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Carcinogenicity of methylmercury chloride in ICR mice: Preliminary note on renal carcinogenesis
Cancer Letters, 12 (1981) 305-310 o Elsevier/North-Holland Scientific Publishers Ltd.
305
CARCINOGENICITY OF METHYLMERCURY CHLORIDE PRELIMINARY NOTE ON RENAL CARCINOGENESIS
KUNITOSHI MITSUMORI*, YASUHIKO SHIRASU Institute
of Environmental
IN ICR MICE:
KEIZOH MAITA, TORU SAITO, SHUJI TSUDA and Toxicology,
Suzuki-cho
2-772,
Kodaira-shi,
Tokyo
187
(Japan) (Received 23 December 1980) (Revised version received 13 February 198 1) (Accepted 16 February 1981)
SUMMARY ICR
mice (60/group) were fed diets containing methylmercury chloride in 0, 15 or 30 ppm for 78 weeks. The majority of mice in the 30-ppm group died due to the neurotoxicity by week 26. The first renal mass was grossly seen in a male of the 15-ppm group at week 58. Histopathology on kidney tissue from all animals surviving after 53 weeks revealed renal tumors in 13 of 16 males of the 15-ppm group in contrast with 1 of 37 males of the control. No renal tumors were seen in the female treated groups and/or control groups. (MMC)
INTRODUCTION
It is well known that organic mercury compounds produce neurological disturbances in man and many experimental animals. Among the investigations to explore other toxic effects of these compounds, mutagenic activities have been suggested in chromosome aberrations [ 7,9], DNA damage [ 31 and dominant lethal [4] studies. However, there is little information available on the carcinogenicity of these compounds, though several long-term toxicity studies have been reported in the rat [ SJO], cat [l] and monkey [ 21. In order to evaluate the chronic toxicity including carcinogenicity of MMC, a long-term feeding study was conducted in mice. After 58 weeks of study, a renal neoplastic mass was grossly observed in 1 male fed a diet containing 15 ppm MMC and the number of cases displaying such a mass increased significantly thereafter. The present study deals with the histological observa-
*To whom all correspondence
should be sent.
306
tions of the kidneys from the mice of all dosage groups which were killed or died after 53 weeks of study. MATERLALS AND METHODS
MMC with 99.3% purity was supplied from Tokyo Kasei Kogyo Co., Ltd., Japan. Male and female SPF ICR mice were obtained from the Shizuoko Agricultural Cooperative Association for Laboratory Animals, Japan, at 4 weeks, and housed 4 to a wire-mesh cage in a barrier system animal room. After a l-week acclimation, both sexes of mice were divided into 3 groups, each consisting of 60 males and 60 females. MMC was blended with a commercial powdered chow MF (Oriental Yeast Ltd., Japan) at 0, 15 or 30 ppm concentrations and fed to the mice for 78 weeks. All animals were examined macroscopically. Kidneys from all dosage groups which died, or became moribund after 53 weeks or were killed at the end of the study were examined histologically (Table 1). Lungs of mice disclosing renal masses were also examined histologically and renal lymph nodes studied when showing any gross abnormalities. These sections were stained with hematoxylin and eosin (H-E). At least 1 longitudinal section from each kidney was routinely examined. RESULTS AND DISCUSSION
Table 1 reveals the fate of mice and incidence of renal masses grossly detectable. Most of the mice in the 30-ppm group of both sexes exhibiting a marked neurotoxic disturbance died or became moribund by week 26. There was also a gradual increase in the number of mice showing neurotoxic signs in the 15-ppm group of either sex with prolongation of the feeding. On necropsy, the number of animals with abnormal kidney, granular and discolored surface indicative of toxic renal impairment, increased in males of the 15-ppm group after 26 weeks. The first renal tumor mass was encountered in a male of this dose group necropsied at week 58. Three of 8 males that died or became moribund during the rest period of the study also disclosed renal masses. Moreover, the final autopsy, held at week 78 revealed 6 cases of renal mass in 8 males. Consequently, a total of 10 of 16 males in the 15-ppm group was grossly identified as renal tumor bearers. All masses were seen unilaterally. No tumor masses were detected on the kidney of females in this dose group and 30-ppm group, as well as animals of both sexes in the control group. Four of the tumor bearers, had a whitish solid, well-demarcated mass less than 2 cm in diameter in the cortex (Fig. 1). The rest (6 mice) had a large multilocular cystic mass measuring 1.8-3.5 cm in diameter. On cut surface, these large masses were composed of white small solid nodules and cysts of various size filled with blackish brown fluid (Fig. 2). The partitions between these cysts were formed by cord-like extensions from those solid nodules.
