Tumorigenicity Study of Sodium Erythorbate Administered Orally to Mice

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1 Hiroshima J. Med. Sci. Vol.38, No.3, , September, 1989 HJM Tumorigenicity Study of Sodium Erythorbate Administered Orally to Mice Kouki NA, Hiroshi AKAMZU, Ryozo ETO, Toshihiro NSHDA, Kazuhiko OHE, Toshihiro KOBUKE, Shigeru NAMBU, Kei MATSUK and Shoji TOKUOKA Department of Pathology, Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima 734, Japan ABSTRACT Sodium erythorbate (SE) was administered at concentrations of 0, 1.25, or 2.5% (maximum tolerated dose, MTD) in the drinking-water to groups of 50 male B6C3F 1 mice respectively. Female groups, each consisting of 50 mice, received SE in the drinking-water at concentrations of 0, 2.5 or 5% (MTD). Treatment continued for 96 wks and the experiment was terminated during wk 110. Tumors were observed at various sites including the liver, hematopoietic system, lung and soft tissue. However, at any of the sites, the tumor incidence, the time to death with tumors or the histological distribution of tumors did not differ significantly from those in the untreated control group. Thus, the present study did not demonstrate a tumorigenic effect of SE on B6C3F 1 mice by means of oral administration. Key words: Tumorigenicity, Sodium elythorbate, Mouse Because of its antioxidation effect, sodium erythorbate (SE) has been used as an additive in foods such as meats and fishes over the last 15 years or more. Recently, SE was found to be a possibly weak mutagen in an Ames' mutagenicity test and a test for chromosomal aberrations 11 l. t is important, therefore, that tests are carried out to determine whether or not SE is a potential carcinogen. The present experiments were carried out to investigate tumorigenicity of orally administered SE in mice. MATERALS AND METHODS Subacute toxicity test: Seventy male and 70 female 8-wk-old B6C3F 1 mice were purchased from Charles River Japan nc. (Atsugi) and divided into six groups. Five groups, each consisting of 10 males and 10 females, were given SE (Organo Co., Ltd.. Toda) ad lib., for 10 consecutive wks, at a level of 0.625, 1.25, 2.5, 5 or 10% in distilled water. The SE used was the grade-specified according to the Japanese Standards of Food Additives. Fresh solution was prepared once every 3 days, and the mice had access ad lib. to a basal diet (CRF-1, Charles River Japan nc.). A control group consisting of male and female mice was given basal diet and distilled water ad lib. All of the mice were housed ten to a plastic cage in an air-conditioned room. At the end of the 10-wk treatment period all surviving mice were killed by ether and autopsied. Chronic toxicity test: B6C3F 1 mice (150 males and 150 females, 8-wk-old) were obtained from Charles River Japan nc. and divided into three groups. The mice in two of three groups were given ad lib. for 96 wks SE in their drinking-water at dose levels that were chosen on the basis of the results of the subacute toxicity test. Doses of 1.25 or 2.5% SE were administered to groups of 50 male mice, respectively. Groups of 50 female mice were given 2.5 or 5% SE. The highest doses given were those that had been determined to be the maximum tolerated dose (MTD) in the subacute toxicity study. Fresh SE solution was prepared every 3 days. All of the mice were given access to the basal diet (CRF-1) ad lib., and a control group consisting of 50 male and 50 female mice was given the basal diet and distilled water ad lib. The mice were housed, ten to a plastic cage, in an air-conditioned room. After the 96-wk treatment period all of the surviving mice were given basal diet and distilled water for a further 14 wks. The amount of drinking-water consumed per cage over 3 consecutive days was measured weekly for the first 10 wks, once every 2 wks for the next 10 wks, and then once every 4 wks until the end of the experiment. Each mouse was weighed once a week for the first 10 wks, once every 2 wks for the next 10 wks, and once every 4 wks until the end of the experiment. Any mouse that was found dead or moribund during the experiment was autopsied. The mice that survived to the end of the 110-wk experimental period (12 males and 32 females) were killed by

