Induction of tumors with cycasin in newborn and preweanling rats.

Comprehensive studies of carcinogenesis in newborn or preweanling SD rats were conducted under various dose schedules of cycasin (CAS: 14901-08-7) administration. When cycasin was given sc to newborn rats at day 0, tumors were detected in more than 80% of rats of both sexes; kidney tumors were by far the most common. The incidences of tumors declined in the older groups, namely, over 60% in both sexes in the 7-day group, 55% in males and 8.3% in females in 14-day rats, and 0% in 21-day groups. By multiple administration, tumor incidences elevated considerably. Administration ip of cycasin also gave rise to tumor induction in newborn rats. A total of 435 kidney tumors found in the experiments were studied pathologically. Most of them were classified as mesenchymal tumor; some of them metastasized. A few other tumors were found in the liver and colon.

Histological conformity of implantation tumors produced by kidney cell lines derived from dimethylnitrosamine-treated rats, with dimethylnitrosamine-induced renal mesenchymal tumors.

The histology of five implantation tumors induced in rats by the deposition of cultured cell lines derived from dimethylnitrosamine (DMN)-treated rats is described and compared with the morphology of the predominant kidney neoplasm induced in vivo by a single high dose of DMN. The cell lines leading to growth upon implantation were long-established, continuously growing cultures obtained either from a DMN-induced renal mesenchymal tumor or from rats treated shortly before with a carcinogenic dose of DMN. The latter cultures had expressed morphological transformation at subcultures 5 or 6. All of the implantation tumors were of mesenchymal type, comprising variously a range of cell forms including fibroblast-like spindle cells, smooth muscle fibers, and "giant" cells, which resembled common aspects of the parent mesenchymal tumors induced in the rat kidney by DMN. Deposition of cells intrarenally illustrated the survival of remnants of preexisting nephrons as epithelial profiles scattered through the proliferating malignant tissue, a feature most characteristic of the parent tumor. The results confirmed the malignant nature of the various cell lines tested, in keeping with their altered behavior in vitro, and they were consistent also with the premise that the in vivo-in vitro system is selecting cells in culture that represent the same target population from which the renal mesenchymal tumors are derived in vivo.

Kidney tumors induced in rats by the antischistosomal drug niridazole.

An increased incidence of kidney tumors was found in MRC rats fed the antischistosomal drug niridazole at four dose levels in the diet. Histologically, the adenomas and adenocarcinomas were solid papillary, clear cell, and tubular types, with the latter type predominating. Seven mesenchymal tumors were found among the 107 renal epithelial neoplasms. Severe nephrosclerosis occurred in both treated and control rats and has been suggested as important in renal carcinogenesis. Niridazole is considered a potent inducer of epithelial kidney tumors.

K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors.

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrectly incorporate opposite deoxyguanine in DNA, then pair with deoxyadenosine during subsequent replication. It appears to preferentially target the 3'-G of 5'-NGGN-3' sequences in mammalian cells in culture to induce G-->A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tumors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone, while N-nitrosomethylurea (NMU) alone or NMU + BrdUrd resulted in incidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treated with BrdUrd for mutations in K-ras exons 1 and 2 and compared the prevalence and spectrum of mutations with those found in comparable tumors induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12-15 months earlier with BrdUrd (three specimens) or NMU (11 specimens) or both agents sequentially (eight specimens) were amplified by PCR. The base sequence of codons 12-13 and 59-63 of K-ras was determined by the dideoxynucleotide method. Sequencing results were confirmed by allele-specific oligonucleotide hybridization. Two of three tumors that appeared in rats given BrdUrd alone contained both a codon 12 GGT-->GAT transition and a codon 61 CAA-->CTA transversion. One tumor induced by NMU alone also showed a codon 12 GGT-->GAT mutation, while only wild type sequence could be demonstrated in the codon 12-13 region in the remaining ten such tumors. Three NMU-induced tumors also showed codon 61 CAA-->CTA mutations, while the remaining tumors had wild type sequence. While the GGT-->GAT transitions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA-->CTA transversions, the overall low prevalence of mutations, and the lack of any difference in mutation spectrum between tumors induced by NMU and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effect of either agent.