307 TABLE 1 INCIDENCE OF RENAL NEOPLASTIC MASSES IN MICE FED DIETS CONTAINING MMC: GROSS FINDINGS Dose group (ppm) 15
0
30
Male
Female
Male
Female
Male
Female
oa/lb O/6
O/l O/6
o/7 O/6
O/6 O/6
o/51 O/6
o/59 O/l
o/10 O/6
o/3 O/6
O/25 O/6
o/12 O/6
o/2 o/o
-
o/13 O/24
o/11 o/33
418 618
o/12 O/18
O/l -
-
-
-
-
-
O/SO
O/60
10160
O/60
O/60
O/60
Died and moribund killing by week 26 Interim killing at week 26 Died and moribund killing by week 52 Interim killing at week 62 Died and moribund killing by week 78 Final killing at week 78
Total a No. of mice with renal mass. b No. of mice examined.
As shown in Table 2, histological examinations revealed renal tumors in 13 of 16 males of the 15ppm group. Of these cases, the renal tumors in 10 cases which had a gross mass were classified as adenocarcinoma. In addition, 1 case which became moribund at week 72 disclosed a small foci of adenocarcinoma in the renal cortex. The remaining 2 animals, which became moribund at week 59 or 69, had an adenoma in unilateral kidney. Adenoma also found in the other side of the kidney from 3 animals which had renal mass unilaterally when killed at the end of the study. Most adenocarcinomas observed were solid in appearance with delicate stroma, partly showing trabecular pattern (Fig. 3). There were occasional areas disclosing obvious renal tubular structure. In multicystic tumors, cystic, papillary (Fig. 4) and solid pattern co-existed. These tumors were composed of polyhedral, columnar or cuboidal cells with eosinophilic cytoplasms and marked cellular and nuclear polymorphism. Mitoses were occasionally noted. In some of the tumors with a large diameter, the surrounding parenchyma was compressed and tumor cell infiltration of adjacent tissues could be sometimes found. There were severe hemorrhagic necrotic foci and hemorrhages into the cyst at the portions showing marked papillary proliferation. No metastases to the lung or renal lymph nodes were seen. Besides these malignant tumors, adenomas found in the 15-ppm male group were composed of columnar cells with large eosinophilic cytoplasms and a few atypical nuclei, disclosing tubular arrangement. In the control male group, 1 small solid
308
309 TABLE 2 INCIDENCES AND HISTOLOGICAL TYPES OF RENAL TUMORS IN MICE THAT WERE KILLED OR DIED AFTER 53 WEEKS Sex and dietary level (ppm) Males 0 15 30 Females 0 15 30
No.of mice with renal tumors (%)
No. of mice examined
Total
Adenocarcinoma
Adenoma
37 16 1
l(3) 13 (Sl)b 0
0 11 (69)b 0
l(3) 5 (31)s 0
44 30 0
0 0 0
0 0 0
0 0 0
a P < 0.01; bP < 0.001 (Fisher’s exact test), as compared to the controls.
adenoma with trabecular pattern was found histologically among 24 survivors. No renal tumors were seen in males of the 30-ppm group and females of the treated and control groups. Renal tumors are less commonly seen in mice [ 5,6]. In the present study, the incidence of the renal epithelial tumors in the 15-ppm male group which were killed or died after 53 weeks apparently exceeded that in the control. The results might indicate that MMC is a renal carcinogen in mice, In order to clarify the total picture of MMC renal carcinogenicity, histological examinations on the mice which were killed or died by week 52 are under way. In addition to the present chronic study, another 24month chronic feeding study with MMC at the dietary levels of 0, 0.4, 2 or 10 ppm has been already completed in mice. Further detailed studies on the carcinogenicity of this compound are now in progress. REFERENCES 1 Charbonneau, S.M., Munro, I.C., Nera, E.A., Armstrong, F.A.J., WiIIes, R.F., Bryce, F. and Nelson, R.F. (1976) Chronic toxicity of methylmercury in the adult cat. Interim report. Toxicology, 5,337-349.
