17
Mutation Research, 48 (1977) 17--28
© Elsevier/North-Holland Biomedical Press
STUDIES ON MUTAGENICITY OF IRRADIATED SUGAR SOLUTIONS IN TYPHIMURIUM
SALMONELLA
A.S. A I Y A R and V. S U B B A R A O
Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Bombay 400 085 (India)
(Received January 1st, 1976) (Revision received June 15th, 1976) (Accepted June 27th, 1976)
Summary Irradiated sugar solutions are mutagenic towards S a l m o n e l l a t y p h i m u r i u m , the effect being dose-dependent up to 2.0 Mrad. At all doses, ribose solution exhibited greater mutagenicity than did sucrose solution. The mutagenic effect was observed only in dividing cells and appears to be directly related to the growth rate. A larger proportion of revertants was observed after incubation with irradiated sugar solution for a period of 4 h than for 24 h. Irradiation of the sugar solutions in the frozen conditions was effective in completely preventing the development of mutagenic potential. Post-irradiation storage of the sugar solutions for a prolonged period (25 weeks) also minimized their mutagenic effect. The irradiated sugar solutions gave rise to both missense and frame-shift (addition as well as deletion) types of mutation; ribose was more effective in inducing the latter type. The irradiated sugar solutions failed to show a mutagenic response in the host-mediated assay with mice as the mammalian host.
Introduction Among the many manifestations of toxicity resulting from exposure of cell cultures to irradiated media are those relating to damage to the genetic material. That sugar solutions exposed to ionizing radiations could cause genetic abnormalities was first indicated by Ehrenberg [8] who observed increased frequency of chlorophyll mutations in the M2 generation of barley seeds exposed to irradiated glucose. Various kinds of deviation in chromosomal structure and function have been reported subsequently by several investigators in cells from plant sources as well as mammalian organisms after treatment with irradiated sugar solutions [15,21]. There is a relative paucity of published in-
18 formation relating to the mutagenic property of irradiated carbohydrate solutions towards microorganisms; as against reports of irradiated media inducing mutations in Escherichia coli [6,23] and yeast [23], the lack of such an effect in lysogenic bacteria [5], E. coil [13] and S. typhimurium [25] is also documented. Reports on genetic effects in cells arising from an interaction with irradiated sugar components in their culture media have evoked considerable interest visa-vis their implications for the safety assessment of irradiated foods. The evaluation of genetic effects in the mammalian organism poses considerable difficulties, and studies with microorganisms may therefore be of significance, at least as a screening procedure. The studies described in this paper were undertaken to assess the possible mutagenicity of irradiated sugar solutions towards S. typhimurium. In addition to the experiments in vitro, a host-mediated assay [10] with mice has been carried out in an attempt to assess the significance of the findings with the mammalian organism. Materials and methods
Chemicals Sucrose and glucose, as well as various inorganic salts for the growth medium were of "AnalaR" grade, obtained from BDH Chemicals Ltd., Poole (England). Both sugars were certified to contain less than 0.0001% of either Fe or heavy metals. Ribose and L-histidine were from Sigma Chemical Company, St. Louis (USA), sodium thio-glycollate was a product of Nutritional Biochemicals Corporation, Cleveland (USA), and nutrient broth and agar of DIFCO Laboratories, Detroit, Michigan (USA). Millipore filters (HA, pore size 0.45 X 10 -6 M) were obtained from Millipore Corporation, Bedford, Massachusetts (USA). Liquid paraffin of reagent quality was purchased locally. Microorganisms Most of the experiments reported were carried out with S. typhimurium 434 His-, which has been used earlier for other studies to assess the effects of irradiated sugar solutions on growth and oxidative metabolism [24]. Three other mutants of the same organism developed by Dr. Bruce N. Ames, University of California, Berkeley (USA), as tester strains [1], have also been used. All these are histidine auxotrophs, differing in the nature of their induced mutation, and are designated as G-46 (base pair substitution), C-3076 (frame-shift with added base pair), and C-207 (frame-shift with base-pair deletion). The strain 434 (His-) has been identified in this laboratory as a missense mutant. Irradiation o f sugar solutions and growth o f Salmonella Solutions of the sugars were prepared in glass-distilled water and irradiated at room temperature with 6°Co gamma-rays in a Gamma Cell-220 (Atomic Energy of Canada Ltd., Canada) at a dose rate of 7--8 krad/min. When the sugar solutions were irradiated in air, they were contained in either test-tubes or conical flasks kept open to the atmosphere. For irradiation in the absence of oxygen the sugar solutions were purged with high purity nitrogen gas for 10 min, sealed in ampoules under nitrogen and then irradiated. Irradiation of the
