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Cell culture tumor promotion experiments with saccharin, phorbol myristate acetate and several common food materials
Cancer Letters, 10 (1980) 0 Blsevier/North-Holland
27-32 Scientific
27 Publishers
Ltd.
CELL CULTURE TUMOR PROMOTION EXPERIMENTS WITH SACCHARIN, PHORBOL MYRISTATE ACETATE AND SEVERAL COMMON FOOD MATERIALS*
ANDREW
SIVAK**
and ALICE S. TU
Bio/Medical Sciences Section, Arthur D. Little, Inc., Cambridge, MA 02140 (U.S.A.) (Received (Accepted
28 March 1980) 3 April 1980)
SUMMARY
The BALB/c-3T3 cell neoplastic transformation system was modified to examine the tumor promoting activity of a set of substances. Following initiation of the target cells with 3-methylcholanthrene, treatment of the cultures with phorbol myristate acetate (0.01 pg/ml; 1.5 X lO_” M) during the remainder of the 4-week assay interval resulted in a marked increase in both spontaneous and initiated Type III transformed foci. In contrast, a similar treatment with saccharin at 20, 100 or 500 pg/ml (0.08, 0.4 or 2.1 X lo-’ M)’did not influence the occurrence of Type III transformed foci and did not result in a promoting response. Sodium ascorbate (2.53 X 10s3 M) and L-tryptophan (2.45 X lo-’ M) almost completely inhibited both spontaneous and initiated Type III transformed foci. Calcium pantothenate (2.10 X 10e3 M) exhibited a marginal promoting effect. Under the conditions of this study in which the classical tumor promoter phorbol myristate acetate was highly active in promoting Type III transformed foci, saccharin was not active as either a direct transforming or promoting agent at doses up to 5 orders of magnitude higher.
INTRODUCTION
The finding of a low, but statistically significant, increase in bladder tumors in male rats of the second generation fed high doses of saccharin (5% of the diet) [l] has resulted in regulatory concern and wide public *This work, conducted Control Council.
at Arthur D. Little, Inc., was funded by members of the Calorie
**Address all correspondence
to: Andrew Sivak, Bio/Medical Little, Inc., Acorn Park, Cambridge, MA 02140, U.S.A.
Sciences Section,
Arthur D.
debate over its safety in humans. The available epidemiological data [ 6,13,16] do not provide any substantial evidence for a direct linkage between saccharin consumption and a significant increase in bladder cancer in humans. The absence of a carcinogenic effect in a number of other feeding studies in rats [ 61, the observations that there is no evidence for metabolic conversion of saccharin in rats or humans [ 4,141 and the equivocal results obtained in several rapid mutagenesis assays [ 71 do not provide a strong expierimental basis to ascertain whether normal levels of saccharin consumption pose a health risk. Indeed, the low frequency of tumors found in the 2-generation study and the knowledge that the urinary tracts of the test animals were under extreme physiological stress at the high doses of saccharin employed in this study suggest that the induction of tumors was the result of secondary, modifying events rather than a direct carcinogenic stimulus. Two published in vivo studies lend support to this view. Hicks [ 91 found that rats given single transurethral bladder doses of N-methyl-N-nitrosourea (MNU) at 1.5 mg, and then fed saccharin in the diet for up to 2 years at 2.0 or 4.0 g/kg/day, bore significantly more bladder tumors than rats given MNU alone. Because of the unstable chemical nature of the initiating agent and the later report of uncertainty with respect to the actual concentrations used in the various test groups [lo], the results of this study should be viewed with caution. In contrast, Cohen et al. [ 41 appear to have demonstrated rather clearly in an experiment using FANFT, a bladder carcinogen for rats when administered orally, that saccharin fed in the diet alter a dose regimen of FANFT that is weakly carcinogenic, induces a significant increase in both the incidence and severity of tumors of the bladder. These studies indicate that saccharin may be acting in a tumor-promoting manner, possibly as a result of unusually severe physiological pressures occurring in the bladder rather than in the usual sense of the tumor promoting phenomenon as a specific cellular response. In a report that appears to support the vie& that saccharin may be acting as a tumor promotor, Mondal and Heidelberger [ 121 reported that saccharin induced a promoting effect at 100 pg/ml in the medium of C3H-lOT,,, cells in culture following an initiating treatment with 3-methylcholanthrene. This dose of saccharin was the only dose tested. However, no promoting effect with saccharin was observed following initiation with ultraviolet light exposure even though the phorbol esters, the only class of chemicals reported to have a promoting type of activity in this particular cell system, were quite active after either 3-methylcholanthrene or ultraviolet light initiating stimuli. In order to obtain additional information bearing on the issue of the potential for tumor-promoting activity of saccharin, we performed several experiments with the BALB/c-3T3 cell neoplastic transformation system, which, like the C3H-lOT,,, system, also exhibits a promoting type of response with phorbol esters. Cultures of the l-13 line of BALB/c-3T3 cells were initiated with 3-methylcholanthrene (0.1 pg/ml. 72 h) using the protocol