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats.

Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any well-known organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.

Differential effects of phorbol ester tumor promoters on 3-methylcholanthrene-induced epithelial and mesenchymal skin tumorigenesis.

The effects of TPA, PDD, PDB, PDA, or MEZ on epithelial and mesenchymal skin tumors induced by a s.c. injection of MCA were studied histologically. Group-I mice received only MCA. At 6 weeks after MCA injection, mice in groups II to VII received acetone, 1.8 nmol TPA, PDD, PDB, PDA, or 6.1 nmol MEZ respectively in 0.1 ml acetone twice weekly until tumor development. Alterations in skin tumor induction patterns were also studied in animals that had been exposed to TPA or acetone for 10 weeks prior to s.c. injection of MCA. Exposure of mouse skin to TPA before or after carcinogen administration increased 2- to 3.5-fold, the incidence of carcinoma and mixed tumors of epithelial and mesenchymal histogenesis. The average time of tumor induction decreased in mice treated with MCA + TPA and 100% of the test animals in the TPA + MCA group developed tumors. In contrast, TPA-related phorbol esters inhibited skin tumor development, particularly trichoepithelioma and fibrosarcoma and increased the average time of tumor induction.

Nephroblastoma and renal mesenchymal tumor induced in rats by N-nitrosoethyl- and N-nitrosomethylurea.

Rats treated prenatally with N-nitrosoethylurea or with N-nitrosomethylurea developed nephroblastomas, renal mesenchymal tumors and epithelial tumors (adenomas and carcinomas) of the kidneys. 15 nephroblastomas and 59 mesenchymal tumors were examined histologically. Nephroblastomas were encapsulated growths composed of dark cells, forming primitive tubules similar to those seen in the rat embryonal kidney and in human Wilms' tumor. Mesenchymal renal tumors showed an infiltrative growth and consisted of fibroblast-like cells, smooth muscles and angiomatous areas with the engulfed pre-existing tubules. These growths are similar to the mesenchymal renal tumors induced in the rat by N-nitrosodimethylamine. Nephroblastoma and mesenchymal renal tumor are considered to be separated entities, the first corresponsing to the epithelial variant of human Wilms' tumor and the second to congenital mesoblastic nephroma.

S-100 protein and glial fibrillary acidic protein (GFAP) in experimentally induced and spontaneous tumours of peripheral nerves in BDVI rats. Light microscopic and immunohistochemical study.

Twenty-five ethylnitrosourea (ENU) induced and 24 spontaneous tumours of peripheral nerves as well as 28 spontaneous mesenchymal tumours in BDVI rats were studied by light microscopy and immunohistochemically for the presence of S-100 protein (S-100). Early ENU-induced schwannomas*) representing the thickenings of nerves showed weak or negative S-100 immunoreactivity. S-100 positivity (both in the cytoplasm and nucleus) was observed in all large ENU-induced tumours found in animals dying or killed at advanced age. Immunostaining was present in both cystic and solid areas of schwannomas. S-100 positivity was found in 20 of 24 spontaneous schwannomas: 14 of 20 positive tumours contained cysts. Twenty-seven schwannomas (12 ENU-induced and 15 spontaneous) were studied for the presence of glial fibrillar acid protein (GFAP) and 13 were positive (7 had cystic areas). GFAP-positivity was relatively high in 4 tumours (3 ENU-induced and 1 spontaneous); these tumours also showed intense S-100 reactivity. Immunoreactivity for S-100 occurred more frequently and was much more intense than that for GFAP. The incidence of spontaneous peripheral nerve tumours in BDVI males reached 4%, cystic schwannomas being the most frequent type. All spontaneous mesenchymal tumours except lipoma (S-100 positive) were negative for S-100 protein and for GFAP.