19 sugar solution in the frozen state was achieved b y keeping the samples (containers) in dry ice before and during irradiation. For irradiation at neutral pH, the sugar solutions were prepared in 10 -~ M potassium phosphate buffer (pH 7.0). Unless otherwise stated, the sugar solutions were irradiated just before their incorporation into the growth medium. For assessment of the effect of postirradiation storage, the sugar solutions were stored open to air at 6--8°C for up to 25 weeks. The cells of the organism were pre-incubated in nutrient broth medium at 37°C for 18 h, harvested b y centrifugation and then washed. The medium used in these studies was a salt--glucose medium (M9 medium) [16] containing 0.002% of L-histidine. Mutagenicity studies in vitro The cells of S. typhimurium were incubated in the growth medium containing an equal volume of either the unirradiated or irradiated sugar solution (1%). The incubations were generally carried o u t at 37°C for a period of 4 h, except for the studies relating to the effects of: (1) time of incubation (up to 24 h) and (2) temperature of incubation (also at 4°C). The cell suspensions were centrifuged, the sedimented cells washed thoroughly and then plated on either fully supplemented or histidineless medium to determine total survivors and revertants, respectively. The histidineless plates contained histidine (1 /~g/ml, i.e. 2.5% of the usual requirement), so as to yield a background lawn facilitating the expression of the reversion mutation. The plates were incubated at 37°C for 48 h and were then scored. Host-mediated assay with mice Swiss albino male mice (15--20 g) were used as the mammalian system for testing of mutagenicity in vivo by the host-mediated assay. The host-mediated assay [10] was carried o u t both after a single dose feeding and after chronic oral administration of the irradiated sugar solution for one or t w o weeks. For the single dose study, the mice were given 2 ml of unirradiated or irradiated (2 Mrad) sugar solution (1%). At 2 h after the oral administration of the sugar solution, two ml of a saline suspension of the microorganism in the exponential phase of growth (about 2 × 108 cells/ml), was introduced into the peritoneal cavity of each mouse. The cells were allowed to grow for 3 h, and after the peritoneum had been flushed with 1 ml saline the cells were withdrawn under sterile conditions through a tuberculin syringe. The number of revertants (histidine independence) was scored as mentioned for the mutagenicity studies in vitro. Some of the mice were given orally 1 ml of the 1% sugar solution (unitradiated for control group and 2 Mrad irradiated for experimental group) each day for one and t w o weeks. The irradiated sugar solution was administered within a period of 7 days after its irradiation. After an interval of 3 h from the last dosing with the sugar solution, the microorganisms were incorporated into the host animal, as described earlier. The subsequent procedures for estimating the mutagenic response are identical with those described for the single-dose study.
20 Results and discussion The mutagenicity of irradiated sugar solutions towards S. typhimurium 434 (His-) was studied both in dividing (37°C) and non
Mutation Research, 48 (1977) 17--28
© Elsevier/North-Holland Biomedical Press
STUDIES ON MUTAGENICITY OF IRRADIATED SUGAR SOLUTIONS IN TYPHIMURIUM
SALMONELLA
A.S. A I Y A R and V. S U B B A R A O
Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Bombay 400 085 (India)
(Received January 1st, 1976) (Revision received June 15th, 1976) (Accepted June 27th, 1976)
Summary Irradiated sugar solutions are mutagenic towards S a l m o n e l l a t y p h i m u r i u m , the effect being dose-dependent up to 2.0 Mrad. At all doses, ribose solution exhibited greater mutagenicity than did sucrose solution. The mutagenic effect was observed only in dividing cells and appears to be directly related to the growth rate. A larger proportion of revertants was observed after incubation with irradiated sugar solution for a period of 4 h than for 24 h. Irradiation of the sugar solutions in the frozen conditions was effective in completely preventing the development of mutagenic potential. Post-irradiation storage of the sugar solutions for a prolonged period (25 weeks) also minimized their mutagenic effect. The irradiated sugar solutions gave rise to both missense and frame-shift (addition as well as deletion) types of mutation; ribose was more effective in inducing the latter type. The irradiated sugar solutions failed to show a mutagenic response in the host-mediated assay with mice as the mammalian host.