29
for carcinogen exposure usually employed for transformation assays. On the seventh day of the culture, and with each biweekly medium renewal thereafter, the agents to be tested for promoting activity were added to the medium. The chemicals tested were sodium saccharin, sodium ascorbate. L-tryptophan, and calcium pantothenate. Four weeks after the termination of MCA treatment the experiments were stopped by fixing the cells with methanol after medium removal and rinsing with phosphate-buffered saline. The plates were then stained with 10% Giemsa, and Type III foci were identified by examination of each plate using a dissecting microscope (4-40X ). RESULTS
The results of 2 separate experiments are shown in Fig. 1. Several conclusions are apparent from these data. First, the induction of neoplastic transformation by MCA alone and its enhancement by phorbol myristate acetate, the potent tumor-promoting agent for mouse skin, was clearly shown in both studies. Second, saccharin over a wide cencentration range (0.08-2.1 mM), which did not exhibit toxicity with respect to reduction of cell number or altered cellular morphology, did not by itself induce neoplastic transformation. Further, saccharin did not exhibit a promoting effect by markedly enhancing either the spontaneous transformation or the low level of transformation induced by the initiating treatment with 0.1 pg/ml of MCA. The increase in transformation induced with 20 pg/ml saccharin in MCA-initiated plates of Experiment I was not reproduced in Experiment II, nor was the spontaneous transformation enhanced in Experiment I. Higher concentrations of saccharin (100 and 500 pg/ml) appeared to inhibit slightly the transformation induced by the initiating dose of MCA. In Experiment I, the addition of sodium ascorbate or L-tryptophan at 500 pg/ml (2.53 mM and 2.45 mM, respectively) almost completely restricted the appearance of either spontaneous Type III foci or those induced by MCA (0.1 pg/ml). These concentrations exhibited no general toxicity to the cells over the course of the exposure period. The results with L-tryptophan are of interest since Cohen et al. [4] found that the DL-form of this amino acid incorporated at 2 and 5% into the diet of rats previously treated with FANFT resulted in an enhanced occurrence of bladder tumors. Based on this finding it was concluded that tryptophan may act as a tumor promoter for bladder carcinogenesis, possibly as a result of abnormal levels of tryptophan metabolites. It is not known whether the BALB/c-3T3 fibroblastic cells have the necessary enzyme complement to generate a significant quantity of tryptophan metabolites, and the reasons for the reduction in the occurrence of neoplastic transformation by this amino acid and sodium ascorbate remain unknown. In addition to these 2 naturally occurring dietary substances which inhibited transformation, calcium pantothenate (1.5 mM) was tested in the promoting protocol and was found to enhance both spontaneous and induced
UNINITIATED
MCA
INITIATED
Z@ml)
I
EXPT. 1
r
EXPT. 2
-
-
Fig. 1. Response of BALB/c-3T3 cells to promotion by saccharin, phorbol myristate acetate and calcium pantothenate. Sets of plates, 20 per group, were plated with 10,000 BALB/c-3T3 cells from trypsinized log phase parent cultures. One day later, sets were treated with 3-methylcholanthrene (MCA) (0.1 pg/ml) or left untreated. One set was treated with MCA at 2.0 pg/ml. Seventy-two hours later, the plates were all renewed with fresh medium containing no carcinogen. After an additional 72-h, the medium containing agents to be tested for promoting activity wasadded to both control and MCA (0.1 pg/ml) treated dishes with the exception that one control and MCA (0.1 pg) set and the 2 pg/ml MCA set received control medium. The plates were refed twice weekly and stained with Giemsa on the twenty-eighth day after removal of the carcinogen. Type III foci were identified by scanning plates under a dissecting microscope. The numbers of plates scored for each set were 9-18 for Experiment I and 14-22 for Experiment II.
transformation in Experiment I. In the repeat experiment, the effect observed was marginal. The reasons for this discrepancy are not presently apparent, although calcium ion metabolism has a marked influence on the transformed phenotype-in cell culture systems [ 21.