2 136 K. nai et al ether and autopsied. At autopsy all visceral organs and any tumors were weighed and examined grossly and microscopically. The tumor incidence and the time to death with tumors in the treated groups were compared with those in the untreated control group by either chi-square or t-test. n cases of either hepatocellular tumor or subcutaneous sarcoma, the time adjusted analysis on tumor incidence was performed according to the method described by Peto et al1 3 l. RESULTS Subacute toxicity test Six male and one female mice in the group that were given the highest dose of SE, 10%, died by the end of the first wk of treatment. The average rate of weekly body-weight gain of male mice given 5% SE was less than 90% that of the control. While female mice given 5% SE showed larger amounts of body-weight gain than that of the control through the experiment period. On the basis of these results, the maximum tolerated dose (MTD) of SE in drinking-water was estimated to be 2.5% for male mice and 5% for female mice. Histological examination of all the major visceral organs in mice that had been given SE concentrations higher than the MTD showed marked atrophy of liver cells, marked atrophy of splenic lymphoid follicles and hydropic degeneration of renal tubular epithelium. No significant changes were seen in the visceral organs of control mice or mice that were given the MTD or lower concentration of SE. Chronic toxicity test The average daily intakes of drinking-water and SE per mouse, which was calculated from all of amounts measured, are shown in Table 1. There was no significant difference between groups in the amount of water intake. Consequently, the total SE intake by male mice in the group given a higher dose of SE was about 1.5 times greater than that of male mice of the lower dose groups. For female mice, SE intake by the higher dose group was about 1.8 times greater than that of the lower dose group. The average body-weights of treated mice remained fairly stable throughout the treatment period and were generally similar to those of the controls (Fig. 1). The survival percentages are shown in Fig. 2. Nine male and seven female mice were excluded when determining the percent survivals because of accidental death during the early experimental period (within wk 21). The mice that were regarded to be 'effective' for data analysis were those that survived beyond wk 43, when the first animal in the study died with a tumor. Nine male and seven female mice were excluded from the 'effective' numbers because au- Table 1. ntake of water and sodium erythorbate by mice Sex Group Water intake SE intake % ml/day mg/day (g/kg, body wt/day) Male (3.3) (4.9) Female (4.0) (7.0) SE = (g) 50 (a) Sodium erythorbate ~ 7- -, -,, - ~..,,.,.,;:; "...,_. r- ~! \ 30 ' 10 (g) 50 ( b) 30 y' " ""'"; Fig. 1. Average body-weight of (a) groups of 50 male B6C3F 1 mice given 0(--), 1.25( oe ) or 2.5( - - )% sodium erythorbate, and (b) groups of 50 female B6C3F 1 mice given 0(--), 2.5( 80 ) or 5(- - -)% sodium erythorbate. tolysis prevented a precise histological examination. Consequently, as shown in Table 2, of the SEtreated mice 81 % (81/100) of the starting male population and 90% (90/100) of the female population were 'effective'. The percentages of 'effective' mice were very similar to the controls: 76% (38/50) of the male and 90% (45/50) of the female control populations were 'effective'. The tumor incidence and the time to death with tumors are shown in Table 2. The tumor incidence in the male groups appeared to increase with increasing SE dose, although no statistical difference was noted. However, as shown in Table 2, a con-

