[Tumour induction by ethylnitrosourea in the central nervous system]. / Inducción de tumores en el sistema nervioso central mediante etilnitrosourea.

INTRODUCTION: Experimental central nervous system (CNS) tumours have been proposed as a useful model for the study of oncogenesis, epiphenomena related to cancer and for the design of new therapeutic strategies. DEVELOPMENT: The administration of chemical substances is one of the most commonly-used methods to induce CNS neoplasms. N-ethyl-N-nitrosourea (ENU) belongs to the nitrosourea family, a wide group of alkylating agents that are able to induce brain tumours in litters after transplacentary administration at the 15th day of pregnancy. This nitrogenous urea compound has a high mutation inducibility affecting the expression of oncogenes such as p53, neu/erbB-2 and Ras. Prenatal exposition of Sprague Dawley rats to ENU induces intra-axial tumours of glial lineage and extra-axial malignant schwannomas. Although the precise mechanism of tumour induction is unclear, it is known to affect cell differentiation of primitive neuroepithelium from the subventricular plate generating oligodendrogliomas, astrocytomas, mixed gliomas or ependimomas. CONCLUSION: The transplacentary administration of ENU induces the development of gliomas and schwannomas that are similar to those found in humans. Animal models are necessary and useful for further studies to get an early diagnosis and to establish correct therapeutic indications.

Inducción de tumores en el sistema nervioso central mediante etilnitrosourea / Tumour induction by ethylnitrosourea in the central nervous system

Introducción. La utilización de modelos experimentalesin vivo de tumores en el sistema nerviosos central (SNC) ha traídoimportantes avances a la neurooncología. Estos modelos animaleshan permitido el estudio de procesos de oncogénesis, de sus epifenómenosy del diseño de nuevas estrategias terapéuticas. Desarrollo.Existen varios métodos de inducción de neoplasias en el SNC,de los cuales la administración de sustancias químicas es una delas modalidades más utilizadas. La N-etil-N-nitrosourea (ENU) esun agente alquilante capaz de inducir tumores cerebrales en la descendenciade ratas gestantes tras su administración transplacentaria.Se tata de un compuesto nitrogenado de la urea con alto podermutagénico que afecta a la expresión de ciertos oncogenes comop53, neu/erbB-2 y Ras. Mediante la exposición prenatal a ratasSprague Dawley del carcinógeno ENU se inducen tumores intraaxialesde estirpe glial y tumores extraaxiales como los schwannomasmalignos. Aunque se desconoce el mecanismo preciso de inducciónde los tumores gliales, se sabe que afecta a la diferenciaciónde las células neuroepiteliales primitivas de la placa subventricular,lo que genera oligodendrogliomas, astrocitomas, gliomasmixtos o ependimomas. Conclusión. La administración transplacentariade ENU permite obtener gliomas y schwannomas malignossimilares a los encontrados en los humanos. Esto puede ayudaral estudio en profundidad de dichos tumores para llegar a realizarun diagnóstico precoz y asentar unas indicaciones terapéuticasprecisas

Introduction. Experimental central nervous system (CNS) tumours have been proposed as a useful model for thestudy of oncogenesis, epiphenomena related to cancer and for the design of new therapeutic strategies. Development. Theadministration of chemical substances is one of the most commonly-used methods to induce CNS neoplasms. N-ethyl-Nnitrosourea(ENU) belongs to the nitrosourea family, a wide group of alkylating agents that are able to induce brain tumoursin litters after transplacentary administration at the 15th day of pregnancy. This nitrogenous urea compound has a highmutation inducibility affecting the expression of oncogenes such as p53, neu/erbB-2 and Ras. Prenatal exposition of SpragueDawley rats to ENU induces intra-axial tumours of glial lineage and extra-axial malignant schwannomas. Although theprecise mechanism of tumour induction is unclear, it is known to affect cell differentiation of primitive neuroepithelium fromthe subventricular plate generating oligodendrogliomas, astrocytomas, mixed gliomas or ependimomas. Conclusion. The transplacentaryadministration of ENU induces the development of gliomas and schwannomas that are similar to those found inhumans. Animal models are necessary and useful for further studies to get an early diagnosis and to establish correcttherapeutic indications

The antimalarials quinacrine and chloroquine potentiate the transplacental carcinogenic effect of ethylnitrosourea on ependymal cells.