31 DISCUSSION
The absence of a promoting effect with saccharin when tested over a wide dose range in the BALB/c-3T3 neoplastic transformation system, the anomalous behavior of saccharin at a single dose in the promoting assay in the C3H-IOT,,, cell system (enhancement of transformation with MCA initiation; no effect with ultraviolet light initiation) [ 121 and the requirement for heroic doses of saccharin to induce tumors in vivo in rats strongly suggest that the tumor-enhancing effects of saccharin in experimental animals do not occur as a result of specific cellular responses of the type associated with the classical mouse skin phorbol ester tumor promotion model. Rather, the promoting-like effect of saccharin in the NMU and FANFT experiments would appear to be due to the response of the bladder epithelium to the continual extraordinary hyperosmolal and hyperacidic environment following an initial tissue insult from exposure to a strongly cytotoxic carcinogen. Indeed, the scanning electron microscope images reported by Cohen et al. [ 41, as well as the recently reported data on bladder hyperplasia induced by saccharin [8], lend support to this view. In another experimental tumor promotion system, Ito et al. have reported that continual feeding of polychlorinated biphenyls (PCB) to mice following exposure to benzene hexachloride as an initiating agent produced a substantial enhancement of hepatic tumors [ 111. This tumor-promoting effect has been ascribed to a general compensatory hypertrophic response of the liver resulting from the continual abnormal physiological effects induced by exposure to PCB. While the results of animal studies with saccharin should not be taken lightly, they must be analyzed with relevance to human exposure levels, especially if tumor promotion is invoked as a possible explanation for the findings. Based on the information now available, the occurrence of the bladder tumors in the rat experiments appears to be the consequence of an abnormal physiological response to a dose of saccharin several orders of magnitude higher than any expected in humans. Since the process of tumor promotion in the one system best understood, i.e. mouse skin, follows clear dose-response relationships, appears to exhibit a threshold in common with other pharmacological effects, and is reversible under some conditions [3,15], it is necessary to perform the appropriate in vivo experimental studies to determine the dose response of the tumor-enhancing effect of saccharin and relate this in a rational manner to the actual human exposure experience. REFERENCES 1 Arnold, D.L., Moodie, C.A., Grice, H.C., Charbonneau, S.M., Stavric, B., Collins, B.T., McGuire, P.F., Zawidzka, Z.Z. and Munro, I.C. (1980) Long-term toxicity of orthotoluenesulfonamide and sodium saccharin in the rat. Toxicol. Appl. Pharmacol., 52, 113-152. 2 Boynton, A.L. and Whitfield, J.F. (1976) Different calcium requirements for proli-
32
3 4
5
6
7
8 9
10
11
12 13 14 15
16
feration of conditionally and unconditionally tumorigenic mouse cells. Rot. NatI. Acad. Sci. U.S.A., 73(5), 1651-1654. Burns, F.J., Vanderlaan, M., Sivak, A. and Albert, R.E. (1976) Regression kinetics of mouse skin papillomas. Cancer Res., 36.1422-1427. Cohen, S.M., Masayuki, A., Jacobs, J.B. and Friedell, G.H. (1979) Promoting effect of saccharin and DL-Tryptophan in urinary bladder carcinogens. Cancer Res., 39, 1207-1217. Committee for a Study on Saccharin and Food Safety Policy. (1978)Saccharin: technical assessment of risks and benefits: report No. 1. Washington: Assembly of Life Sciences, Institute of Medicine, National Research Council, National Academy of Sciences, 3-l 1 to 3-l 7. Committee for a Study on Saccharin and Food Safety Policy. (1978) Saccharin: technical