3 Tumorigencity of Sodium Erythorbate 137 (%)(a) loot =;--~ (%) (b)... ;~.~-L 1 :.....! '--,... : 1_, L..L..;-c-1 1_, t!!!"'!::;"""-'.''"'"""'"""''''"""'"'"'"'"' i...,f,,..,:.,..!l_l_ i...,. L---,.. """C.1-1-, '""1..,... l-1 -.., 1 :..... ~... c.~ llo : L_L_ - : llo Fig. 2. Percent survival of (a) groups of 50 male B6C3F 1 mice given 0(--), 1.25(... ) or 2.5( - -)% sodium erythorbate, and (b) groups of 50 female B6C3F 1 mice given 0(--), 2.5( oeu ) or 5(- - -)% sodium erythorbate. siderable difference of the mean time to death in 'effective' mice between the two groups necessitated further time adjusted analysis of the tumor incidence. n the female groups, the tumor incidences of the treated groups were higher than the control group, although no statistical difference was noted. As shown in Table 3, the tumors observed in the male mice were hepatocellular tumors, subcutaneous sarcoma, adenoma and carcinoma of the lung, and lymphoma/leukemia. However, it was apparent that the difference of tumor incidence was shown at hepatocellular tumor and subcutaneous sarcoma. Therefore, the time adjusted analysis of tumor incidence was performed on these two tumors. All of the hepatocellular tumors including carcinoma and adenoma were observed in the incidental context. Z-value was calculated 0.76% from T-value (2.306) and variance (9.2). On the other hand, all of the subcutaneous sarcoma were observed in the fatal context. Z-value was calculated 0.56 from T value (1.4) and variance (4.608). Consequently, it was apparent that each of these two tumors had no positive trend of dose-related tumorigenicity. n the female mice, the tumor with the highest incidence was lymphoma/leukemia, as shown in Table 3. However, there was no difference in incidence between the treated groups and the control group. The average weights of major visceral organs in effective mice without any tumor rate are shown in Table 4. For male mice, the weights of heart and brain seemed to show dose-dependent reduction. For female mice, the weights of heart, lungs, kidney and brain were significantly different between the higher dose group and the control group. Histological examination showed no significant difference between the treated groups and the control group. DSCUSSON Abe et al2l conducted a study in which 2.5% and 1.25% SE drinking-water was administered to F344 rats for 104 wks in order to determine the chronic toxicity of SE and in particular the carcinogenicity of SE. n this study carcinogenicity was not demonstrated. Fitzhugh and Nelson 3 l reported on the chronic toxicity of sodium-free erythorbic acid from SE, and carcinogenicity was not observed. However, these are all studies using rats and no reports have been made on findings obtained from mice. n the present experiment, the tumor incidence appeared to be significantly higher in the male 2.5% dose-group when compared to that of the male control. n this regard, the results of the time adjusted analysis on tumor incidence, described by Peto et al1 3 l, confirmed that neither the incidence of hepatocellular tumor nor subcutaneous sarcoma was affected by the given SE doses. Table 2. Number of effective mice and mice with tumor Sex Group No. of Mean time to death in % effective mice" effective miceb week Male ± ± ± 2oc1 Female ± ± ± 12d a: Effective mice are those that survived beyond the 43 wk. b: Mean ± standard deviation c: Significantly different from 0% group at p<0.05 by chi-square test d: Significantly different from 0% group at p<0.01 by t-test No. of mice with tumor (%) 9 (24) 14 (37) 19 (44) 10 (22) 16 (36) 15 (33) Mean time to death in mice with tumorb week 84 ± ± ± ± ± 6d 101 ± 13

