Transplacental Administration Of Ethylnitrosourea Research Articles

Distinctive expression of midkine in the repair period of rat brain during neurogenesis: immunohistochemical and immunoelectron microscopic observations.

Distinctive expression of midkine (MK) was observed during the repair period of fetal brain neuroepithelium. MK is a heparin-binding growth factor that occurs as a product of a retinoic acid-inducible gene, and has a molecular mass of 13 kDa. MK expression was examined immunohistochemically and by immunoelectron microscopy during a period of repair in developing rat brain at the neurogenesis stage. Injury was induced in rat fetuses by transplacental administration of ethylnitrosourea (ENU) on embryonic Day (E) 16, and histological changes were examined up to 48 hr thereafter (i.e., up to E 18). In normal rat fetuses, MK immunostaining was observed in the cytoplasm and radial and horizontal processes of all cells in the neuroepithelium (NE), subventricular zone (SV), and intermediate zone (IMZ). In ENU-administered brains, cells in the NE, SV, and IMZ were damaged severely, especially 16-24 hr after ENU administration. The remaining neuroepithelial cells, with the exception of those in M-phase and the tips of processes at the ventricular surface, were negative for MK immunohistochemistry 16-24 hr after the administration of ENU. Forty-eight hours after the administration, the cytoplasm and processes of cells in the NE, SV, and IMZ were MK immunopositive. Our previous data reported that the cell cycle of most NE cells is synchronized to the S-phase 16 hr after ENU administration and to the M-phase at 24 hr, and many NE cells were recovered 48 hr after ENU administration. The previous results taken together with the present results indicate that: (1) MK expression does not increase during the repair period of the NE, being different from adults; (2) MK expression is likely to be suppressed at S-phase according to the condition of the NE; and (3) MK expression is not essential for every cell cycle phase of NE cells; but (4) is necessary to maintain the M-phase of NE cells.

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.

Expression of calbindin D-28k and parvalbumin in cerebral cortical dysgenesis induced by administration of ethylnitrosourea to rats at the stage of neurogenesis.

It has been reported that transplacental administration of ethylnitrosourea (ENU), which is cytotoxic immediately after administration, to rat fetuses at the neurogenesis stage induces dysgenesis of the cerebral cortex, characterized by neuronal sparseness and architectural irregularity. In the present study, we examined the topographic distribution of neurons containing 5-bromo-2-deoxyuridine (BrdU), and those containing calbindin D-28k (CaBP) and parvalbumin (PV), most of latter two are considered to be interneurons located in particular layers of the normal cerebral cortex in rats with experimentally induced cerebral cortical dysgenesis. Pregnant Wistar albino rats were given a single transplacental administration of ENU on embryonic day 16, followed 4, 8, 16, 24, 36, or 48 h later by a single intraperitoneal injection of BrdU. The pups were killed 10 weeks after birth. In the normal cerebral cortex, BrdU-immunopositive neurons showed an inside-out pattern according to the time of BrdU injection, whereas in ENU-treated rats the topographic localization of the BrdU-immunopositive neurons was irregular and the inside-out pattern was disrupted. Although the number of CaBP- and PV-immunopositive neurons was lower in ENU-treated animals, no topographic difference was evident between the normal and the dysgenetic cerebral cortices. These findings indicate that the expression of CaBP and PV in the neurons of the rat cerebral cortex is extrinsic, and depends on the position of the neurons rather than on the time of their formation or on genetic control. This suggests the existence of re-regulation of the expression of CaBP and PV in the developing brain, which may be one of the effective mechanisms by which the cerebral cortex can maintain its normal function in spite of cytoarchitectural abnormality.

Process of repair in the neuroepithelium of developing rat brain during neurogenesis: chronological and quantitative observation of DNA-replicating cells