Quinacrine and chloroquine, two widely used antimalarials, bind strongly to deoxyribonucleic acid, thus preventing mutagenesis. We studied a possible chemoprotective effect of these substances on carcinogenesis of the nervous system induced in Wistar rats by transplacental administration of ethylnitrosourea. One experimental group consisted of rats born from mothers treated with quinacrine prior to prenatal exposure to ethylnitrosourea; a second group consisted of rats chronically treated with chloroquine after prenatal exposure to ethylnitrosourea. When compared with controls, no significant differences were observed in tumor incidence. However, early tumor growth was observed in both rats treated with quinacrine (P < 0.0004) and rats treated with chloroquine (P < 0.02). These differences were due mostly to rapid development of ependymomas of the spinal cord. Our results suggest that quinacrine and chloroquine do not prevent the structural alterations induced in DNA by ethylnitrosourea, which lead, in the long term, to a high incidence of neoplasms in the nervous system. Moreover, the antimalarials studied seem to promote the carcinogenic effects of ethylnitrosourea on ependymal cells.

Ganglioside biosynthetic gene expression in experimental mouse brain tumors.

The genes for cytidine monophospho-N-acetylneuraminic acid hydroxylase (NeuAc-H) and beta-1,4-N-acetylgalactosaminyl transferase (GalNAc-T) were examined using reverse transcription-PCR in two experimental mouse brain tumors, EPEN and CT-2A. NeuAc-H is required for the synthesis of gangliosides containing N-glycolylneuraminic acid, whereas GalNAc-T is required for the synthesis of ganglioside GM2. The genes were analyzed in solid tumors grown in vivo and in tumor cells grown in vitro. NeuGc-containing gangliosides are abundant in cells of the mouse immune system, including macrophages, but are undetectable in normal mouse brain. GM2 is expressed in both neural and nonneural mouse cells and tissues. The EPEN tumor cells synthesize only ganglioside GM3, whereas the CT-2A tumor cells synthesize GM3, GM2, GM1, and GD1a. NeuAc-H gene expression was detected in both solid tumors grown in vivo but was undetectable in either tumor cell line. The presence or absence of NeuAc-H gene expression in the tumor tissues and cells correlates with the presence or absence, respectively, of NeuGc-containing gangliosides. Differences in GalNAc-T gene expression between the solid tumors and the cultured tumor cells correlate with the expression of ganglioside GM2. The findings suggest that the differences in ganglioside biosynthetic gene expression between brain tumors grown in vivo and in vitro are associated with the presence or absence, respectively, of tumor-infiltrating host cells.

Synaptophysin expression in "ependymal tumors" induced by ethyl-nitrosourea in rats.

Synaptophysin expression was studied in seven "ependymomas" induced by transplacental administration of ethyl-nitrosourea in rats. In all the cases, strong positivity for synaptophysin was found on tumor cells. This finding supports previous studies suggesting that ENU-induced brain tumors considered to be ependymal neoplasms, are, in fact, primitive neuroectodermal tumors.

Effects of catecholamine and serotonin in central nervous system in newborn mice with special reference to neural crest cells; presumptive evidence of neural crest origin.

Catecholamine and serotonin injected i.p. to new-born mice caused gliomas, ependymomas and choroid plexus papilloma together with multiple APUDomas within 48 hours after injection. We consider that an error in neural crest DNA may be caused through an adenylate-cyclic AMP system and Ca++ during replication and/or transcription. Multiple brain tumors caused by catecholamine and serotonin give the presumptive evidence that stem cells of neuroglia may be of neural crest origin.

Immunohistochemical characterization of rat central and peripheral nerve tumors induced by ethylnitrosourea.