Fig. 1. Solid and well-demarcated neoplastic mass in the renal cortex. Fig. 2. Renal multilocular cystic containing many cysts and solid nodules. Fig. 3. Microscopic feature of the renal adenocarcinoma’in the neoplastic mass shown in Fig. 1. The tumor cells are arranged in a solid pattern, showing cellular and nuclear polymorphism. H-E x173. Fig. 4. Renai adenocarcinoma showing papillary proliferation of atypical columnar H-E x153.
cells.
310 2 Ikeda, Y., Tobe, M., Kobayashi, K., Suzuki, S., Kawasaki, Y. and Yonemaru, H. (1973) Long-term toxicity study of methylmercuric chloride in monkeys. First report. Toxicology, 1, 361-375. 3 Kanematsu, N., Hara, M. and Kada, T. (1980) Ret assay and mutagenicity studies on metal compounds. Mutat. Res., 77, 109-116. 4 Khera, KS. (1973) Reproductive capability of male rats and mice treated with methyl mercury. Toxicol. Appl. Pharmacol., 24, 167-177. 5 Percy, D.H. and Jonas, A.M. (1971) Incidence of spontaneous tumors in CDR-1 HaM/ ICR mice. J. Natl. Cancer Inst., 46, 1045-1065. 6 Prejean, J.D., Peckham, J.C., Casey, A.E., Griswold, D.P., Weisburger, E.K. and Weisburger, J.H. (1973) Spontaneous tumors in Sprague-Dawley rats and Swiss mice. Cancer Res., 33, 2768-2773. 7 RameI, C. (1967) Genetic effects of organic mercury compounds. Hereditas, 57, 445-447. 8 Shirasu, Y., Usui, T., Mitsumori, K., Takahashi, K., Matano, 0. and Goto, S. (1978) Chronic toxicity studies with methylmercury chloride in rats. In: Proceedings of the First International Congress on Toxicology, pp. 481. Editors: G.L. Plaa and W.A.M. Duncan. Academic Press, New York. 9 Skerfring, S., Hansson, K. and Lindsten, J. (1970) Chromosome breakage in humans exposed to methyl mercury through fiih consumption. Arch. Environ. Health, 21, 133-139. 10 Verschuuren, H.G., Kroes, R., Den Tonkelaar, E.M., Rerkvens, J.M., Helleman, P.W., Rauws, A.G., Schuller, P.L. and van Esch, G.J. (1976) Toxicity of methylmercury chloride in rats III, Long-term toxicity study. Toxicology, 6,107-123.
305
CARCINOGENICITY OF METHYLMERCURY CHLORIDE PRELIMINARY NOTE ON RENAL CARCINOGENESIS
KUNITOSHI MITSUMORI*, YASUHIKO SHIRASU Institute
of Environmental
IN ICR MICE:
KEIZOH MAITA, TORU SAITO, SHUJI TSUDA and Toxicology,
Suzuki-cho
2-772,
Kodaira-shi,
Tokyo
187
(Japan) (Received 23 December 1980) (Revised version received 13 February 198 1) (Accepted 16 February 1981)
SUMMARY ICR
mice (60/group) were fed diets containing methylmercury chloride in 0, 15 or 30 ppm for 78 weeks. The majority of mice in the 30-ppm group died due to the neurotoxicity by week 26. The first renal mass was grossly seen in a male of the 15-ppm group at week 58. Histopathology on kidney tissue from all animals surviving after 53 weeks revealed renal tumors in 13 of 16 males of the 15-ppm group in contrast with 1 of 37 males of the control. No renal tumors were seen in the female treated groups and/or control groups. (MMC)
INTRODUCTION
It is well known that organic mercury compounds produce neurological disturbances in man and many experimental animals. Among the investigations to explore other toxic effects of these compounds, mutagenic activities have been suggested in chromosome aberrations [ 7,9], DNA damage [ 31 and dominant lethal [4] studies. However, there is little information available on the carcinogenicity of these compounds, though several long-term toxicity studies have been reported in the rat [ SJO], cat [l] and monkey [ 21. In order to evaluate the chronic toxicity including carcinogenicity of MMC, a long-term feeding study was conducted in mice. After 58 weeks of study, a renal neoplastic mass was grossly observed in 1 male fed a diet containing 15 ppm MMC and the number of cases displaying such a mass increased significantly thereafter. The present study deals with the histological observa-
*To whom all correspondence
should be sent.