assessment of risks and benefits: report No. 1. Waahington: Assembly of Life Sciences, Institute of Medicine, National Research Council, Nationai Academy of Sciences, 3-17 to 3-35. Committee for a Study on Saccharin and Food Safety Policy. (1978) Saccharin: technical assessment of risks and benefits: report No. 1. Washington: Assembly of Life Sciences, Institute of Medicine, National Research Council, National Academy of Sciences, 3-37 to 3-44. Fukuahima, S. and Cohen, S.M. (1980) Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res., 40, 734-736. Hicks, R.M. and Chowaniec, J. (1977) The importance of synergy between weak carcinogens in the induction of bladder cancer in experimental animals and humans. Cancer Res., 37,2943-2949. Hicks, R.M., Chowaniec, J. and Wakefield, J. (1978) Carcinogenesis, Vol. 2: Mechanisms of Tumor Promotion and Cocarcinogenesis. pp. 475489. Editors: T.J. Slaga, A. Sivak and R.K. Boutwell, Raven Ress, New York. Ito, N., Nagasaki, H., Arai, M., Sugihsra, S. and Makiura, S. (1973) Histologic and ultrastructural studies on the hepatocarcinogenicity of benzene hexachloride in mice. J. Nat]. Cancer Inst., 51, 817-825. Mondal, S., Brankow, D.W. and Heidelberger, C. (1978) Enhancement of oncogenesis in CSH-lOT,,, mouse embryo cell culture by saccharin. Science, 201,1141-1142. Morrison, A.S. and Buring, J.E. (1980) Artificial sweeteners and cancer of the lower urinary tract. N. Engl. J. Med., 302(10), 537-541. Sweatman, T.W. and Perwick, A.G. (1979) Saccharin metabolism and tumorigenicity. Science, 205, 1019-1020. Van Duuren, B.L., Sivak, A., Segal, A., Seidman, I. and Katz, C. (1973) Dose response studies with a pure tumor-promoting agent, phorbol my&ate acetate. Cancer Res., 33,2166-2172. Wynder, E.L. and Stellman, S.D. (1980) Artificial sweetener use and bladder cancer: a case control study. Science 207 (4436) 1214-1216.
27-32 Scientific
27 Publishers
Ltd.
CELL CULTURE TUMOR PROMOTION EXPERIMENTS WITH SACCHARIN, PHORBOL MYRISTATE ACETATE AND SEVERAL COMMON FOOD MATERIALS*
ANDREW
SIVAK**
and ALICE S. TU
Bio/Medical Sciences Section, Arthur D. Little, Inc., Cambridge, MA 02140 (U.S.A.) (Received (Accepted
28 March 1980) 3 April 1980)
SUMMARY
The BALB/c-3T3 cell neoplastic transformation system was modified to examine the tumor promoting activity of a set of substances. Following initiation of the target cells with 3-methylcholanthrene, treatment of the cultures with phorbol myristate acetate (0.01 pg/ml; 1.5 X lO_” M) during the remainder of the 4-week assay interval resulted in a marked increase in both spontaneous and initiated Type III transformed foci. In contrast, a similar treatment with saccharin at 20, 100 or 500 pg/ml (0.08, 0.4 or 2.1 X lo-’ M)’did not influence the occurrence of Type III transformed foci and did not result in a promoting response. Sodium ascorbate (2.53 X 10s3 M) and L-tryptophan (2.45 X lo-’ M) almost completely inhibited both spontaneous and initiated Type III transformed foci. Calcium pantothenate (2.10 X 10e3 M) exhibited a marginal promoting effect. Under the conditions of this study in which the classical tumor promoter phorbol myristate acetate was highly active in promoting Type III transformed foci, saccharin was not active as either a direct transforming or promoting agent at doses up to 5 orders of magnitude higher.
INTRODUCTION
The finding of a low, but statistically significant, increase in bladder tumors in male rats of the second generation fed high doses of saccharin (5% of the diet) [l] has resulted in regulatory concern and wide public *This work, conducted Control Council.
at Arthur D. Little, Inc., was funded by members of the Calorie
**Address all correspondence
to: Andrew Sivak, Bio/Medical Little, Inc., Acorn Park, Cambridge, MA 02140, U.S.A.