The neuroepithelium (NE) of the cerebrum of the developing brain has been reported to `regenerate' within a certain period after being injured. To clarify the process of repair of the NE during neurogenesis, we chronologically examined the number of cells in the neocortex, and the rate of DNA-replicating cells and the mitotic figure ratio in the NE of rats after injury. The injury was induced by transplacental administration of ethylnitrosourea (ENU), which is cytotoxic immediately after administration, to the pregnant rats on embryonic day 16. The number of living and pyknotic cells in a 220- μm width of the neocortex was evaluated in each of three groups, such as NE, subventricular zone (SVZ)+intermediate zone (IMZ)+subplate (SP), and cortical plate (CP). The 5-bromo-2-deoxyuridine (BrdU)-labeling index and the mitotic index were examined 4, 8, 16, 24, 36, and 48 h after ENU administration. Up to 16 h after ENU administration, the number of living cells per 220 μm width in the NE and SVZ+IMZ+SP decreased, but increased in CP. From 16 to 24 h, the living cell number per 220 μm width in the NE and CP was unchanged, but increased in the SVZ+IMZ+SP. From 24 to 36 h, the living cell number per 220 μm width increased in all the groups, NE, SVZ+IMZ+SP and CP. From 36 to 48 h, the living cell number per 220 μm width in the NE was unchanged, but increased in the SVZ+IMZ+SP and CP. The BrdU-labeling index reached its nadir at 8 h, but markedly increased by 16 h, and then decreased to the control level by 36 h. No mitotic figures were observed at 16 h after administration, but a significant increase in mitotic index was noted at 24 h, and after which it decreased to almost the control value by 36 h. These findings indicate: (i) that temporary arrest of neuroepithelial cell cycle occurs in the G 1-phase from 4 to 8 h after ENU administration, (ii) that the cell cycle synchronizes in the S-phase at 16 h, (iii) that a proportion of neuroepithelial cells of rat fetal neocortex at the neurogenesis stage return to neuroepithelial cell proliferation stage, to repair the NE, and (iv) that regulation of the cell kinetics of neuroepithelial cells depends on the number of cells in a certain width of NE during regeneration.

Immunohistochemical observations on rat radial glia: relationship with the origin of ethylnitrosourea-induced tumors.

Gliomas induced in the rat by transplacental administration of ethylnitrosourea (ENU) are intensely immunoreactive for vimentin and scarcely for glial fibrillary acidic protein (GFAP). Since tumoral transformation takes place during the late fetal and early postnatal period, the sequential expression of the two glial antigens has been investigated in this age period in ENU-treated and control rats. Immunohistochemical and immunoelectron microscopical methods have been employed. Vimentin was widely expressed starting from embryonal day 14 (E 14) in the processes of radial glia; as long as radial glia was present, vimentin decorated it. GFAP was, at earliest, observed at E 20 and expressed by glial cells with a stellate, i.e., mature shape. No GFAP-positive radial process was observed. No difference was found between ENU-treated and control rats. Since ENU is most effective in producing tumors when administered at the 16-17th day of fetal life, vimentin-positive radial glia is a candidate target of ENU. The similarity of intermediate filament pattern between radial glia in the late fetal life and tumors induced by transplacental ENU suggests that radial glia might be the cell of origin.

Glomeruloid blood vessels in ethylnitrosourea-induced rat gliomas. Histological and immunohistochemical studies.

Glomeruloid blood vessels (GBVs), a characteristic histological feature of most human malignant gliomas, were recognized with high incidence in autochthonous rat gliomas induced by transplacental administration of ethylnitrosourea. To evaluate some of the biological properties of these GBVs, we carried out a study using histological methods and immunohistochemical staining for glial fibrillary acidic protein, factor VIII-related antigen (VIII Ag) and bromodeoxyuridine (BrdUrd). Of 22 animals with large, massively growing gliomas in the CNS, GBVs including conglomerate aggregations of small blood vessels with endothelial hyperplasia and strong VIII Ag expression were observed in 13 large gliomas histologically consisting of primitive neuroepithelial neoplasms (PNN; so called ependymoma) and mixed-type gliomas in combination with astrocytoma and PNN or anaplastic astrocytoma. The anaplastic gliomas in our series were devoid of GBVs. These findings indicate that GBV formation takes place in a histological variety of experimental gliomas. Furthermore, the GBVs were frequently associated with the vasculo-mesenchymal stroma in the parent gliomas, suggesting an intimate relationship with the morphogenesis of GBVs. In addition, it was shown that the GBVs had a higher BrdUrd-labelling index than that of other blood vessels in gliomas and also that of neoplastic cells in most parent gliomas, except for anaplastic gliomas. Based on these results, the possible mechanism of GBV morphogenesis is discussed with regard to the roles of macromolecules in the induction and regulation of GBVs.

Phenobarbital lacks promoting activity for neurogenic tumors in F344 rats transplacentally exposed to ethylnitrosourea.

A chronic rodent study in F344 rats was conducted to investigate the promoting ability of phenobarbital (PB) on neurogenic tumors initiated by transplacental administration of ethylnitrosourea (ENU). Pregnant F344 rats were given a single intravenous dose of 3.5 mg ENU/kg or vehicle on the twentieth day of gestation. A total of 192 male offspring were divided into four groups: ENU-PB, ENU-control, PB-control, and control-control. Rats in ENU-PB and PB-control groups received 0.05% PB in their drinking water from four to 78 weeks of age. Nervous system tumors were induced only in animals exposed to ENU. The difference in the incidence of neuroectodermal tumors in rats that were ENU initiated only (13/37; 35%) compared to the incidence in rats that were initiated and given PB (13/57; 23%) was not statistically significant (p greater than 0.05). ENU-control and ENU-PB treatment groups exhibited no differences in tumor multiplicity or tumor latency. These results demonstrate that PB lacks promoting activity for neurogenic tumors in F344 male rats transplacentally exposed to ENU.