Ethylnitrosourea-induced central and peripheral nerve tumors in Sprague-Dawley rats were tested for GFAP (Glial Fibrillary Acidic Protein), S-100 protein, NSE (Neuron Specific Enolase) and Anti-Leu 7 (HNK-1) immunoreactivity utilizing the ABC method (avidin-biotin-complex) for GFAP, S-100 protein and NSE, and the PAP method (peroxidase-antiperoxidase) for Anti-Leu 7. Peripheral nerve neurinomas were consistently positive for S-100 protein and consistently negative for GFAP and Anti-Leu 7. Neurinomas would occasionally exhibit positive staining for NSE (2 of 55 tumors). The staining intensity for S-100 protein varied from strongly positive in differentiated neurinomas to weakly positive in anaplastic tumors. Neoplastic and reactive astrocytes exhibited positive staining for both S-100 protein and GFAP. Variation in the GFAP staining intensity of glial tumors correlated with the degree of differentiation as anaplastic tumors did not stain with the same intensity as their more differentiated counterparts. Oligodendrogliomas exhibited occasional immunoreactivity to S-100 protein (3 of 36 tumors). NSE reactivity in oligodendrogliomas was rarely observed (1 tumor in 36) and immunoreactivity against GFAP or Anti-Leu 7 was consistently absent. Anti-Leu 7 and NSE proved to be of little value in the classification of ENU-induced neural tumors.

Promoting effect of 12-O-tetradecanoylphorbol-13-acetate on neurogenic microtumors initiated by transplacental exposure to N-ethyl-N-nitrosourea.

The promoting effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the occurrence of neurogenic microtumors in SD-JCL rats initiated transplacentally with N-ethyl-N-nitrosourea (ENU) was investigated. The treatment with TPA induced earlier occurrence of microtumors than as compared with the initiation alone. Thus, TPA has tumor-promoting activity on the formation of neurogenic microtumors in rats prenatally exposed to ENU.

Ependymoblastoma associated with prenatal exposure to diphenylhydantoin and methylphenobarbitone.

Ependymoblastoma developed in a 28-month-old girl whose epileptic mother took diphenylhydantoin and methylphenobarbitone throughout pregnancy. The child was also shown to be a genetic carrier for ornithine transcarbamylase deficiency, an x-linked inborn error of urea cycle metabolism. The possibility of transplacental carcinogenesis should be considered, as other juvenile embryonic tumors such as neuroblastoma, melanotic neuroectodermal tumor, and mesenchymoma have been reported in offspring after diphenylhydantoin use by the mother during pregnancy.

The murine ependymoblastoma: growth pattern and survival in C57Bl/6J mice.

The murine ependymoblastoma is a transplantable tumor of cerebral origin. The growth pattern and survival times of the murine ependymoblastoma implanted peripherally and intracranially in non nude C57Bl/6J mice have been found to be predictable and consistent when examined by means of Tumor Cell Dose Assessment (end point solution), Tumor Growth and Survival Assessment. The results suggest that a greater tumor cell dose is required to generate peripheral tumor take than brain tumor take. This difference may result from a greater immunologic response to tumor implanted peripherally than into the immunologically privileged brain.

[Interest of glioma 26 in the selected chemotherapy of intracranial tumors (author's transl)]. / Intérêt du gliome 26 pour la sélection des chimothérapies des tumeurs intracraniennes.

Glioma 26 (G26) is a chemically induced ependymoblastoma of mouse. It was used through sub-cutaneous and/or intracranial injection in 7 experiences which showed that this experimental model was able to select drugs acting on intracranial tumors : nitrosourea (CCNU), VM 26, procarbazine (PCB). Combination chemotherapy (VM 26-CCNU ; VM 26 CCNU-PCB) was more active than monochemotherapy in 2 out of 3 experiences. It seems that the variability of the results is at least partly due to the important tumoral necrosis in tumors treated by combination chemotherapy and to technical problems. The latters can be avoided by the mechanical dispersion of tumor cells and by their injection through a stereotaxic frame. Selection of drugs is easier and cheaper to perform with G 26 than with other in vivo and in vitro experimental models. The main problem for all models remains their lack of specificity.

The immunocytochemical localization of GFA protein in experimental murine CNS tumors.

