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Tumor induction by N-nitroso compounds in bivalve mollusks unio pictorum
Cancer Letters. 4 (1978) 349--354 © Elsevier/North-Holland Scientific Publishers Ltd.
349
TUMOR INDUCTION BY N-NITROSO COMPOUNDS IN BIVALVE MOLLUSKS UNIO PICTOR UM
V.V. KHUDOLEY and O.A. SYRENKO Laboratory of Chemical Carcinogenic Agents, N.N. Petrov Research Institute of Oncology of the USSR Ministry o f Public Health, 68 Leningradskaya Street, Pesochny-2, Leningrad 188646 (U.S.S.R.) (Received 14 November 1977) (Revised version received 14 April 1978) (Accepted 17 April 1978)
SUMMARY
Diethylnitrosamine (DENA) and dimethylnitrosamine (DMNA) dissolved in tank water induced neoplasms in bivalve mollusks Unio pictorum. DENA induced tumors in 18% of the animals exposed to 200 ppm and 68% of those exposed to 400 ppm; average latent periods were 71 and 61 days, respectively. DMNA induced tumors in 8% of the animals exposed to 200 ppm for 51 days and 27% of those exposed for 152 days with average latent periods of 85 and 81 days, respectively. Neoplasms were observed in the digestive gland (basophilic ceil tumors; 38 cases), the hemopoietic system (hemocytoblastosis; 12 cases) and the kidney (poorly differentiated carcinoma; 1 case). It is expedient to use mollusks, both for testing of N-nitroso compounds and as a biologic indicator of hydrospheric pollution.
INTRODUCTION
Determination of the sensitivity of various animal species to carcinogenic agents is vital for use in testing chemical compounds for tumorigenic activity and for biologic indication of environmental pollution [2]. In this respect, fish [6,8] and amphibia [3] are best among the aquatic animals studied. Spontaneous neoplasms have long been known to occur in mollusks [4,7,9]. Despite numerous attempts to induce tumors in mollusks with chemical
Address correspondence to: Dr. Veniamin V. Khudoley, N.N. Petrov Research Institute of Oncology, Leningrad 188646, U.S.S.R. Abbreviations: DENA, diethylnitrosamine ; DMNA, dimethylnitrosamine.
350 carcinogens, positive results have been obtained with polycyclic aromatic hydrocarbons only [5]. The purpose of this investigation was to evaluate the possibility of t u m o r induction in mollusks by nitrosamines. MATERIALS AND METHODS Experiments were carried out on 350 mature adult freshwater mollusks Unio pictorum obtained from a commercial breeder. The animals were kept in 10-1 glass tanks with aeration at 18--20°C, and a suspension of fly larvae and yeast was given. Three series of experiments were performed. In 2 series the maximal endurable doses of carcinogenic nitrosamines were used. Mollusks were exposed to nitrosamines dissolved in water. Series 1. In group 1 , 1 1 5 animals were exposed to DENA for 64 days at a concentration of 200 ppm. In group 2, 45 mollusks received DENA at a concentration of 400 ppm for 62 days. After exposure animals were placed in fresh water w i t h o u t carcinogenic contamination. The animals of these groups were observed for 173 days. Series 2. In group 1, DMNA was given to 30 mollusks at a concentration of 200 ppm for 51 days. In group 2, 60 animals received DMNA in the same concentration for 152 days. The maximal length of observation in both groups was 152 days. Series 3. One hundred control mollusks were kept under similar conditions w i t h o u t the carcinogen and observed for 310 d a y s . Morphologic examination was performed on t h e digestive gland, gonads, intestine and gills from animals dying or killed. The specimens were fixed in 4% formalin or Bouin's fluid. Sections were stained with hematoxylin and eosin, axan and van Gieson's solution. RESULTS The number of animals tested, the dose levels of the compounds, the duration of the experiments and the incidence and latency of the neoplasms are shown in Table 1. Changes in behavior, including decreased m o t o r and filtration activities, were manifested on the first and subsequent weeks. The morphological alterations in series 1 and 2 were similar and developed predomin a n t l y in the digestive gland. The histologic structure of the digestive gland in bivalve mollusks is very much the same as t h a t of the liver in vertebrates; however, there are certain pecularities. The digestive gland consists of numerous digestive diverticula. The epithelial cells of these structures are made up of 'basophilic' and digestive 'eosinophilic' cells. Thin connective tissue is located between diverticula (Figure la). The toxic action of nitrosamines was manifested within 2--4 weeks. Microscopic examination of the digestive gland revealed focal necrosis and a simultaneous development of dystrophic processes. Beginning at 3--4 weeks, diffuse o r
351 TABLE 1 TUMOR INDUCTION BY N-NITROSO COMPOUNDS IN BIVALVE MOLLUSKS U. P1C TO R U M
Series/group
Number of animals
Dose (ppm)
Duration Duration of exposure of experiment (days) (days)
Average latency of t u m o r induction (days)
Tumor incidence a
1/1 DENA 1/2 DENA 2/1DMNA 2/2 DMNA 3 Control
115 45 30 60 100
200 400 200 200
64 62 51 152
71 61 85 81
16/87 (18%) 13/19 (68%) 2/24 (8%) 12/45 (27%)
173 173 152 152 310
a Number of mollusks with tumors/number of mollusks surviving at time of first tumor induction.