Chemotherapy in intracerebrally transplanted tumors induced with transplacental administration of ethylnitrosourea in rats.

Four tumors of the spinal cord were induced with ethylnitrosourea in rats by transplacental administration and transplanted into the brains of animals of the same strain. One of these intracerebrally grafted tumor lines (G-XIII) was followed up over the first 10 passages and treated with CCNU and other alkylating drugs. The results were compared with findings in an earlier established line (G-XII) in passage 12 and 59, which in the first instance was sensitive to CCNU. The CCNU application prolonged survival in treated animals in various treatment schedules in the first 10 intracerebrally grafted generations of the tumor up to 59%. Induction times of tumors became increasingly shorter. The susceptibility of early passages was similar in both lines. Its loss in late passages went together with diffuse growth of the tumor and reticulin fiber production. In addition, glial fibrillary acid protein expression and formation of intermediate filaments in perivascular tumor cells was lost.

The chronology of lesion repair in the developing rat brain: biological significance of the pre-existing extracellular space.

We observed the histological peculiarities of the repair process in a destructive lesion of the developing rat brain during neurogenesis. Degeneration was induced selectively in certain cells of the proliferating phase in the rat fetal neopallium on embryonic day 16 by transplacental administration of ethylnitrosourea. Successive elimination of necrotic cells and the restoration process were observed. The repair process was divided into the following steps: elimination of individually affected cells by phagocytes in the pre-existing extracellular space; successive restoration of the disintegrated area by cells which differentiated from remaining matrix cells. No reactive gliosis, fibrosis, abnormal vascularization or infiltration of granulocytes and lymphocytes was observed at any time. The thinned neopallium on postnatal day 21 revealed only a small number and abnormal distribution of the cortical neurons. It may be assumed that the fetal brain owes its unique repair features to the presence of a vast extracellular space under normal conditions. In this pre-existing extracellular space, every kind of cell seems to exist separately without the intracellular adhesions characteristic of the adult brain. When degeneration occurs in certain cells the phagocytes would be able to eliminate the degenerate cells completely in this space without having to break intercellular adhesions. As a result, after the completion of cell elimination, the injured brain is restored to its original state with no cell reaction, giving the appearance of a small brain with normal-looking histological architecture, save only for the sparseness of cells.

Distribution pattern of DNA template activity in the developing cerebellar cortex in rats and its alteration after transplacental administration of ethylnitrosourea.

The ultracytochemical acridine orange(AO) method has been employed to demonstrate DNA template activity in the developing cerebellar cortex in rats and its alteration after transplacental administration of ethylnitrosourea (ENU). Electron microscopic studies revealed that AO binds to DNA exclusively within the active extended euchromatin portion of the cell nucleus of the proliferating external granular layer. No or only a few AO reaction products were visible in the non-proliferating cell nuclei of the internal granular layer and of Purkinje cells. With increasing cellular differentiation of granule cells in the cerebellar cortex the number of AO positive cells as well as the number of AO reaction products per cell showed a progressive decrease. A single i.p. injection of 50 mg/kg of ENU in rats on day 15 to 21 of gestation resulted in occasional cell damage to the proliferating external granule cells of developing cerebellar cortex at 4 and 7 days after birth. Damaged cells of the external granular layer were found to have no or only a small number of AO reaction products in their nuclei. In further development from 14 to 28 days the cerebellar cortex of offspring from ENU-treated mother showed no abnormality and exhibited no definite alteration in distribution pattern of AO reaction products.

Initiation of focal hyperplastic hepatic lesions by transplacental administration of ethylnitrosourea in rats of F1 generation, and no transmission of the effect to F2 and F3 generations.

Administration of ethylnitrosourea (ENU) to pregnant rats in the late stage of gestation has been known to have a mainly neurocarcinogenic action on the progeny. However, when transplacental administration of ENU was combined in postnatal life with a brief dietary exposure to 2-acetylaminofluorene (2-AAF) and a two-thirds partial hepatectomy, numerous focal hyperplastic lesions of gamma-glutamyltranspeptidase (GGT)-positive hepatocytes emerged in the liver of the progeny. On the other hand, rats of F2 generation born of F1 parents and F3 rats born of F2 parents showed no increase in the incidence of hepatic lesions after treatment with the same dietary regimen. It was suggested that direct interaction between fetal liver cells and the carcinogen is important for occurrence of initiated hepatocytes and that the initiation effect on rat liver by ENU does not persist in the descendants of F2 and F3 generations.