Immunocytochemical localization of GFA protein in formalin-fixed, paraffin-embedded tissue sections by the peroxidase-antiperoxidase method of Sternberger was used to study experimental murine CNS tumors. Transplacental tumor induction in rats by ethylnitrosourea produced oligodendrogliomas and mixed gliomas in the cerebrum and spinal cord, and malignant Schwannomas of the trigeminal nerve. A methylcholanthrene-induced mouse "ependymoblastoma" inoculated intracerebrally in normal and in toxoplasma-infected mice was also studied. A positive reaction of GFA protein antibody was seen in the astrocytic portion of the mixed gliomas; the oligodendrogliomas, the malignant Schwannomas and the mouse "ependymoblastoma" were negative. Staining for GFA protein delineated the astrocytic reaction of neural tissue adjacent to the tumors. The reaction was markedly intensified in the brains of mice infected with toxoplasma. Additionally, ependymal cells near the tumors stained positively for GFA protein; normal ependyma at a distance from tumor remained negative. The technique, which combines a high degree of specificity with great sensitivity and is readily adaptable to routinely processed tissue, should prove a valuable tool in experimental oncology of the central nervous system.

Methylcholanthrene induced murine primitive neuroectodermal tumor: ultrastructure and nuclear RNA polymerase activity.

The histology, ultrastructure, and nuclear RNA polymerase activity are described in a murine primitive neuroectodermal tumor derived by serial transplantation from a tumor originally induced with methylcholanthrene and classified as an ependymoblastoma. The light microscope and ultrastructural studies show that this tumor does not contain the distinguishing morphological features of differentiated ependymal cells which are also commonly seen in human ependymomas. One outstanding feature is the size and number of the nucleoli. The mean number of nucleoli/nucleus is 4 which is two to four times that of the normal neuroglial cell. The nucleolar diameter is about twice that found in normal neuroglial cells. The nucleolar diameter is about twice that found in normal neuroglial cells. The nuclear RNA synthesizing activity is the highest of the chemically induced animal tumors we have studied. The alpha amanitin inhibition is the lowest seen in any of these tumors which suggests that RNA polymerases inhibited by alpha amanitin contribute less to the total nuclear RNA synthesis. Adriamycin significantly inhibits the nuclear RNA polymerase activity of this tumor.

Studies on experimental malignant nerve sheath tumors maintained in tissue and organ culture systems. III. Melanin pigment and melanogenesis in experimental neurogenic tumors: a reappraisal of the histogenesis of pigmented nerve sheath tumors.

Four melanin pigment-containing intracranial tumors were found in three Long-Evans rats in the course of experimental oncogenesis by transplacental ethylnitrosourea (ENU). One of them was a leptomeningeal melanoma. Aside from the presence of scattered melanin-pigmented cells, the other three had the typical histological features of ENU-induced malignant nerve sheath tumors. Two of the three tumors were studied by electron microscopy and in tissue and organ culture systems. One of them demonstrated progressive melanogenesis in vitro; the other failed to produce more melanin and showed increasing differentiation, with a Schwannoma-like pattern by light microscopy. Melanosomes and premelanosomes were identified in both tumors by electron microscopy; the other fine structural features were those of malignant Schwannomas. These observations are relevant to the controversy on the histogenesis of pigmented nerve sheath tumors occasionally encountered in man and on the relationship of these tumors to pigmented nevi. The findings in the present study support the view of Masson that neoplastic nerve sheath cells are capable of melanogenesis.

Oncogenic response of rats with x-ray-induced microcephaly to transplacental ethylnitrosourea.

The relation of congenital malformations to tumor development was examined. Pregnant Sprague-Dawley rats were given 200 rads of X-rays on the 15th or 16th day of gestation and injections of 10 mg ethylnitrosourea (ENU)/kg 1-4 days later, or they were irradiated or injected only. Surviving weanlings that had been irradiated had micrencephaly and other malformations. Offspring exposed to ENU only had no external deformities. By 15 months of age 16.7% of the offspring exposed to X-rays and ENU prenatally had developed neurogenic tumors, whereas 62.2% of those exposed to ENU alone had developed tumors. Those only irradiated had no tumors. Both of the former groups developed oligodendrogliomas, mixed gliomas, ependymomas, and schwannomas, but the first manifestations of tumors occurred later in the group receiving the combined treatment. This delay persisted furing the subsequent period of the study.