Sciences Section,
Arthur D.
debate over its safety in humans. The available epidemiological data [ 6,13,16] do not provide any substantial evidence for a direct linkage between saccharin consumption and a significant increase in bladder cancer in humans. The absence of a carcinogenic effect in a number of other feeding studies in rats [ 61, the observations that there is no evidence for metabolic conversion of saccharin in rats or humans [ 4,141 and the equivocal results obtained in several rapid mutagenesis assays [ 71 do not provide a strong expierimental basis to ascertain whether normal levels of saccharin consumption pose a health risk. Indeed, the low frequency of tumors found in the 2-generation study and the knowledge that the urinary tracts of the test animals were under extreme physiological stress at the high doses of saccharin employed in this study suggest that the induction of tumors was the result of secondary, modifying events rather than a direct carcinogenic stimulus. Two published in vivo studies lend support to this view. Hicks [ 91 found that rats given single transurethral bladder doses of N-methyl-N-nitrosourea (MNU) at 1.5 mg, and then fed saccharin in the diet for up to 2 years at 2.0 or 4.0 g/kg/day, bore significantly more bladder tumors than rats given MNU alone. Because of the unstable chemical nature of the initiating agent and the later report of uncertainty with respect to the actual concentrations used in the various test groups [lo], the results of this study should be viewed with caution. In contrast, Cohen et al. [ 41 appear to have demonstrated rather clearly in an experiment using FANFT, a bladder carcinogen for rats when administered orally, that saccharin fed in the diet alter a dose regimen of FANFT that is weakly carcinogenic, induces a significant increase in both the incidence and severity of tumors of the bladder. These studies indicate that saccharin may be acting in a tumor-promoting manner, possibly as a result of unusually severe physiological pressures occurring in the bladder rather than in the usual sense of the tumor promoting phenomenon as a specific cellular response. In a report that appears to support the vie& that saccharin may be acting as a tumor promotor, Mondal and Heidelberger [ 121 reported that saccharin induced a promoting effect at 100 pg/ml in the medium of C3H-lOT,,, cells in culture following an initiating treatment with 3-methylcholanthrene. This dose of saccharin was the only dose tested. However, no promoting effect with saccharin was observed following initiation with ultraviolet light exposure even though the phorbol esters, the only class of chemicals reported to have a promoting type of activity in this particular cell system, were quite active after either 3-methylcholanthrene or ultraviolet light initiating stimuli. In order to obtain additional information bearing on the issue of the potential for tumor-promoting activity of saccharin, we performed several experiments with the BALB/c-3T3 cell neoplastic transformation system, which, like the C3H-lOT,,, system, also exhibits a promoting type of response with phorbol esters. Cultures of the l-13 line of BALB/c-3T3 cells were initiated with 3-methylcholanthrene (0.1 pg/ml. 72 h) using the protocol
29
for carcinogen exposure usually employed for transformation assays. On the seventh day of the culture, and with each biweekly medium renewal thereafter, the agents to be tested for promoting activity were added to the medium. The chemicals tested were sodium saccharin, sodium ascorbate. L-tryptophan, and calcium pantothenate. Four weeks after the termination of MCA treatment the experiments were stopped by fixing the cells with methanol after medium removal and rinsing with phosphate-buffered saline. The plates were then stained with 10% Giemsa, and Type III foci were identified by examination of each plate using a dissecting microscope (4-40X ). RESULTS