focal hyperplasia of the digestive gland were noted, accompanied by nodular proliferations composed of pleomorphic basophilic cells. These alterations were associated with an increase in cell layers (from 1 to 2 or 3). The cells were enlarged and rounded with prominent nuclei and delicate basophilic cytoplasm. The first neoplasms were observed after 36--39 days in DENA experiments and after 40--55 days in DMNA experiments. At that time 87 and 19 mollusks survived, respectively. The liver of these mollusks was enlarged (1.5--2 times), nodular, brown and of firm consistency. The tumors developed on a background of dystrophic and hyperplastic lesions. In such cases the structure of the digestive gland was altered and the shape and size of diverticula varied. Basophilic cells obstructed the diverticular lumen, displacing eosinophilic digestive cells. These proliferations were characterized by destruction and invasion of t u m o r cells on diverticular walls, atypia and polymorphism of cell elements with formation of giant cells showing light basophilic cytoplasm and large nuclei. Atypical mitotic figures and multinuclear cells were encountered (Fig. lb). These atypical proliferative lesions were marked in 26 mollusks in series 1 and-in 12 in series 2 and were classified as "basophilic cell tumors.' Simultaneously, increased activity and numbers of hemocytes were observed in the connnective tissue between diverticula. Excessive proliferation of hemocytes was found in 10 animals of the DENA experiment and 2 with DMNA • (usually along with basophilic cell tumors). The size and configuration of these cells varied from small blood cells with wrinkled nuclei to gigantic basophilic cells. Bright, f o a m y cytoplasm and large nuclei could also be noted. Multifocal proliferations of the cells o f the hematopoietic tissue were located predominantly in the digestive gland, intestine, gills and gonads. These lesions were calssified as hemocytoblastosis (Fig. lc).
352
Fig. 1. (a) Normal structure of the digestive gland in control mollusk Unio pictorum. H and E × 230. (b) Basophilic cell t u m o r of the digestive gland. DENA. 39 days. H and E × 230. (c) Hemocytoblastosis in the digestive gland. DENA. 61 days. H and E × 900. (d) Poorly differentiated carcinoma of the kidney. DMNA. 63 days. H and E × 900.
353 In one case (series 2) along with hemocytoblastosis, a kidney neoplasm occurred. This t u m o r (0.5 × 0.2 cm) consisted of a mixture of muscular and glandular structures with extraordinarily pleomorphic eosinophilic cells. These cells were long, fusiform or polygonal and had large round or ovoid nuclei. Mitotic figures were frequent. Based on morphologic criteria, the neoplasm was classified as a poorly differentiated carcinoma (Fig. ld). In one case (series 1) focal hyperplasia of the gill tissue was revealed. Tumors developed in up to 68% of the animals treated with DENA and 27% of those treated with DMNA. Thirty-eight neoplasms were located in the digestive gland, 12 in the hematopoietic system and 1 in the kidney. The average latent period of t u m o r development was 63 days with DENA and 83 days with DMNA. In the control series no tumors of preneoplastic lesions were found in the digestive gland or other organs. DISCUSSION The present study demonstrated induction of neoplasms in mollusks by 2 nitrosamines. Similar studies on mollusks have not been previously described in the literature. The mollusk tumors had morphologic and clinical characteristics similar to hepatic neoplasms induced b y chemical carcinogens in fish and amphibians. Tumors arose primarily from the digestive glands on a background of diffuse and focal proliferation of basophilic cells. They were invasive and characterized b y extraordinary pleomorphisms and atypia. A correlation between the dosage and the incidence of tumors was observed (see Table 1). In a number of cases tumors of the hematopoietic tissue were also noted. These neoplasms were similar to those occurring spontaneously in hematopoietic tissues of mollusks [ 1 ]. However, such spontaneous neoplasms were not found in our control groups. It is believed that the development of these tumors in mollusks is due to water pollution b y carcinogenic compounds [9]. Our experiments showed mollusks Unio pictorum to be highly sensitive to the neoplastic action of nitrosamines. Therefore, mollusks may be used to test for the presence of soluble carcinogenic agents and possible hydrospheric pollutants. ACKNOWLEDGEMENTS The authors express thanks to the Chief of the Laboratory of Chemical Carcinogenic Agents, Prof. G.B. Pliss, to collaborators M.A. Zabezhinsky, N.N. Vlasov and V.V. Eliseev for their useful advice, to Mrs. N.I. Shalumovich for illustrations and to Mrs. A.D. Lipkovich and Miss K.I. Molchanova for translation and preparation of the manuscript. REFERENCES 1 Frierman, E.M. and Andrews, J.D. (1976) Occurrence of hematopoietic neoplasms in Virginia oysters (Crassostrea virginica). J. Natl. Cancer Inst., 56,319--324.