306
tions of the kidneys from the mice of all dosage groups which were killed or died after 53 weeks of study. MATERLALS AND METHODS
MMC with 99.3% purity was supplied from Tokyo Kasei Kogyo Co., Ltd., Japan. Male and female SPF ICR mice were obtained from the Shizuoko Agricultural Cooperative Association for Laboratory Animals, Japan, at 4 weeks, and housed 4 to a wire-mesh cage in a barrier system animal room. After a l-week acclimation, both sexes of mice were divided into 3 groups, each consisting of 60 males and 60 females. MMC was blended with a commercial powdered chow MF (Oriental Yeast Ltd., Japan) at 0, 15 or 30 ppm concentrations and fed to the mice for 78 weeks. All animals were examined macroscopically. Kidneys from all dosage groups which died, or became moribund after 53 weeks or were killed at the end of the study were examined histologically (Table 1). Lungs of mice disclosing renal masses were also examined histologically and renal lymph nodes studied when showing any gross abnormalities. These sections were stained with hematoxylin and eosin (H-E). At least 1 longitudinal section from each kidney was routinely examined. RESULTS AND DISCUSSION
Table 1 reveals the fate of mice and incidence of renal masses grossly detectable. Most of the mice in the 30-ppm group of both sexes exhibiting a marked neurotoxic disturbance died or became moribund by week 26. There was also a gradual increase in the number of mice showing neurotoxic signs in the 15-ppm group of either sex with prolongation of the feeding. On necropsy, the number of animals with abnormal kidney, granular and discolored surface indicative of toxic renal impairment, increased in males of the 15-ppm group after 26 weeks. The first renal tumor mass was encountered in a male of this dose group necropsied at week 58. Three of 8 males that died or became moribund during the rest period of the study also disclosed renal masses. Moreover, the final autopsy, held at week 78 revealed 6 cases of renal mass in 8 males. Consequently, a total of 10 of 16 males in the 15-ppm group was grossly identified as renal tumor bearers. All masses were seen unilaterally. No tumor masses were detected on the kidney of females in this dose group and 30-ppm group, as well as animals of both sexes in the control group. Four of the tumor bearers, had a whitish solid, well-demarcated mass less than 2 cm in diameter in the cortex (Fig. 1). The rest (6 mice) had a large multilocular cystic mass measuring 1.8-3.5 cm in diameter. On cut surface, these large masses were composed of white small solid nodules and cysts of various size filled with blackish brown fluid (Fig. 2). The partitions between these cysts were formed by cord-like extensions from those solid nodules.
307 TABLE 1 INCIDENCE OF RENAL NEOPLASTIC MASSES IN MICE FED DIETS CONTAINING MMC: GROSS FINDINGS Dose group (ppm) 15
0
30
Male
Female
Male
Female
Male
Female
oa/lb O/6
O/l O/6
o/7 O/6
O/6 O/6
o/51 O/6
o/59 O/l
o/10 O/6
o/3 O/6
O/25 O/6
o/12 O/6
o/2 o/o
-
o/13 O/24
o/11 o/33
418 618
o/12 O/18
O/l -
-
-
-
-
-
O/SO
O/60
10160
O/60
O/60
O/60
Died and moribund killing by week 26 Interim killing at week 26 Died and moribund killing by week 52 Interim killing at week 62 Died and moribund killing by week 78 Final killing at week 78
Total a No. of mice with renal mass. b No. of mice examined.
As shown in Table 2, histological examinations revealed renal tumors in 13 of 16 males of the 15ppm group. Of these cases, the renal tumors in 10 cases which had a gross mass were classified as adenocarcinoma. In addition, 1 case which became moribund at week 72 disclosed a small foci of adenocarcinoma in the renal cortex. The remaining 2 animals, which became moribund at week 59 or 69, had an adenoma in unilateral kidney. Adenoma also found in the other side of the kidney from 3 animals which had renal mass unilaterally when killed at the end of the study. Most adenocarcinomas observed were solid in appearance with delicate stroma, partly showing trabecular pattern (Fig. 3). There were occasional areas disclosing obvious renal tubular structure. In multicystic tumors, cystic, papillary (Fig. 4) and solid pattern co-existed. These tumors were composed of polyhedral, columnar or cuboidal cells with eosinophilic cytoplasms and marked cellular and nuclear polymorphism. Mitoses were occasionally noted. In some of the tumors with a large diameter, the surrounding parenchyma was compressed and tumor cell infiltration of adjacent tissues could be sometimes found. There were severe hemorrhagic necrotic foci and hemorrhages into the cyst at the portions showing marked papillary proliferation. No metastases to the lung or renal lymph nodes were seen. Besides these malignant tumors, adenomas found in the 15-ppm male group were composed of columnar cells with large eosinophilic cytoplasms and a few atypical nuclei, disclosing tubular arrangement. In the control male group, 1 small solid
308
309 TABLE 2 INCIDENCES AND HISTOLOGICAL TYPES OF RENAL TUMORS IN MICE THAT WERE KILLED OR DIED AFTER 53 WEEKS Sex and dietary level (ppm) Males 0 15 30 Females 0 15 30
No.of mice with renal tumors (%)
No. of mice examined
Total
Adenocarcinoma
Adenoma
37 16 1
l(3) 13 (Sl)b 0
0 11 (69)b 0
l(3) 5 (31)s 0
44 30 0
0 0 0
0 0 0
0 0 0
a P < 0.01; bP < 0.001 (Fisher’s exact test), as compared to the controls.