[Modifying effects of changes connected with pregnancy on 1,2-dimethylhydrazine-induced carcinogenesis in rats]. / Modifitsiruiushchee vliianie izmenenii, sviazannykh s beremennost'iu, na kantserogenez, indutsirovannyi 1,2-dimetilgidrazinom, u krys.

The modifying effect of pregnancy and lactation on carcinogenesis induced by single injection of 1,2-dimethylhydrazine to female rats 30 days before mating has been studied. The total incidence of malignant tumours in rats with pseudopregnancy, single pregnancy, pregnancy and lactation, repeated pregnancies was lower in comparison with virgin animals (75, 44, 68, 59 and 93%, respectively). Colon adenocarcinoma incidence in rats with single pregnancy or repeated pregnancies was lower than that in virgin rats (42, 49 and 76%, respectively). Protective effect was observed mainly in descending colon. The mesenchymal kidney tumours were not developed at all in rats with single pregnancy. In virgin animals it was 31%. The inhibition of tumour incidence in the liver (cholangioma, cholangiocellular carcinoma) was observed in rats with single pregnancy or pregnancy and lactation in comparison with virgin control (3, 5 and 24%, respectively).

Comparison of the tumorigenic potential of liver and kidney tumors induced by N-nitrosodimethylamine.

The aim of the study was to determine the tumorigenic potential of two cell lines established from N-nitrosodimethylamine induced rat hepatocarcinoma (HeDe) and mesenchymal renal tumors (NeDe). The basis of the distinction is that human cancers are known to overexpress facilitative GLUT transporters and TGF-beta1 protein. These proteins are linked to the increased metabolic energy consumption indicating uncontrolled growth and proliferation. We have assayed not only the expression of GLUT-1, GLUT-3 and TGF-beta1 proteins, but also the uptake of 2-fluoro-[18F]-2-deoxy-D-glucose (18FDG), a tracer for cancer diagnosis. Western blot analysis and whole body autoradiography were used to measure the 18FDG uptake of tumor cells. Elevated 18FDG uptake was measured in both tumor cell lines. Whole body autoradiography provided evidence that the uptake of 18FDG was lower in the necrotic inner part than in the more vascularized outer parts of primary hepatocarcinoma and mesenchymal renal tumors. GLUT-1 overexpression in hepatocarcinoma tumor, and high levels of GLUT-3 were found in the NeDe cell line and in the mesenchymal renal tumor. TGF-beta-1 was overexpressed in hepatocarcinoma and mesenchymal renal tumors. In vitro and in vivo parameters support the view that the tumorigenic potential of cancer cells cannot be determined by the expression of a single parameter such as the expression of either GLUT-1, GLUT-3 or 18FDG uptake. Besides the tumorigenic potential of the hepatocarcinoma, the high metabolic activity of the renal tumor indicated by its 18FDG uptake, GLUT-3 and TGF-beta1 expression, the mesenchymal renal tumor induced by N-nitroso-dimethylamine is not a benign, but an an aggressive renal carcinoma.

Malignant mesenchymoma and birth defects. Prenatal exposure to phenytoin.

Malignant mesenchymoma developed in an 18-year-old patient with phenytoin-associated cleft lip and palate. Although these conditions may be related by chance, the possibility of transplacental carcinogenesis by phenytoin should be considered, especially since neuroblastoma was reported recently in two children with phenytoin-induced malformations. Following combination chemotherapy for metastases, the patient experienced a 7-year disease-free interval, which is consistent with recent improvement in the treatment of soft-tissue sarcomas.

Experimental models for the sequential analysis of chemically-induced renal carcinogenesis.