Introduction Among the many manifestations of toxicity resulting from exposure of cell cultures to irradiated media are those relating to damage to the genetic material. That sugar solutions exposed to ionizing radiations could cause genetic abnormalities was first indicated by Ehrenberg [8] who observed increased frequency of chlorophyll mutations in the M2 generation of barley seeds exposed to irradiated glucose. Various kinds of deviation in chromosomal structure and function have been reported subsequently by several investigators in cells from plant sources as well as mammalian organisms after treatment with irradiated sugar solutions [15,21]. There is a relative paucity of published in-
18 formation relating to the mutagenic property of irradiated carbohydrate solutions towards microorganisms; as against reports of irradiated media inducing mutations in Escherichia coli [6,23] and yeast [23], the lack of such an effect in lysogenic bacteria [5], E. coil [13] and S. typhimurium [25] is also documented. Reports on genetic effects in cells arising from an interaction with irradiated sugar components in their culture media have evoked considerable interest visa-vis their implications for the safety assessment of irradiated foods. The evaluation of genetic effects in the mammalian organism poses considerable difficulties, and studies with microorganisms may therefore be of significance, at least as a screening procedure. The studies described in this paper were undertaken to assess the possible mutagenicity of irradiated sugar solutions towards S. typhimurium. In addition to the experiments in vitro, a host-mediated assay [10] with mice has been carried out in an attempt to assess the significance of the findings with the mammalian organism. Materials and methods
Chemicals Sucrose and glucose, as well as various inorganic salts for the growth medium were of "AnalaR" grade, obtained from BDH Chemicals Ltd., Poole (England). Both sugars were certified to contain less than 0.0001% of either Fe or heavy metals. Ribose and L-histidine were from Sigma Chemical Company, St. Louis (USA), sodium thio-glycollate was a product of Nutritional Biochemicals Corporation, Cleveland (USA), and nutrient broth and agar of DIFCO Laboratories, Detroit, Michigan (USA). Millipore filters (HA, pore size 0.45 X 10 -6 M) were obtained from Millipore Corporation, Bedford, Massachusetts (USA). Liquid paraffin of reagent quality was purchased locally. Microorganisms Most of the experiments reported were carried out with S. typhimurium 434 His-, which has been used earlier for other studies to assess the effects of irradiated sugar solutions on growth and oxidative metabolism [24]. Three other mutants of the same organism developed by Dr. Bruce N. Ames, University of California, Berkeley (USA), as tester strains [1], have also been used. All these are histidine auxotrophs, differing in the nature of their induced mutation, and are designated as G-46 (base pair substitution), C-3076 (frame-shift with added base pair), and C-207 (frame-shift with base-pair deletion). The strain 434 (His-) has been identified in this laboratory as a missense mutant. Irradiation o f sugar solutions and growth o f Salmonella Solutions of the sugars were prepared in glass-distilled water and irradiated at room temperature with 6°Co gamma-rays in a Gamma Cell-220 (Atomic Energy of Canada Ltd., Canada) at a dose rate of 7--8 krad/min. When the sugar solutions were irradiated in air, they were contained in either test-tubes or conical flasks kept open to the atmosphere. For irradiation in the absence of oxygen the sugar solutions were purged with high purity nitrogen gas for 10 min, sealed in ampoules under nitrogen and then irradiated. Irradiation of the
19 sugar solution in the frozen state was achieved b y keeping the samples (containers) in dry ice before and during irradiation. For irradiation at neutral pH, the sugar solutions were prepared in 10 -~ M potassium phosphate buffer (pH 7.0). Unless otherwise stated, the sugar solutions were irradiated just before their incorporation into the growth medium. For assessment of the effect of postirradiation storage, the sugar solutions were stored open to air at 6--8°C for up to 25 weeks. The cells of the organism were pre-incubated in nutrient broth medium at 37°C for 18 h, harvested b y centrifugation and then washed. The medium used in these studies was a salt--glucose medium (M9 medium) [16] containing 0.002% of L-histidine. Mutagenicity studies in vitro The cells of S. typhimurium were incubated in the growth medium containing an equal volume of either the unirradiated or irradiated sugar solution (1%). The incubations were generally carried o u t at 37°C for a period of 4 h, except for the studies relating to the effects of: (1) time of incubation (up to 24 h) and (2) temperature of incubation (also at 4°C). The cell suspensions were centrifuged, the sedimented cells washed thoroughly and then plated on either fully supplemented or histidineless medium to determine total survivors and revertants, respectively. The histidineless plates contained histidine (1 /~g/ml, i.e. 2.5% of the usual requirement), so as to yield a background lawn facilitating the expression of the reversion mutation. The plates were incubated at 37°C for 48 h and were then scored. Host-mediated assay with mice Swiss albino male mice (15--20 g) were used as the mammalian system for testing of mutagenicity in vivo by the host-mediated assay. The host-mediated assay [10] was carried o u t both after a single dose feeding and after chronic oral administration of the irradiated sugar solution for one or t w o weeks. For the single dose study, the mice were given 2 ml of unirradiated or irradiated (2 Mrad) sugar solution (1%). At 2 h after the oral administration of the sugar solution, two ml of a saline suspension of the microorganism in the exponential phase of growth (about 2 × 108 cells/ml), was introduced into the peritoneal cavity of each mouse. The cells were allowed to grow for 3 h, and after the peritoneum had been flushed with 1 ml saline the cells were withdrawn under sterile conditions through a tuberculin syringe. The number of revertants (histidine independence) was scored as mentioned for the mutagenicity studies in vitro. Some of the mice were given orally 1 ml of the 1% sugar solution (unitradiated for control group and 2 Mrad irradiated for experimental group) each day for one and t w o weeks. The irradiated sugar solution was administered within a period of 7 days after its irradiation. After an interval of 3 h from the last dosing with the sugar solution, the microorganisms were incorporated into the host animal, as described earlier. The subsequent procedures for estimating the mutagenic response are identical with those described for the single-dose study.
20 Results and discussion The mutagenicity of irradiated sugar solutions towards S. typhimurium 434 (His-) was studied both in dividing (37°C) and non