adenoma with trabecular pattern was found histologically among 24 survivors. No renal tumors were seen in males of the 30-ppm group and females of the treated and control groups. Renal tumors are less commonly seen in mice [ 5,6]. In the present study, the incidence of the renal epithelial tumors in the 15-ppm male group which were killed or died after 53 weeks apparently exceeded that in the control. The results might indicate that MMC is a renal carcinogen in mice, In order to clarify the total picture of MMC renal carcinogenicity, histological examinations on the mice which were killed or died by week 52 are under way. In addition to the present chronic study, another 24month chronic feeding study with MMC at the dietary levels of 0, 0.4, 2 or 10 ppm has been already completed in mice. Further detailed studies on the carcinogenicity of this compound are now in progress. REFERENCES 1 Charbonneau, S.M., Munro, I.C., Nera, E.A., Armstrong, F.A.J., WiIIes, R.F., Bryce, F. and Nelson, R.F. (1976) Chronic toxicity of methylmercury in the adult cat. Interim report. Toxicology, 5,337-349.
Fig. 1. Solid and well-demarcated neoplastic mass in the renal cortex. Fig. 2. Renal multilocular cystic containing many cysts and solid nodules. Fig. 3. Microscopic feature of the renal adenocarcinoma’in the neoplastic mass shown in Fig. 1. The tumor cells are arranged in a solid pattern, showing cellular and nuclear polymorphism. H-E x173. Fig. 4. Renai adenocarcinoma showing papillary proliferation of atypical columnar H-E x153.
cells.
310 2 Ikeda, Y., Tobe, M., Kobayashi, K., Suzuki, S., Kawasaki, Y. and Yonemaru, H. (1973) Long-term toxicity study of methylmercuric chloride in monkeys. First report. Toxicology, 1, 361-375. 3 Kanematsu, N., Hara, M. and Kada, T. (1980) Ret assay and mutagenicity studies on metal compounds. Mutat. Res., 77, 109-116. 4 Khera, KS. (1973) Reproductive capability of male rats and mice treated with methyl mercury. Toxicol. Appl. Pharmacol., 24, 167-177. 5 Percy, D.H. and Jonas, A.M. (1971) Incidence of spontaneous tumors in CDR-1 HaM/ ICR mice. J. Natl. Cancer Inst., 46, 1045-1065. 6 Prejean, J.D., Peckham, J.C., Casey, A.E., Griswold, D.P., Weisburger, E.K. and Weisburger, J.H. (1973) Spontaneous tumors in Sprague-Dawley rats and Swiss mice. Cancer Res., 33, 2768-2773. 7 RameI, C. (1967) Genetic effects of organic mercury compounds. Hereditas, 57, 445-447. 8 Shirasu, Y., Usui, T., Mitsumori, K., Takahashi, K., Matano, 0. and Goto, S. (1978) Chronic toxicity studies with methylmercury chloride in rats. In: Proceedings of the First International Congress on Toxicology, pp. 481. Editors: G.L. Plaa and W.A.M. Duncan. Academic Press, New York. 9 Skerfring, S., Hansson, K. and Lindsten, J. (1970) Chromosome breakage in humans exposed to methyl mercury through fiih consumption. Arch. Environ. Health, 21, 133-139. 10 Verschuuren, H.G., Kroes, R., Den Tonkelaar, E.M., Rerkvens, J.M., Helleman, P.W., Rauws, A.G., Schuller, P.L. and van Esch, G.J. (1976) Toxicity of methylmercury chloride in rats III, Long-term toxicity study. Toxicology, 6,107-123.