Experimental hematogenic metastases of malignant schwannoma in the rat.

To study development, growth patterns and morphology of metastatic schwannomas in an animal model, adult Lewis rats were injected intravenously with tissue culture cells derived from a malignant schwannoma induced in a female LEW/mai rat by transplacental administration of ethylnitrosourea (ENU). Groups of animals received a single injection of 0.1 x 10(5); 0.5 x 10(5); 1.0 x 10(5); 5.0 x 10(5); 10 x 10(5) or 100 x 10(5) cells via the femoral vein. The injections resulted in the development of metastatic tumors, primarily involving the lung in all animals. Lung metastases were observed in the form of microscopic and macroscopic peripheral subpleural tumors, central peribronchial and perivascular tumors, parenchymal tumors, diffuse intraalveolar infiltrates and intravascular tumor emboli which were growing. Animals receiving high dosages had a greater variety of forms of the metastatic growth in the lungs than those given low dosages. The animals which received the highest dose had the greatest degree of total lung tumor burden. In 36 of the 60 animals injected, metastatic tumors also developed in the pericardium. Thirteen animals developed metastatic tumors in the kidney, heart, ovary, adrenal, intestine or skeletal muscles. As the cell dosages were increased, more organs became involved, but no tumors developed in the brain.

Ultrastructural findings in so-called ependymal rat tumors induced by transplacental administration of ethylnitrosourea (ENU).

Tumors of the central nervous system (CNS) in Sprague-Dawley and Lewis rats were induced transplacentally by a single intraperitoneal (IP) injection of 10–25 mg/kg ethylnitrosourea (ENU) on days 16–20 of gestation. Light-microscopic examination revealed that 22% of these tumors could be diagnosed as ependymomas, anaplastic ependymomas (ependymoblastomas), or mixed tumors revealing both oligodendroglioma and anaplastic ependymoma tissues if the criteria of accepted classifications were followed. Electron-microscopic examination, however, demonstrated that the ependymoma and the anaplastic ependymoma-like tissue in ENU-induced tumors lacked ependymal features, such as basal bodies, cilia, complicated junctional complexes, microvilli, etc. This tissue type was repetitious, always being composed of cells arranged in groups, cords, and rosette-like (pseudorosette) formations. In the pseudorosettes, the cell nuclei were polarized at the periphery and the cytoplasm contained numerous polyribosomes, occasional short microtubules and usually a few small dense-core vesicles. The center of the pseudorosettes showed numerous slender interdigitating processes interconnected by maculae adherens. The tips of these processes showed vesicular degeneration. The cells arranged in groups or cords and perivascular rosettes revealed identical ultrastructure, but they were not polarized. The present findings indicate that the socalled ENU-induced ependymomas and anaplastic ependymomas are not true ependymal tumors, but rather primitive neuroepithelial neoplasms with some features of oligodendroglioma.

Ultrastructure and Actin Distribution in Neoplastic Neurogenic Cells in Culture

The Ultrastructure of 13 neoplastic cell culture lines derived from BD IX‐rat brain was investigated in relation to growth pattern, the degree of aneuploidy, and tumourigenicity. Neoplastic transformation was induced by transplacental administration of ethylnitrosourea (ENU). The neoplastic cell culture lines were either obtained from solid neuroectodermal tumours (V‐lines), or developed in culture after transfer of fetal rat brain cells shortly after exposure to ENU in vivo (BT‐lines). By transmission electron microscopy the following ultrastructural characteristics were found in both types of neoplastic cell lines: varying numbers of microtubules, few or no microfilament bundles and a variable amount of 10 nm (glial) filaments, as well as atypical nuclear structure. High numbers of 10 nm filaments were found in strongly aneuploid lines. By immunofluorescence with anti‐serum against actin, the paucity of microfilament bundles was also visualized. In most cell lines a diffuse cytoplasmic actin fluorescence was found, but sometimes actin‐containing bundles appeared as irregular streaks. For comparison, secondary cells from untreated fetal rat brains were also investigated. They showed parallel actin bundles throughout the cytoplasm, but these disappeared when the cells were induced to »differentiate« to an astrocyte‐like morphology by addition of a protein fraction from adult brains. A diffuse positive reaction over the whole cytoplasm was then seen, much in the same way as in neoplastic cells.

