (o.ooi g°/o) alone had no apparent deleterious effects on the viability of the cells and the mean survival time of mice (12.4 ± 0.5 days) was not significantly different from the PBS control. When the unirradiated cells were exposed to the radiolytic products of PBS + BHT (o.ooi gO/o) at 800 tad there was no observable effect on cell viability and the mean survival time of the inoculated mouse (12.6 ~ 0. 9 days). On the other hand, whenever B H T (o.ooi g~/o) was present during cell irradiation, there was a very pronounced increase in the survival of C57BL mice. This radiosensitization was absent when a lower concentration of B H T (o.ooi g°/o) was used (Table I). Therefore, there is a threshold concentration (between o.ooi g~/o and o.oooi g ~/o) below which B H T is ineffective. The survival of EL4 inoculated mice is dependent on the number of tumor cells inoculated, i.e., with a higher number of inoculated cells the tumors appear earliel and the survival time is reduced accordingly (Table II). The mean survival data in Table I suggests that B H T radiosensitized by a factor of about two, i.e., in the absence of B H T approximately double the amount of radiation is required to produce an effect which would occur if B H T (o.ooi g ~o) was present during cell irradiation. In a separate in vitro experiment it was observed that the cell viability (trypan blue exclusion test) was not significantly altered for 4 h after irradiation of BHT-treated lymphoma cells. TABLE II S U R V I V A L OF
EL 4 L Y M P H O M A
I N O C U L A T E D MICE &
Number of EL 4 ells inoculated
Survival (days ~= S.D.)
103
20.0 -- I.O
lO4
18.4 + 0.55 15.6 -~ 1 . 8 1
105 IO e 10 T
12.8 ± 0.83 1 2 . 4 4- O . 8 9
a The differences in the mean life-span of IOe cell-injected mice in Table I and Table II are a non-significant statistical variation of two separate experiments. Apparently a fraction of the cells although viable at the time of inoculation are unable to multiply and produce tumors in the mice. By comparing the survival data in Tables I and II, it is possible to get an approximate idea about the fraction of inoculated EL 4 cells that is unable to multiply. For example, the mean survival time for mice inoculated with lO 4 cells is 18.4 -1- 0.5 days (Table II) which approximately equals the survival when lO 6 cells irradiated with 800 rad are inoculated; the same survival is obtained with 400 rad in the presence of B H T (Table I). In feeding experiments B H T (0.5% wt. of daily diet) has been reported to prolong the mean life-span of LAF1 mice by as much as 45 % (ref. 4). This has been attributed to the antioxidant properties of the chemical and would appear to be consistent with the free radical theory of aging in mammals. Based upon these observations, HARMAN4 has suggested that the mean life-span in man could be increased by 5 to 15 years by adding one or more free-radical reaction inhibitors to a properly
SHORT COMMUNICATIONS
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selected d i e t ; a r e c o m m e n d a t i o n u n l i k e l y to be a c c e p t e d b y the r e g u l a t o r y authorities. A l t h o u g h B H T does not seem to p r o d u c e d o m i n a n t lethals I or increase the f r e q u e n c y of r a d i a t i o n - i n d u c e d d o m i n a n t lethals in mice ~ it does h a v e a p r o n o u n c e d sensitizing effect on t h e life-span of 725R w h o l e - b o d y - i r r a d i a t e d mice 1. The W o r l d H e a l t h O r g a n i z a t i o n a n d The F o o d a n d A g r i c u l t u r e O r g a n i z a t i o n of the U n i t e d N a t i o n s as well as the N a t i o n a l R e g u l a t o r y bodies ( F D A in the U.S., F D D in C a n a d a a n d the M i n i s t r y of A g r i c u l t u r e in t h e U.K.) allow an i n t a k e of o.5 m g B H T / k g b o d y weight 8. The a m o u n t s c o n s u m e d d a i l y b y an a d u l t Canadian are est i m a t e d as 89 m g B H T + B H A along with u p to 35 m g P G 3. I n view of the chronic n a t u r e of exposure to m a n a n d t h e clear indications we h a v e now a b o u t the radiosensitizing p r o p e r t y of B H T it is necessary to f u r t h e r e v a l u a t e the p o t e n t i a l h a z a r d s posed b y phenolic a n t i o x i d a n t s . The m e c h a n i s m b y which B H T exposure radiosensitized E L 4 l y m p h o m a cells (or m a t u r e D r o s o p h i l a sperm~, * r e m a i n s to be d e t e r m i n e d . This w o r k was assisted in p a r t b y funds p r o v i d e d b y the Public H e a l t h R e s e a r c h G r a n t No. 6o2-7-158, N a t i o n a l R e s e a r c h Council G r a n t No. A-4892 a n d funds for d e v e l o p m e n t of research b y the F a c u l t y of Medicine, Dalhousie U n i v e r s i t y . *Laboratory of Radiation Biology, and **Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada)
O. P. KAMRA* T. GHOSE** M. MAMMEN**
I CUMMINGS, R. B., Chemically induced changes in the effects of chemical nlutagens in the mouse
Genetics, 64 (197o) SI 4.