The results of 2 separate experiments are shown in Fig. 1. Several conclusions are apparent from these data. First, the induction of neoplastic transformation by MCA alone and its enhancement by phorbol myristate acetate, the potent tumor-promoting agent for mouse skin, was clearly shown in both studies. Second, saccharin over a wide cencentration range (0.08-2.1 mM), which did not exhibit toxicity with respect to reduction of cell number or altered cellular morphology, did not by itself induce neoplastic transformation. Further, saccharin did not exhibit a promoting effect by markedly enhancing either the spontaneous transformation or the low level of transformation induced by the initiating treatment with 0.1 pg/ml of MCA. The increase in transformation induced with 20 pg/ml saccharin in MCA-initiated plates of Experiment I was not reproduced in Experiment II, nor was the spontaneous transformation enhanced in Experiment I. Higher concentrations of saccharin (100 and 500 pg/ml) appeared to inhibit slightly the transformation induced by the initiating dose of MCA. In Experiment I, the addition of sodium ascorbate or L-tryptophan at 500 pg/ml (2.53 mM and 2.45 mM, respectively) almost completely restricted the appearance of either spontaneous Type III foci or those induced by MCA (0.1 pg/ml). These concentrations exhibited no general toxicity to the cells over the course of the exposure period. The results with L-tryptophan are of interest since Cohen et al. [4] found that the DL-form of this amino acid incorporated at 2 and 5% into the diet of rats previously treated with FANFT resulted in an enhanced occurrence of bladder tumors. Based on this finding it was concluded that tryptophan may act as a tumor promoter for bladder carcinogenesis, possibly as a result of abnormal levels of tryptophan metabolites. It is not known whether the BALB/c-3T3 fibroblastic cells have the necessary enzyme complement to generate a significant quantity of tryptophan metabolites, and the reasons for the reduction in the occurrence of neoplastic transformation by this amino acid and sodium ascorbate remain unknown. In addition to these 2 naturally occurring dietary substances which inhibited transformation, calcium pantothenate (1.5 mM) was tested in the promoting protocol and was found to enhance both spontaneous and induced
UNINITIATED
MCA
INITIATED
Z@ml)
I
EXPT. 1
r
EXPT. 2
-
-
Fig. 1. Response of BALB/c-3T3 cells to promotion by saccharin, phorbol myristate acetate and calcium pantothenate. Sets of plates, 20 per group, were plated with 10,000 BALB/c-3T3 cells from trypsinized log phase parent cultures. One day later, sets were treated with 3-methylcholanthrene (MCA) (0.1 pg/ml) or left untreated. One set was treated with MCA at 2.0 pg/ml. Seventy-two hours later, the plates were all renewed with fresh medium containing no carcinogen. After an additional 72-h, the medium containing agents to be tested for promoting activity wasadded to both control and MCA (0.1 pg/ml) treated dishes with the exception that one control and MCA (0.1 pg) set and the 2 pg/ml MCA set received control medium. The plates were refed twice weekly and stained with Giemsa on the twenty-eighth day after removal of the carcinogen. Type III foci were identified by scanning plates under a dissecting microscope. The numbers of plates scored for each set were 9-18 for Experiment I and 14-22 for Experiment II.
transformation in Experiment I. In the repeat experiment, the effect observed was marginal. The reasons for this discrepancy are not presently apparent, although calcium ion metabolism has a marked influence on the transformed phenotype-in cell culture systems [ 21.
31 DISCUSSION
The absence of a promoting effect with saccharin when tested over a wide dose range in the BALB/c-3T3 neoplastic transformation system, the anomalous behavior of saccharin at a single dose in the promoting assay in the C3H-IOT,,, cell system (enhancement of transformation with MCA initiation; no effect with ultraviolet light initiation) [ 121 and the requirement for heroic doses of saccharin to induce tumors in vivo in rats strongly suggest that the tumor-enhancing effects of saccharin in experimental animals do not occur as a result of specific cellular responses of the type associated with the classical mouse skin phorbol ester tumor promotion model. Rather, the promoting-like effect of saccharin in the NMU and FANFT experiments would appear to be due to the response of the bladder epithelium to the continual extraordinary hyperosmolal and hyperacidic environment following an initial tissue insult from exposure to a strongly cytotoxic carcinogen. Indeed, the scanning electron microscope images reported by Cohen et al. [ 41, as well as the recently reported data on bladder hyperplasia induced by saccharin [8], lend support to this view. In another experimental tumor promotion system, Ito