Cross-reactivities of ethylnitrosourea-induced neurogenic rat tumors studied by in vitro assays for cell-mediated immunity

Neurogenic tumors were induced in inbred Wistar rats by the transplacental administration of ethylnitrosourea (ENU). The tumors were transplantable in syngeneic rats and they could easily be grown in vitro. Migration inhibition tests (MIT) were carried out with KCl extracts of several neurogenic tumors, a spontaneous mammary carcinoma and of various normal adult and fetal tissues. Thymocytes and lymph node cells for these tests were obtained from primary tumor hosts, recipients of transplanted tumors and from rats hyperimmunized with Mitomycin C treated tumor cells. An indirect 51 Cr-release test as well as an indirect DNA inhibition test were carried out with culture supernatants derived from mixed cultures of neurogenic tumor cells and sensitized or non-sensitized lymphoid cells. All three assays revealed tumor associated antigens with cross-reactions between various neurogenic tumors. Cross-reactions were also demonstrated by MIT between neurogenic tumors and fetal tissues. A peripheral neurinoma showed cross-reaction with adult brain and a trigeminal nerve neurinoma cross-reacted with the spontaneous mammary carcinoma. None of the neurogenic tumors cross-reacted with adult liver. Thus, ENU induced neurogenic tumors share one or several common antigens which, however, are not tumor specific. The reactivity of lymphoid cells derived from tumor bearing rats was considerably smaller than that of lymphoid cells from hyperimmunized rats. It was also found that the reactivity of lymphoid cells from thymus and lymph nodes was considerably stronger than that of splenocytes.

Chromosome analysis of experimental rat glial tumor(1)--correlation of chromosome changes between glial tumors and fetal brain tissue of rats after a transplacental exposure to ethylnitrosourea.

Chromosomal constitution of 7 rat glial tumors induced by methylnitrosourea (MNU) and ethylnitrosourea (ENU) was investigated. Cells from rat glial tumors showed preferentially complete monosomy for # 3 chromosomes and partial monosomy for # 2 chromosomes. Numerical and morphological changes in # 11 and # 12 chromosomes were also observed. These chromosome aberrations have no relation with histological classification of experimental rat gial tumors. One # 3 chromosome missing in neolplastic glial tumors was constantly observed through a long term culture and had no relation with the appearance of marker chromosome. These chromosome changes were compared with the preferential involvement of # 1 and 2 chromosomes in fetal brain cells of rats shortly after a transplacental administration of ENU.

Light and electron microscopic investigations on a transplantable tumour of the rat, induced by transplacental administration of ENU (ethylnitrosourea).

With a single dose of 80 mg/kg ENU, a tumour originating from the trigeminal nerve was induced transplacentally in the offspring of a Sprague-Dawley rat. The neoplasm diagnosed as a neurinoma, was transplanted over 20 passages to 5 six-month-old Sprague-Dawley rats in each case. During this time, the biological behaviour of the tumour as well as its histological appearance were followed. Ultrastructural observations were made of the 20th passage. The tumour caused multiple metastases in the lung and in the lymph nodes of the neck in 30 animals. Recurrences were formed within a week, metastases of the lung appeared from then on in all cases. During the time of transplantation, the neurinoma dedifferentiated histologically so markedly that it was no longer possible to classify the neoplasm as a neurogenous tomour after 20 passages. Ultrastructurally, poorly differentiated Schwann cells could nevertheless be recognized.

Biological characteristics of peripheral nerve tumors induced with ethylnitrosourea.

Nervous system tumors were induced in the offspring of BD-IX (inbred) and CFE (non-inbred) rats by transplacental administration of ethylnitrosourea. Tumors of the peripheral nervous system (PNS) were obtained in approximately 80% of all animals, and tumors of the central nervous system (CNS) in 16%. Although several peripheral nerves were sites of tumor formation, the trigeminal nerve was the most frequent nerve affected. In male CFE rats, 70% of all PNS tumors affected the trigeminal nerve. The incidence of trigeminal nerve tumors in female BD-IX rats was only 33% of PNS tumors. Light and electron microscopy showed that the PNS tumors are made-up of Schwann cells, and behave like malignant tumors. The tumors began as a proliferation of recognizable Schwann cells, later they lost their basement lamina, destroyed the nerve tissue and invaded neighboring structures, but did not metastasize. Like their human counterparts, they showed “dense” and “loose” areas. In both of these, Schwann cells were recognized as the tumor cells. Trigeminal nerve tumors were transplanted fromin vitro cultured cells or directly from transplanted fresh tumor fragments to isologous rats (BD-IX). Subcutaneous. intraperitoneal and intracerebral transplants were done. The morphology and behavior of the transplanted tumors were similar to the original malignant neurinomas.