354
2 Khudoley, V.V. (1976) Comparative analysisof tumor growtl~.J. Gen. Biol.,37, 242--254 (in Russian). 3 Khudoley, V.V. (1977) Tumor induction by carcinogenic agents in anuran amphibians Rana temporaria. Arch. Geschwulstforsch.,47,385--395. 4 Khudoley, V.V. and Syrenko, O.A. (1977) Tumors in mollusks. Adv. Modern Biol.,84, 128--137 (in Russian). 5 Krieg, K. (1973) Invertebraten in der Geschwulstforschung. Theodor Steinkopff verlag, Dresden. 6 Matsushima, T. and Sugimura, T. (1976) Experimental carcinogenesis in small aquarium fishes. Prog. Exp. Tumor Res., 20,367--379. 7 Pau!ey, G.B. (1969) A critical review of neoplasia and tumor-like lesions in mollusks. Natl. Cancer Inst. Monogr., 31,509--540. 8 Pliss, G.B. and Khudoley, V.V. (1975) Tumor induction by carcinogenic agents in aquarium fish. J. Natl. Cancer Inst., 55,129--136. 9 Rosenfield, A. (1976) Recent environmental studies of neoplasms in marine shellfish. Prog. Exp. Tumor Res., 20,263--274.
349
TUMOR INDUCTION BY N-NITROSO COMPOUNDS IN BIVALVE MOLLUSKS UNIO PICTOR UM
V.V. KHUDOLEY and O.A. SYRENKO Laboratory of Chemical Carcinogenic Agents, N.N. Petrov Research Institute of Oncology of the USSR Ministry o f Public Health, 68 Leningradskaya Street, Pesochny-2, Leningrad 188646 (U.S.S.R.) (Received 14 November 1977) (Revised version received 14 April 1978) (Accepted 17 April 1978)
SUMMARY
Diethylnitrosamine (DENA) and dimethylnitrosamine (DMNA) dissolved in tank water induced neoplasms in bivalve mollusks Unio pictorum. DENA induced tumors in 18% of the animals exposed to 200 ppm and 68% of those exposed to 400 ppm; average latent periods were 71 and 61 days, respectively. DMNA induced tumors in 8% of the animals exposed to 200 ppm for 51 days and 27% of those exposed for 152 days with average latent periods of 85 and 81 days, respectively. Neoplasms were observed in the digestive gland (basophilic ceil tumors; 38 cases), the hemopoietic system (hemocytoblastosis; 12 cases) and the kidney (poorly differentiated carcinoma; 1 case). It is expedient to use mollusks, both for testing of N-nitroso compounds and as a biologic indicator of hydrospheric pollution.
INTRODUCTION
Determination of the sensitivity of various animal species to carcinogenic agents is vital for use in testing chemical compounds for tumorigenic activity and for biologic indication of environmental pollution [2]. In this respect, fish [6,8] and amphibia [3] are best among the aquatic animals studied. Spontaneous neoplasms have long been known to occur in mollusks [4,7,9]. Despite numerous attempts to induce tumors in mollusks with chemical
Address correspondence to: Dr. Veniamin V. Khudoley, N.N. Petrov Research Institute of Oncology, Leningrad 188646, U.S.S.R. Abbreviations: DENA, diethylnitrosamine ; DMNA, dimethylnitrosamine.