2 EPSTEIN, S. S., AND H. SHAFNER, Chemical mutagens in the human environment, Nature, 219
(1968) 385-387 . 3 Food Advisory Bureau, Ottawa, Ont., Canada (Personal communication). 4 HARMAN, D., Agents and Actions, I (1969) 3-8. 5 KAMRA, O. P., Intern. J. Radiation Biol., 23 (1973) 295-297. 6 KAMRA, O. P., AND R. RAJARAMAN, Third Environ. Mutagenic Soc. Meeting, Cherry Hill, N.J. 1972 (Abstract). 7 ]~ERMODE, G. O., Sci. Am., 226 (1972) 15--20. 8 Specifications for the Identity and Purity of Food Additives and Their Toxicological Evaluation, FAO/VVHO Expert Committee on Food Additives, Rept No. 9 (FAO and WHO, Geneva, 19661. 9 STUCKEY, I3. N., in T. E. FURIA (Ed.), CRC Handbook of Food Additives, Chem. Rubber Co., Cleveland, 1968, pp. 209-245. R e c e i v e d F e b r u a r y 9th, 1973
293
Radiosensitization of EL4 lymphoma cells by a commonly used food additive-butylated hydroxytoluene (BET) R e c e n t experiments have suggested t h a t a phenolic a n t i o x i d a n t , B H T , is a p o t e n t radiosensitizer for the sex-linked recessive lethal m u t a t i o n s in the m a t u r e sperm of Drosophila melanogaster although at the c o n c e n t r a t i o n used (o.ooi g %) it appeared to be n o n - m u t a g e n i c a n d non-lethal~, 6. Since phenolic a n t i o x i d a n t s ( B H T along with BHA, a n d PG) are widely used to stabilize fats a n d oils c o n t a i n e d in food 7 a n d are considered harmless 9 when used in low c o n c e n t r a t i o n s it was decided to test the p o t e n t i a l radiosensitizing ability of B H T on mouse E L 4 l y m p h o m a cells. The results presented here are in complete agreement with the earlier Drosophila work~, 6 a n d indicate t h a t B H T is a very p o t e n t radiosensitizer for the E L 4 l y m p h o m a cells. E L 4 l y m p h o m a was originally o b t a i n e d from the Chester B e a t t y Research I n s t i t u t e , London, a n d m a i n t a i n e d in our l a b o r a t o r y b y serial i n t r a p e r i t o n e a l passage in the C57BL mice. Freshly collected E L 4 l y m p h o m a cells were i n c u b a t e d for 30 rain in B H T dissolved in alcohol a n d resuspended in PBS to a final c o n c e n t r a t i o n of o.ooi g°/o or o.oooi g % B H T in 0.2% ethyl alcohol. Controls w i t h o u t B H T were suspended in PBS c o n t a i n i n g 0.2 % ethyl alcohol for the same duration. The cell suspension was exposed to o, 400, 600, 800, iooo, 125o, 15oo, 175o a n d 2000 rad of v-rays from a 137Cs 7 - b e a m (Atomic E n e r g y of C a n a d a Ltd.) at a dose rate of 4.1 rad per second. Groups of five mice were injected i n t r a p e r i t o n e a l l y with l y m p h o m a cells t h a t had been i n c u b a t e d in PBS or PBS + B H T for all the different radiation doses. The viabilit y of the cells in all t r e a t m e n t s was assessed b y t r y p a n blue exclusion test a n d lO 6 viable cells were inoculated per mouse. Some of the t r e a t m e n t s were r e p e a t e d a n d "FABLE I SURVIVALOF C57BL MICE Mean survival time (days 4- S.D.) of groups of mice (5-1o per treatment) inoculated with lO6 EL 4 lymphoma cells pretreated with:
Radiation dose (rad)
PBS
PBS+BHT (o.oor g%)
o 400 600 800 iooo I250 I5oo
I3.8 ± 1.33 I4. 4 :/~ 1,5I 16.8 ± 2.04 I8.6 :t: 1.26 2o. 3 -4- x.7r 24.0 4- 1.65 6 died, mean survival 25.I ± 3.3 days 4 surviving All surviving All surviving 16.8 Jr 2.04
I2. 4:4- 0.47 18.6 :-r 1,5I 25.0 2~ I.O All surviving All surviving All surviving
i75o 20oo 600 800
All surviving All surviving All surviving I6.8 a 4- 1.64 I2.6b ~: 0. 9
a O.OOOI g~0 BHT used. There is no radiosensitization effect at this concentration. b Unirradiated lymphoma cells were exposed to the radiolytic products of o.ooi g% BHT in PBS at 8oo rad. No deleterious effects were observed. A b b r e v i a t i o n s : BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; PBS, phos-
phate-buffered saline; PG, propyl gallate.
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consistently reproducible results on the mean survival time of tumor-inoculated mice were obtained. Table I summarises the details of survival 125 days after tumor cell inoculation. The inoculation of the lO 8 viable E L 4 cells into the peritoneal cavity of C57BL mice resulted in the development of ascites tumor and lethality in about 13.8 -~ 1. 3 days. B H T (o.ooi g°/o) alone had no apparent deleterious effects on the viability of the cells and the mean survival time of mice (12.4 ± 0.5 days) was not significantly different from the PBS control. When the unirradiated cells were exposed to the radiolytic products of PBS + BHT (o.ooi gO/o) at 800 tad there was no observable effect on cell viability and the mean survival time of the inoculated mouse (12.6 ~ 0. 9 days). On the other hand, whenever B H T (o.ooi g~/o) was present during cell irradiation, there was a very pronounced increase in the survival of C57BL mice. This radiosensitization was absent when a lower concentration of B H T (o.ooi g°/o) was used (Table I). Therefore, there is a threshold concentration (between o.ooi g~/o and o.oooi g ~/o) below which B H T is ineffective. The survival of EL4 inoculated mice is dependent on the number of tumor cells inoculated, i.e., with a higher number of inoculated cells the tumors appear earliel and the survival time is reduced accordingly (Table II). The mean survival data in Table I suggests that B H T radiosensitized by a factor of about two, i.e., in the absence of B H T approximately double the amount of radiation is required to produce an effect which would occur if B H T (o.ooi g ~o) was present during cell irradiation. In a separate in vitro experiment it was observed that the cell viability (trypan blue exclusion test) was not significantly altered for 4 h after irradiation of BHT-treated lymphoma cells. TABLE II S U R V I V A L OF
EL 4 L Y M P H O M A
I N O C U L A T E D MICE &
Number of EL 4 ells inoculated
Survival (days ~= S.D.)