4 138 K. nai et al Table 3. ncidence of mice with histological types of tumors Sites Histological types of tumors No. and incidences (%) of mice with tumors in: Males at SE doses of: Females at SE doses of: 0% 1.25% 2.5% 0% 2.5% 5% Liver Hepatocellular carcinoma 2 (5) 4 (11) 4 (9) 1 (2) 2 (4) Hepatocellular adenoma 1 (3) 4 (11) 5 (12) 1 (2) 1 (2) Hemangioma 3 (8) 2 (5) 1 (2) Hematopoietic system Lymphoma/leukemia 2 (5) 1 (3) 4 (9) 5 (11) 7 (16) 5 (11) Lung Alveolar/bronchiolar carcinoma 1 (3) 1 (2) 1 (2) 1 (2) Alveolar/bronchiolar adenoma 4 (11) 4 (9) 1 (2) ntegumentary system Fibrosarcoma 2 (5) Leiomyosarcoma 1 (3) 1 (2) N eurofibrosarcoma 1 (2) Malignant fibrous histiocytoma 1 (3) 1 (3) 2 (5) Osteogenic sarcoma 1 (2) 1 (2) 1 (2) N eurofibroma 1 (2) Lipoma 1 (3) Mammary gland Carcinoma 1 (2) 1 (2) Fibroadenoma 1 (2) Ovary Cystadenoma 1 (2) 1 (2) 1 (2) Uterus Adenocarcinoma 2 (4) Strama! sarcoma 1 (2) Leiomyoma 1 (2) Stomach Squamous cell carcinoma 1 (2) Heart Hemangioma 1 (3) 1 (2) Adrenal gland Pheochromocytoma 1 (3) 1 (2) Spleen Hemangioma 1 (3) SE = Sodium erythorbate Table 4. Final body weight and relative organ weight (% body weight) of mice Sex Group No. of Final body Relative organ weight (% body weight)a % mice weight" g Heart Lungs Liver Kidney Spleen Brain Male ± ± ± ± ± ± ± ± ± ± ± ± ±0.17 l.78±0.39d ±6.3b 0.76±0.21a 1.47± ± ± ±0.64 l.56±0.32b Female ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.lSb ± ±0.24d 2.32±1.0lb 6.41 ±2.14 l.04±0.23c 0.74± ±0.34b a: Mean± standard deviation b: Significantly different from 0% group at p<0.001 by t-test c: Significantly different from 0% group at p<0.01 by t-test d: Significantly different from 0% group at p < 0.05 by t-test Also, the historical control should be reviewed to evaluate tumor incidences in the tumorigenicity study. The incidence of spontaneous hepatocellular tumor in B6C3F 1 mice has been reported to be 21.6% 14 ) and 31.1 % 6 ) and also in our experience with untreated B6C3F 1 mice the incidence of hepatocellular tumor was 24% 12 ) or 38% 10 l. The incidence observed in the mice given SE in the present experiment concurred with previous observations. On the contrary, the incidence observed in the untreated control mice was rather lower than those previously reported incidences. This appears to be due to the short survival time in these control mice. With respect to lymphoma/leukemia, the most common tumor in the females, the incidence of the spontaneous tumor reported heretofore such as 16.8% 14 ) and 27.2% 6 ) are higher than those observed in the treated and untreated control female mice in the present experiment. n our experience the incidence of lymphoma/leukemia in the untreated female mice was 16% 12 ) or 46% 10 l. Since the survival times of mice are not different between the present and the previous experiment, the reason why the incidence of lymphoma/leukemia was low is unclear. t has been pointed out that mutagenicity is a good index of carcinogenicity 11 l, but there is not necessarily good agreement between the two. We have conducted long-term administration experi-

5 Tumorigencity of Sodium Erythorbate 139 ments on mice with a number of substances known to have a mutagenicity 8 9 l, but in most of the cases findings suggestive of carcinogenicity were not detected. On the other hand, the structural formula of SE resembles that of ascorbic acid and SE also possesses the same action of preventing oxidation as ascorbic acid. t has been reported from the past that ascorbic acid is effective in inhibiting mutagenicity and carcinogenicity 5 l. t is interesting to determine whether SE also has such actions. Abe et apl in studying the effect of SE administration