350 carcinogens, positive results have been obtained with polycyclic aromatic hydrocarbons only [5]. The purpose of this investigation was to evaluate the possibility of t u m o r induction in mollusks by nitrosamines. MATERIALS AND METHODS Experiments were carried out on 350 mature adult freshwater mollusks Unio pictorum obtained from a commercial breeder. The animals were kept in 10-1 glass tanks with aeration at 18--20°C, and a suspension of fly larvae and yeast was given. Three series of experiments were performed. In 2 series the maximal endurable doses of carcinogenic nitrosamines were used. Mollusks were exposed to nitrosamines dissolved in water. Series 1. In group 1 , 1 1 5 animals were exposed to DENA for 64 days at a concentration of 200 ppm. In group 2, 45 mollusks received DENA at a concentration of 400 ppm for 62 days. After exposure animals were placed in fresh water w i t h o u t carcinogenic contamination. The animals of these groups were observed for 173 days. Series 2. In group 1, DMNA was given to 30 mollusks at a concentration of 200 ppm for 51 days. In group 2, 60 animals received DMNA in the same concentration for 152 days. The maximal length of observation in both groups was 152 days. Series 3. One hundred control mollusks were kept under similar conditions w i t h o u t the carcinogen and observed for 310 d a y s . Morphologic examination was performed on t h e digestive gland, gonads, intestine and gills from animals dying or killed. The specimens were fixed in 4% formalin or Bouin's fluid. Sections were stained with hematoxylin and eosin, axan and van Gieson's solution. RESULTS The number of animals tested, the dose levels of the compounds, the duration of the experiments and the incidence and latency of the neoplasms are shown in Table 1. Changes in behavior, including decreased m o t o r and filtration activities, were manifested on the first and subsequent weeks. The morphological alterations in series 1 and 2 were similar and developed predomin a n t l y in the digestive gland. The histologic structure of the digestive gland in bivalve mollusks is very much the same as t h a t of the liver in vertebrates; however, there are certain pecularities. The digestive gland consists of numerous digestive diverticula. The epithelial cells of these structures are made up of 'basophilic' and digestive 'eosinophilic' cells. Thin connective tissue is located between diverticula (Figure la). The toxic action of nitrosamines was manifested within 2--4 weeks. Microscopic examination of the digestive gland revealed focal necrosis and a simultaneous development of dystrophic processes. Beginning at 3--4 weeks, diffuse o r
351 TABLE 1 TUMOR INDUCTION BY N-NITROSO COMPOUNDS IN BIVALVE MOLLUSKS U. P1C TO R U M
Series/group
Number of animals
Dose (ppm)
Duration Duration of exposure of experiment (days) (days)
Average latency of t u m o r induction (days)
Tumor incidence a
1/1 DENA 1/2 DENA 2/1DMNA 2/2 DMNA 3 Control
115 45 30 60 100
200 400 200 200
64 62 51 152
71 61 85 81
16/87 (18%) 13/19 (68%) 2/24 (8%) 12/45 (27%)
173 173 152 152 310
a Number of mollusks with tumors/number of mollusks surviving at time of first tumor induction.
focal hyperplasia of the digestive gland were noted, accompanied by nodular proliferations composed of pleomorphic basophilic cells. These alterations were associated with an increase in cell layers (from 1 to 2 or 3). The cells were enlarged and rounded with prominent nuclei and delicate basophilic cytoplasm. The first neoplasms were observed after 36--39 days in DENA experiments and after 40--55 days in DMNA experiments. At that time 87 and 19 mollusks survived, respectively. The liver of these mollusks was enlarged (1.5--2 times), nodular, brown and of firm consistency. The tumors developed on a background of dystrophic and hyperplastic lesions. In such cases the structure of the digestive gland was altered and the shape and size of diverticula varied. Basophilic cells obstructed the diverticular lumen, displacing eosinophilic digestive cells. These proliferations were characterized by destruction and invasion of t u m o r cells on diverticular walls, atypia and polymorphism of cell elements with formation of giant cells showing light basophilic cytoplasm and large nuclei. Atypical mitotic figures and multinuclear cells were encountered (Fig. lb). These atypical proliferative lesions were marked in 26 mollusks in series 1 and-in 12 in series 2 and were classified as "basophilic cell tumors.' Simultaneously, increased activity and numbers of hemocytes were observed in the connnective tissue between diverticula. Excessive proliferation of hemocytes was found in 10 animals of the DENA experiment and 2 with DMNA • (usually along with basophilic cell tumors). The size and configuration of these cells varied from small blood cells with wrinkled nuclei to gigantic basophilic cells. Bright, f o a m y cytoplasm and large nuclei could also be noted. Multifocal proliferations of the cells o f the hematopoietic tissue were located predominantly in the digestive gland, intestine, gills and gonads. These lesions were calssified as hemocytoblastosis (Fig. lc).