103
20.0 -- I.O
lO4
18.4 + 0.55 15.6 -~ 1 . 8 1
105 IO e 10 T
12.8 ± 0.83 1 2 . 4 4- O . 8 9
a The differences in the mean life-span of IOe cell-injected mice in Table I and Table II are a non-significant statistical variation of two separate experiments. Apparently a fraction of the cells although viable at the time of inoculation are unable to multiply and produce tumors in the mice. By comparing the survival data in Tables I and II, it is possible to get an approximate idea about the fraction of inoculated EL 4 cells that is unable to multiply. For example, the mean survival time for mice inoculated with lO 4 cells is 18.4 -1- 0.5 days (Table II) which approximately equals the survival when lO 6 cells irradiated with 800 rad are inoculated; the same survival is obtained with 400 rad in the presence of B H T (Table I). In feeding experiments B H T (0.5% wt. of daily diet) has been reported to prolong the mean life-span of LAF1 mice by as much as 45 % (ref. 4). This has been attributed to the antioxidant properties of the chemical and would appear to be consistent with the free radical theory of aging in mammals. Based upon these observations, HARMAN4 has suggested that the mean life-span in man could be increased by 5 to 15 years by adding one or more free-radical reaction inhibitors to a properly
SHORT COMMUNICATIONS
295
selected d i e t ; a r e c o m m e n d a t i o n u n l i k e l y to be a c c e p t e d b y the r e g u l a t o r y authorities. A l t h o u g h B H T does not seem to p r o d u c e d o m i n a n t lethals I or increase the f r e q u e n c y of r a d i a t i o n - i n d u c e d d o m i n a n t lethals in mice ~ it does h a v e a p r o n o u n c e d sensitizing effect on t h e life-span of 725R w h o l e - b o d y - i r r a d i a t e d mice 1. The W o r l d H e a l t h O r g a n i z a t i o n a n d The F o o d a n d A g r i c u l t u r e O r g a n i z a t i o n of the U n i t e d N a t i o n s as well as the N a t i o n a l R e g u l a t o r y bodies ( F D A in the U.S., F D D in C a n a d a a n d the M i n i s t r y of A g r i c u l t u r e in t h e U.K.) allow an i n t a k e of o.5 m g B H T / k g b o d y weight 8. The a m o u n t s c o n s u m e d d a i l y b y an a d u l t Canadian are est i m a t e d as 89 m g B H T + B H A along with u p to 35 m g P G 3. I n view of the chronic n a t u r e of exposure to m a n a n d t h e clear indications we h a v e now a b o u t the radiosensitizing p r o p e r t y of B H T it is necessary to f u r t h e r e v a l u a t e the p o t e n t i a l h a z a r d s posed b y phenolic a n t i o x i d a n t s . The m e c h a n i s m b y which B H T exposure radiosensitized E L 4 l y m p h o m a cells (or m a t u r e D r o s o p h i l a sperm~, * r e m a i n s to be d e t e r m i n e d . This w o r k was assisted in p a r t b y funds p r o v i d e d b y the Public H e a l t h R e s e a r c h G r a n t No. 6o2-7-158, N a t i o n a l R e s e a r c h Council G r a n t No. A-4892 a n d funds for d e v e l o p m e n t of research b y the F a c u l t y of Medicine, Dalhousie U n i v e r s i t y . *Laboratory of Radiation Biology, and **Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada)
O. P. KAMRA* T. GHOSE** M. MAMMEN**
I CUMMINGS, R. B., Chemically induced changes in the effects of chemical nlutagens in the mouse
Genetics, 64 (197o) SI 4.
2 EPSTEIN, S. S., AND H. SHAFNER, Chemical mutagens in the human environment, Nature, 219
(1968) 385-387 . 3 Food Advisory Bureau, Ottawa, Ont., Canada (Personal communication). 4 HARMAN, D., Agents and Actions, I (1969) 3-8. 5 KAMRA, O. P., Intern. J. Radiation Biol., 23 (1973) 295-297. 6 KAMRA, O. P., AND R. RAJARAMAN, Third Environ. Mutagenic Soc. Meeting, Cherry Hill, N.J. 1972 (Abstract). 7 ]~ERMODE, G. O., Sci. Am., 226 (1972) 15--20. 8 Specifications for the Identity and Purity of Food Additives and Their Toxicological Evaluation, FAO/VVHO Expert Committee on Food Additives, Rept No. 9 (FAO and WHO, Geneva, 19661. 9 STUCKEY, I3. N., in T. E. FURIA (Ed.), CRC Handbook of Food Additives, Chem. Rubber Co., Cleveland, 1968, pp. 209-245. R e c e i v e d F e b r u a r y 9th, 1973