In order to discriminate non-specific toxicity from the early precursor lesions of neoplasia, emphasis in these studies has been on the use of models requiring only a single administration of chemical. Our interests have focussed on three neoplastic entities in the kidney, renal mesenchymal neoplasia, renal cell carcinoma, and nephroblastoma. Dimethylnitrosamine administered as a single intraperitoneal injection to immature female Wistar rats, pre-conditioned for several days with a no-protein/sugar-only diet, has been used for investigating the complex morphological nature of renal mesenchymal tumors, their pathogenesis and the development of cell culture correlates. The near 100% tumor incidence and its facility for cell culture manipulation makes this a particularly potent model for studying chemical carcinogenesis and the evolution of cell transformation. Discovery that the rat kidney response to DMN was biphasic with respect to the time of treatment led to the subsequent development of a high incidence system for inducing renal adenocarcinoma, using older rats. Renal cell carcinomas could also be induced in mice by a single intravenous injection of streptozotocin. The tumor frequency in female CBA/H/T6J mice was almost 100%, providing a new model for the investigation of renal carcinogenesis in this species. Nephroblastoma has been a poorly comprehended neoplasm in both lower animals and man because of the lack of a high incidence model in conventional laboratory mammals. Recently, we have exploited an increased spontaneous predisposition of the Nb rat to nephroblastoma using a single intraperitoneal dose of N-ethylnitrosourea in pregnant females on day 18 of gestation, producing a frequency of 50% for this tumor type. More potent however, was a system which utilized the partially inbred IIIVO/J strain of rabbit using the same carcinogen and transplacental route of administration. The resultant incidence of nephroblastomas in the progeny was in excess of 90%, and like their counterparts in man, the neoplasms developed rapidly and had a potential for distant metastasis. Each one of these animal models is suitable for the sequential tracing of tumor pathogenesis, and in depth analysis of the biochemical and molecular mechanisms involved in the initiation and formation of different types of renal cancer.

Induction of mesenchymal neoplasms by DMBA implantation in deep lingual submucosa of male albino rats.

45 male albino rats were used to study the effect of deep submucosal implantation of DMBA on lingual carcinogenesis. The results showed development of different mesenchymal neoplasms (fibroma, fibrosarcoma and leiomyosarcoma). So, this method of implantation could not be used as an ideal animal model for production of single known neoplasm as had been investigated by many investigators before.

Additive effect in the induction of kidney tumours in rats treated with dimethylnitrosamine and ethylmethanesulphonate.

Wistar rats were treated with a single dose of 30 mg/kg of DMN or with single doses of 100, 200 or 300 mg/kg of EMS. Tumours of the kidney developed in a few animals receiving EMS and in 33% of the male and 63% of the female rats treated with DMN alone. In the animals receiving DMN and, 8 hours later, a single dose of 100, 200 or 300 mg/kg of EMS, an additive effect was observed in the induction of kidney tumours. This additive effect was more pronounced in female than in male rats. Morphologically, the tumours were of epithelial and mesenchymal type with a preponderance of the former type. The significance of alkylation of the nucleic acids of the kidney observed with these two compounds is discussed in relation to the present findings.

Ultrastructural study of Ni3S2-induced tumors in rats.

An ultrastructural study was conducted on a total of 24 tumors selected from 70 tumors induced by intramuscular injection of Ni3S2 in Fischer and Hooded rats. Well differentiated rhabdomyosarcomas were predominantly composed of strap-like cells containing numerous myofilaments occasionally forming S-line-like structures, whereas poorly differentiated rhabdomyosarcomas were made up mainly of round or oval cells with abundant cytoplasm. Embryonal rhabdomyosarcomas contained cells resembling the mast cells in myxomatous areas as well as spindle-shaped myoblast-like cells. Mesenchymal tumors exhibited morphological characteristics of the lymphoid cells. The tumor cells from leiomyosarcoma-like tumors showed myoblast-like appearance. Tumors diagnosed as fibrosarcoma-like tumors at the light microscopic level demonstrated the ultrastructural characteristics of fibrosarcomas. The possibility of a multicentric origin of the tumor cells in Ni3S2-induced tumors has been discussed.