et al. have reported that continual feeding of polychlorinated biphenyls (PCB) to mice following exposure to benzene hexachloride as an initiating agent produced a substantial enhancement of hepatic tumors [ 111. This tumor-promoting effect has been ascribed to a general compensatory hypertrophic response of the liver resulting from the continual abnormal physiological effects induced by exposure to PCB. While the results of animal studies with saccharin should not be taken lightly, they must be analyzed with relevance to human exposure levels, especially if tumor promotion is invoked as a possible explanation for the findings. Based on the information now available, the occurrence of the bladder tumors in the rat experiments appears to be the consequence of an abnormal physiological response to a dose of saccharin several orders of magnitude higher than any expected in humans. Since the process of tumor promotion in the one system best understood, i.e. mouse skin, follows clear dose-response relationships, appears to exhibit a threshold in common with other pharmacological effects, and is reversible under some conditions [3,15], it is necessary to perform the appropriate in vivo experimental studies to determine the dose response of the tumor-enhancing effect of saccharin and relate this in a rational manner to the actual human exposure experience. REFERENCES 1 Arnold, D.L., Moodie, C.A., Grice, H.C., Charbonneau, S.M., Stavric, B., Collins, B.T., McGuire, P.F., Zawidzka, Z.Z. and Munro, I.C. (1980) Long-term toxicity of orthotoluenesulfonamide and sodium saccharin in the rat. Toxicol. Appl. Pharmacol., 52, 113-152. 2 Boynton, A.L. and Whitfield, J.F. (1976) Different calcium requirements for proli-
32
3 4
5
6
7
8 9
10
11
12 13 14 15
16
feration of conditionally and unconditionally tumorigenic mouse cells. Rot. NatI. Acad. Sci. U.S.A., 73(5), 1651-1654. Burns, F.J., Vanderlaan, M., Sivak, A. and Albert, R.E. (1976) Regression kinetics of mouse skin papillomas. Cancer Res., 36.1422-1427. Cohen, S.M., Masayuki, A., Jacobs, J.B. and Friedell, G.H. (1979) Promoting effect of saccharin and DL-Tryptophan in urinary bladder carcinogens. Cancer Res., 39, 1207-1217. Committee for a Study on Saccharin and Food Safety Policy. (1978)Saccharin: technical assessment of risks and benefits: report No. 1. Washington: Assembly of Life Sciences, Institute of Medicine, National Research Council, National Academy of Sciences, 3-l 1 to 3-l 7. Committee for a Study on Saccharin and Food Safety Policy. (1978) Saccharin: technical assessment of risks and benefits: report No. 1. Waahington: Assembly of Life Sciences, Institute of Medicine, National Research Council, Nationai Academy of Sciences, 3-17 to 3-35. Committee for a Study on Saccharin and Food Safety Policy. (1978) Saccharin: technical assessment of risks and benefits: report No. 1. Washington: Assembly of Life Sciences, Institute of Medicine, National Research Council, National Academy of Sciences, 3-37 to 3-44. Fukuahima, S. and Cohen, S.M. (1980) Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res., 40, 734-736. Hicks, R.M. and Chowaniec, J. (1977) The importance of synergy between weak carcinogens in the induction of bladder cancer in experimental animals and humans. Cancer Res., 37,2943-2949. Hicks, R.M., Chowaniec, J. and Wakefield, J. (1978) Carcinogenesis, Vol. 2: Mechanisms of Tumor Promotion and Cocarcinogenesis. pp. 475489. Editors: T.J. Slaga, A. Sivak and R.K. Boutwell, Raven Ress, New York. Ito, N., Nagasaki, H., Arai, M., Sugihsra, S. and Makiura, S. (1973) Histologic and ultrastructural studies on the hepatocarcinogenicity of benzene hexachloride in mice. J. Nat]. Cancer Inst., 51, 817-825. Mondal, S., Brankow, D.W. and Heidelberger, C. (1978) Enhancement of oncogenesis in CSH-lOT,,, mouse embryo cell culture by saccharin. Science, 201,1141-1142. Morrison, A.S. and Buring, J.E. (1980) Artificial sweeteners and cancer of the lower urinary tract. N. Engl. J. Med., 302(10), 537-541. Sweatman, T.W. and Perwick, A.G. (1979) Saccharin metabolism and tumorigenicity. Science, 205, 1019-1020. Van Duuren, B.L., Sivak, A., Segal, A., Seidman, I. and Katz, C. (1973) Dose response studies with a pure tumor-promoting agent, phorbol my&ate acetate. Cancer Res., 33,2166-2172. Wynder, E.L. and Stellman, S.D. (1980) Artificial sweetener use and bladder cancer: a case control study. Science 207 (4436) 1214-1216.