352
Fig. 1. (a) Normal structure of the digestive gland in control mollusk Unio pictorum. H and E × 230. (b) Basophilic cell t u m o r of the digestive gland. DENA. 39 days. H and E × 230. (c) Hemocytoblastosis in the digestive gland. DENA. 61 days. H and E × 900. (d) Poorly differentiated carcinoma of the kidney. DMNA. 63 days. H and E × 900.
353 In one case (series 2) along with hemocytoblastosis, a kidney neoplasm occurred. This t u m o r (0.5 × 0.2 cm) consisted of a mixture of muscular and glandular structures with extraordinarily pleomorphic eosinophilic cells. These cells were long, fusiform or polygonal and had large round or ovoid nuclei. Mitotic figures were frequent. Based on morphologic criteria, the neoplasm was classified as a poorly differentiated carcinoma (Fig. ld). In one case (series 1) focal hyperplasia of the gill tissue was revealed. Tumors developed in up to 68% of the animals treated with DENA and 27% of those treated with DMNA. Thirty-eight neoplasms were located in the digestive gland, 12 in the hematopoietic system and 1 in the kidney. The average latent period of t u m o r development was 63 days with DENA and 83 days with DMNA. In the control series no tumors of preneoplastic lesions were found in the digestive gland or other organs. DISCUSSION The present study demonstrated induction of neoplasms in mollusks by 2 nitrosamines. Similar studies on mollusks have not been previously described in the literature. The mollusk tumors had morphologic and clinical characteristics similar to hepatic neoplasms induced b y chemical carcinogens in fish and amphibians. Tumors arose primarily from the digestive glands on a background of diffuse and focal proliferation of basophilic cells. They were invasive and characterized b y extraordinary pleomorphisms and atypia. A correlation between the dosage and the incidence of tumors was observed (see Table 1). In a number of cases tumors of the hematopoietic tissue were also noted. These neoplasms were similar to those occurring spontaneously in hematopoietic tissues of mollusks [ 1 ]. However, such spontaneous neoplasms were not found in our control groups. It is believed that the development of these tumors in mollusks is due to water pollution b y carcinogenic compounds [9]. Our experiments showed mollusks Unio pictorum to be highly sensitive to the neoplastic action of nitrosamines. Therefore, mollusks may be used to test for the presence of soluble carcinogenic agents and possible hydrospheric pollutants. ACKNOWLEDGEMENTS The authors express thanks to the Chief of the Laboratory of Chemical Carcinogenic Agents, Prof. G.B. Pliss, to collaborators M.A. Zabezhinsky, N.N. Vlasov and V.V. Eliseev for their useful advice, to Mrs. N.I. Shalumovich for illustrations and to Mrs. A.D. Lipkovich and Miss K.I. Molchanova for translation and preparation of the manuscript. REFERENCES 1 Frierman, E.M. and Andrews, J.D. (1976) Occurrence of hematopoietic neoplasms in Virginia oysters (Crassostrea virginica). J. Natl. Cancer Inst., 56,319--324.
354
2 Khudoley, V.V. (1976) Comparative analysisof tumor growtl~.J. Gen. Biol.,37, 242--254 (in Russian). 3 Khudoley, V.V. (1977) Tumor induction by carcinogenic agents in anuran amphibians Rana temporaria. Arch. Geschwulstforsch.,47,385--395. 4 Khudoley, V.V. and Syrenko, O.A. (1977) Tumors in mollusks. Adv. Modern Biol.,84, 128--137 (in Russian). 5 Krieg, K. (1973) Invertebraten in der Geschwulstforschung. Theodor Steinkopff verlag, Dresden. 6 Matsushima, T. and Sugimura, T. (1976) Experimental carcinogenesis in small aquarium fishes. Prog. Exp. Tumor Res., 20,367--379. 7 Pau!ey, G.B. (1969) A critical review of neoplasia and tumor-like lesions in mollusks. Natl. Cancer Inst. Monogr., 31,509--540. 8 Pliss, G.B. and Khudoley, V.V. (1975) Tumor induction by carcinogenic agents in aquarium fish. J. Natl. Cancer Inst., 55,129--136. 9 Rosenfield, A. (1976) Recent environmental studies of neoplasms in marine shellfish. Prog. Exp. Tumor Res., 20,263--274.