Transplacental induction of neurogenic tumors in BD IX rats by intragastric administration of ethylnitrosourea precursors.

The concurrent daily intragastric administration of ethylurea at two dose levels (50 mg/kg and 100 mg/kg bodyweight) together with one dose level of sodium nitrite (50 mg/kg bodyweight) by a stomach tube to pregnant BD IX rats from day 15 to day 22 of gestation resulted in the induction of neurogenic tumors in all offspring. Since both ENU-precursors alone do not produce neurogenic tumors, these results are evidence of ENU formation from its precursors under the influence of gastric juice. Differences in the survival time and the incidence of tumors at both dose levels were not significant. The amount of ethylnitrosourea synthesized in the animals was very close at both dose levels, and was dependent on the amount of sodium nitrite applied. The experimental results are consistent with the conclusion, that the rat fetuses had been exposed to a total amount of about 60 mg/kg ethylnitrosourea. Neurogenic tumors dominated with 98% incidence over the non-neurogenic. The incidence of neurogenic tumors per rat was high (6.0 for Group I and 6.7 for Group II). Neurogenic tumors were equally distributed among the central and peripheral nervous systems. The neurogenic tumors induced with the precursors of ethylnitrosourea were morphologically similar in all aspects to those induced with the carcinogen itself and could be classified as oligodendroglioma, astrocytoma, mixed glioma, anaplastic glioma, glioependymoma, ependymoma, and neurinoma. Three unusual tumors were found: one early anaplastic "septum ependymoma" in the dorsal column of the spinal cord, and two special mixed tumors of the cranial nerves, i.e. a neurinoma with portions of an oligodendroglioma and a neurinoma with parts of an invasive ependymoma.

In vitro growth of neoplastic rat glial cells.

An ependymoma was induced in the brain of a BD-IX rat by repeated doses of MNU. In vitro grown cells of the tumor were fibroblasts and stellate cells considered to be glial. Reimplantation of these cells into the brain of another BD-IX rat resulted a tumor growing in the meninges. This tumor was then repeatedly transplanted over several passages. Its gross morphology resembled that of a sarcoma; however, glial in cells could be demonstrated with silver methods. A cell line derived from the first transplanted tumor group proved to contain S-100 protein in traces in the first passages in vitro. Explanted cells of the primary ependymoma were cloned. Two clones were established, RGL 1 and RGL 2. The cells of both clones resembled morphologically the stellate elements of the primary explant. RGL 1 contained S-100 protein. It was cloned again in the 16th subpassage. Four clones were established. Only 1 contained S-100 protein in traces. The clone RGL 1 had a subdiploid karyotype with a modal number of 39 and a large submetacentric marker. It was subcloned in the 25th passage with two clones, both having a karyotype of 41 chromosomes.

Histopathological studies of the nervous system tumors in rats induced by N-nitroso-methyl-urea.

Histopathological examinations were made on tumors of the nervous system induced in rats of Donryu strain by weekly intravenous injections with N-nitroso-methyl-urea (NMU) or by a single administration of NMU through the mothers. A total of 176 neural and nonneural neoplasms were produced in this study. It was suggested that the fetal nervous system of Donryu rats was also highly susceptible to the oncogenic effects of NMU. Of these tumors produced, those of the peripheral nervous system amounted to 121, comprising 68.7% of the total number of the neoplasm. Microscopically, most of the nerve tumors showed the histology corresponding to that of human neurinomas. Many tumors, however, disclosed more or less anaplastic cytological appearance. Fifteen gliomas were produced in the brain and spinal cord. Microscopically, they were classified into mixed glioma, oligondendroglioma and anaplastic astrocytoma. The commonest brain tumors produced in rats from intravenously treated group were periventricular mixed gliomas, while gliomas in rats from transplacentably treated group showed an isomorphic histology with a close resemblance to that of oligodendroglioma.

Increased survival of mouse ependymoblastoma hosts previously treated with dehistonized tumor chromatin.

Half of the mice treated with dehistonized chromatin (protein-DNA complex) from a transplantable mouse ependymoblastoma before subcutaneous challenge with the syngeneic tumor survived twice as long as untreated animals.