during the process of gastric cancer development in rats with the use of N-methyl-N'-nitrosoguanidine (MNNG) have reported observing neither promoting action nor inhibiting action, but Fukushima et al 4 l have reported that SE administration during the process of bladder cancer development with N butyl-n-(4-hydroxybutyl)nitrosamine (BEN) has a promoting action as in the case of ascorbic acid. t has been pointed out that there is an organ specificity in the action of cancer promotion 7 l. Further study must be made on the co-carcinogenic action of SE. n the present study, a better survival of the mice given SE, both male and female, was observed. The final body weights in the treated group, both male and female, were larger than those of the control group, however the relative weights of major visceral organs were smaller than those of the control group. Accordingly, it seems that there is no evidence of a direct effect of SE towards better survival, and it should be considered that survival in the control mice was too short. ACKNOWLEDGEMENT This work was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare of Japan. (Received May 15, 1989) (Accepted July 13, 1989) REFERENCES 1. Abe,., Saito, S., Hori, K., Suzuki, M. and Sato, H Effects of erythorbate on N-methyl-N' nitrosoguanidine-induced stomach carcinogenesis in F344 rats. Sci.Rep.Res.nst.Tohoku Univ. 30: Abe,., Saito, S., Hori, K., Suzuki, M. and Sato, H Sodium erythorbate is not carcinogenic in F344 rats. Exp.Molec.Pathol. 41: Fitzhugh, 0.G. and Nelson, A.A Subacute and chronic toxicities of ascorbyl palmitates. Proc.Soc.Exp.Biol.Med. 61: , 4. Fukushima, S., maida, K., Kurata, Y., Shibata, M. and Mori, S Promoting activities of sodium erythorbate and ethoxyquin but not of ascorbic acid in 2-stage bladder carcinogenesis. Proc.Jpn.Cancer Assoc. 42nd Annual Meet., P Guttenplan, J.B Mechanisms of inhibition by ascorbate of microbial mutagenesis induced by N nitrosocompounds. Cancer Res. 38: Haseman, J.K., Huff, J. and Borrman, G.A Use of historical control data in carcinogenicity studies in rodents. Toxicol.Pathol. 12: maida, K., Fukushima, S., Shirai, T., Masui, T., Ogiso, T. and to, N Promoting activities of butylated hydroxyanisole, butylated hydroxytoluene and sodium L-ascorbate on forestomach, and urinary bladder carcinogenesis initiated with methylnitrosourea in F344 rats. Gann 75: nai, K., Aoki, Y. and Tokuoka, S Chronic toxicity of sodium nitrite in mice, with reference to its tumorigenicity. Gann 70: nai, K., Aoki, Y., Akamizu, H., Eto, R., Nishida, T. and Tokuoka, S Tumorigenicity study of butyl and isobutyl p-hydroxybenzoates administered orally to mice. Fd.Chem.Toxic. 23: nai, K., Kobuke, T., Nambu, S., Takemoto, T., Kou, E, Nishina, H., Fujihara, M., Yonehara, S., Suehiro, S., Tsuya, T., Horiuchi, K. and Tokuoka, S Hepatocellular tumorigenicity of butylated hydroxytoluene administered orally to B6C3F 1 mice. Jpn.J.Cancer Res.(Gann) 79: Kawachi, T Development of technics based on mutagenicity for screening carcinogens. n "Annual Report of the Cancer Research''. The Ministry of Health and Welfare, Tokyo, Japan. 12. Kobuke, T., nai, K., Nambu, S., Ohe, K., Takemoto, T., Matsuki, K., Nishina, H., Huang, l.b. and Tokuoka, S Tumorigenicity study of disodium glycyrrhizinate administered orally to mice. Fd.Chem.Toxic. 23: Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S., Peto, J., Richards, S. and Wahrendorf, J Guidelines for simple, sensitive significance tests for carcinogenic effects in longterm animal experiments. n: Long-term and shortterm screening assays for carcinogens: A critical appraisal. ARC Monogr.; suppl. 2: , 14. Ward, J.M., Goodman, D.G., Squire, R.A., Chu, K.C. and Linhart, M.S Neoplastic and nonneoplastic lesions in aging (C57BL/6NxC3H/HeN)F 1 (B6C3F 1 ) mice. J.Natl.Cancer nst. 63: