Differentiation of choroid plexus ependymal cells into astrocytes after grafting into the pre-lesioned spinal cord in mice.

Choroid plexus epithelial cells represent a continuation of, and have the same origin as, ventricular ependymal cells, and are regarded as modified ependymal cells. To extend previous studies of the use of choroid plexus ependymal cell (CPEC) grafting for nerve regeneration in the spinal cord, we investigated the capacity of cultured choroid plexus ependymal cells to differentiate into other types of glial cells in the spinal cord tissue. The choroid plexuses were excised from the fourth ventricle of green fluorescent protein (GFP)-transgenic mice and the cells were dissociated and cultured for 4-6 weeks. CPECs were harvested from the monolayer cultures and injected into the pre-lesioned spinal cords of wild-type mice of the same strain using a Hamilton syringe. One week after injection, some GFP-positive transplanted cells became immunohistochemically positive for glial fibrillary acidic protein (GFAP) but negative for neurofilament and myelin basic protein. All the GFAP-positive transplanted cells were negative for vimentin. Two weeks after grafting, immunoelectron microscopy showed that the GFP-positive transplanted cells that had gained GFAP immunoreactivity contained numerous bundles of intermediate filaments, a morphological characteristic similar to that of astrocytes, and were in close contact with adjacent host tissue. These results indicate that, when grafted into the spinal cord, at least some cultured choroid plexus ependymal cells have the capacity to differentiate into astrocytes.

Grafting of choroid plexus ependymal cells promotes the growth of regenerating axons in the dorsal funiculus of rat spinal cord: a preliminary report.

Nerve regeneration in the central nervous system has been studied by grafting various tissues and cells. In the present study, we demonstrated that choroid plexus ependymal cells can promote nerve regeneration when grafted into spinal cord lesions. The choroid plexus was excised from the fourth ventricle of adult rats (Wistar), minced into small fragments, and grafted into the dorsal funiculus at the C2 level in adult rat spinal cord from the same strain. Electron microscopy and fluorescence histochemistry showed that ependymal cells of the grafted choroid plexus intimately interacted with growing axons, serving to support the massive growth of regenerating axons. CGRP-positive fibers closely interacted with grafted ependymal cells. HRP injection at the sciatic nerve showed that numerous HRP-labeled regenerating fibers from the fasciculus gracilis extended into the graft 7 days after grafting. This regenerating axons from the fasciculus gracilis was maintained for at least 10 months, with some axons elongating rostrally into the dorsal funiculus. Evoked potentials of long duration were recorded at a level ca. 5 mm rostral to the lesion in the rats 8 to 10 months after grafting. These findings indicate that choroid plexus ependymal cells have the ability to facilitate axonal growth in vivo, suggesting that they may be a promising candidate as graft for the promotion of nerve regeneration in the spinal cord.

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro.

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1-10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against beta-tubulin III and S-100 beta, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 +/- 14, 395 +/- 15 and 565 +/- 12 microM on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS.MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenesis Study Group.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS.MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

Effects of tonic neck reflex on optokinetic nystagmus in rabbits.

In this study, rabbits with black-pigmented eyes were suspended in air with none of their limbs touching the ground, and surrounded by a full-field optokinetic drum. The drum was rotated counterclockwise at 60 degrees/s. Horizontal eye movement was recorded by electronystagmograph with the animals in three postures: 1) initial posture, head and trunk aligned along the same body axis, 2) trunk rotated clockwise by 60 degrees with head stationary, and 3) trunk rotated counterclockwise by 60 degrees with head stationary. The recordings were repeated after cervical nerve roots were sectioned unilaterally. The speed of the slow phase of nystagmus was significantly faster in the second than in the initial posture, but significantly slower in the third than in the initial posture. These effects of trunk rotation on the speed of the slow phase were not seen when the side of cervical nerve section was extended. The results indicate that smooth following eye movement is influenced by the neck reflex. It is accelerated when the head turns in the same direction as the visual field, whereas it is inhibited when the head turns in the opposite direction. Proprioceptors of the extended neck appear to account for the effects observed.

Acrylamide; induction of DNA damage, chromosomal aberrations and cell transformation without gene mutations.

The genotoxic potential of acrylamide monomer (AA), a compound familiar as a raw material of polyacrylamide electrophoresis gel, was extensively investigated in vitro. The results were clear cut: AA did not induce any gene mutations in Salmonella/microsome test systems (TA98, TA100, TA1535, TA1537), Escherichia coli/microsome assay (WP2 uvrA-) up to a dose of 50 mg AA/plate, or in HPRT-locus in Chinese hamster V79H3 cells (AA, 1-7 mM, 24 h treatment). On the other hand, AA showed a strong positive response: (a) in a Bacillus subtilis spore-rec assay (DNA damage) at 10-50 mg/disc, (b) to a chromosomal structural change test (AA, 2-5 mM, 24 h treatment), (c) to a polyploidy test (AA, 1-5 mM, 24 h treatment) in Chinese hamster V79H3 cells, (d) to a cell transformation assay in mouse BALB/c3T3 cells (AA, 1-2 mM, 72 h treatment). Sister chromatid exchange was also weakly but significantly induced by AA (AA, 1-2.5 mM, 24 h treatment) in Chinese hamster V79H3 cells. Carcinogenic potential of AA was reported in mice and rats several years ago. AA thus seems to be a typical clastogenic rodent carcinogen without any gene mutation potential. Furthermore, this experiment showed for the first time positive response of AA to a microbial test system (B. subtilis spore-rec assay).

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.

Establishment of lung fibroblastic cell lines from a non-human primate Tupaia belangeri and their use in a forward gene mutation assay at the hypoxanthine-guanine phosphoribosyl transferase locus.

The cells obtained from a lung of a new-born male Tupaia belangeri were maintained in mass culture for greater than 400 days. After 55 population doubling levels (100 days in culture), three cell lines were separately established; these lines showed constant growth properties. One line, designated as T-23, was used for a mutation assay. The T-23 cells showed an absolute plating efficiency of 30-50%, and a population doubling time of 18-19 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells had a modal chromosome number of 62 (pseudodiploid) with the loss of a chromosome and the gain of an unidentified one. T-23 cells, like human cells, were much more susceptible to ouabain than mouse cells but relatively less susceptible to 8-azaguanine, while, unlike human cells, they were less sensitive to 6-thioguanine (6TG). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was less, but 4-nitroquinoline-1-oxide (4NQO) was more toxic to T-23 cells than to human or mouse cells. Benzo[a]pyrene-induced toxicity was almost comparable among the cell types. For the mutation assay, we chose 6TG-resistance (100 microM) as a marker. The optimal expression time (8-13 days) and cell density at selection to eliminate metabolic cooperation (2 x 10(4) cells/60-mm dish) were determined. Some of the cells selected with 6TG showed less than 0.4% of the total incorporation of [14C]hypoxanthine into wild-type cells, suggesting the mutants under selection were affected at the hypoxanthine-guanine phosphoribosyl transferase locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of 6-thioguanine-resistant mutation and sister chromatid exchanges in Chinese hamster V79 cells with forty chemical and physical agents.

The induction of sister chromatid exchanges (SCE) and mutation at the hypoxanthine-guanine phosphoribosyl transferase locus and toxicities of 40 different chemical and physical agents were examined on Chinese hamster V79 cells. These agents included mono-, di-, tri-, and polyfunctional alkylating agents, intercalators, gamma-rays, and UV light irradiation. Mutation was measured as resistance to 6-thioguanine and toxicity as loss of cell-plating efficiency. SCE were examined 29 hr after treatment. With the agents examined, a highly positive correlation (r = 0.89) existed between SCE-inducing and mutagenic potencies, when expressed as increase in the number per a unit dose over the control values. But the great difference of the ratios of mutagenic potencies versus SCE-inducing potencies among agents was observed, the maximal difference in the ratios being about 200-fold. The agents that showed the higher values of the ratio (agents producing more mutations than SCE) were bleomycin, cobalt-60 gamma-rays, all ethylating agents (N-ethyl-N-nitrosourea, N-ethyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and diethylsulfate), N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, isopropyl methanesulfonate, intercalating acridine compounds (2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]-acridine X 2HCl and 2-methoxy-6-chloro-9-[3-(chloroethyl)-aminopropylamino]acridine 2HCl) and UV light at 254 nm. The agents that showed the lower values (agents producing more SCE than mutations) were platinum compounds (cis-diamminedichloro-platinum and trans-diamminedichloroplatinum), epoxides (epichlorohydrin, styrene oxide, and diepoxybutane) and aziridines (mitomycin C, decarbamoyl mitomycin C, tris(1-aziridinyl)phosphine sulfide, triethylenemelamine, and carboquone). The agents that showed the intermediate values included all methylating agents (N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and dimethyl sulfate), N-(2-hydroxyethyl)ethyleneimine, beta-propiolactone, treatment of 8-methoxypsoralen plus near-UV light irradiation at 352 nm, 4-nitroquinoline-1-oxide, quinacrine mustard, sodium sorbate, cigarette tar, and diesel tar. For most agents that induced SCE, the toxicity dependency of induced SCE was rather biphasic; increase in SCE was steep at low to moderate toxicity and less at moderate to high toxicity. At equitoxic doses, the agents showed great difference in induction of SCE.

Induction of 8-azaguanine-resistant mutation and neoplastic transformation of hamster embryonic cells by coadministration of sodium nitrite and aminopyrine.

Hamster embryos in utero on the 11th or 12th day of gestation were treated simultaneously with aminopyrine (Ap) and sodium nitrite (NaNO2) by oral administration of the compounds to the mothers by stomach tube. For measurement of induction of 8 AG-resistant mutations, the embryonic cells from treated and control mothers were cultured in MEM plus 10% FBS for 72 h and then selected in medium containing 10 or 20 microgram/ml of 8 AG. The number of 8 AG-resistant colonies was markedly increased after co-administration of Ap and NaNO2, and slight induction of mutations was also observed in cells from mothers given NaNO2 alone. This treatment also caused morphological or malignant transformation of cultured cells. About 5- to 6-fold increase in the number of transformed colonies was observed in cells from mothers given Ap plus NaNO2. Cells from the transformed colonies produced tumors when implanted into the cheek pouches of young golden hamsters. These tumors were diagnosed as pleomorphic fibrosarcomas. Similar results were obtained with cells from embryos treated transplacentally with NDMA as positive controls. A single transplacental oral application of Ap at 200 mg/kg or of NaNO2 had only slight biological actions to the cultured embryonic cells. NDMA was produced in the stomach of animals treated simultaneously with Ap and NaNO2. A small amount of NDMA was also detected in the stomach after a single dose of NaNO2.

Transplacental mutagenesis of products formed in the stomach of golden hamsters given sodium nitrite and morpholine.

Hamster embryos were exposed in utero to the action of sodium nitrite (NaNO2) and morpholine (Mo) administered simultaneously by stomach tube to the mothers on the 11th or 12th day of pregnancy. Embryo cells were examined for chromosomal aberrations, micronuclear formation, morphological or malignant transformation and drug resistance mutations. For detection of induced mutations, the embryo cells were cultured in normal medium for 72 h and then transferred to medium containing 10 or 20 micrograms/ml of 8-azaguanine (8AG) or 1m7 ouabain (Oua). The number of 8AG-, Ouaresistant colonies was markedly increased after administration of NaNO2 and Mo. The embryonic fibroblasts also showed a markedly increased frequency of micronucleation and a slight increase in chromosome aberrations. This treatment also caused morphological or malignant transformation of fetal cells. After cultivation in vitro, cells from some transformed colonies produced tumors when inoculated into the cheek pouch of young golden hamsters. Orally administered N-nitroso-morpholine (N-Mo), as a positive control, had the same transplacental biological actions on embryonic fibroblasts. However, transplacentally Mo alone was ineffective. A single administration of 500 mg/kg NaNO2 had only slight biological effects. N-Mo was produced in the stomachs of animals treated simultaneously with NaNO2 and Mo. A small amount of a nitrosamine, N-nitrosodimethylamine (DMN), was detected in the stomach after a single dose of NaNO2.

Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster.

Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 microgram/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose-dependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNO2 did not induce any biological change in cultured embryonic fibroblasts.

Chromosome breakage and neoplastic transformation of Syrian golden hamster embryonic cells in tissue culture by transplacental application of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2).

Hamster embryos were treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) in vivo (in the mother) by transplacental application. The fetuses were isolated 24 h after administration of the AF-2 and cultured. Within the first 24h of primary culture, some parts of the cells were treated with colcemide for 3 h so that mitotic cells could be observed in the first cell cycle in vitro. Cultured embryonic fibroblasts in metaphase plates showed a marked dose-dependence in chromosomal aberrations. Transplacental application of AF-2 also caused slightly dose-dependent morphological transformation. When some transformed colonies were cloned and transferred to the hamster cheek pouch, these cells produced tumors in the host animals. This new in vivo--in vitro combination assay system is considered to be useful for detection of environmental potential carcinogens.

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant Syrian hamsters.

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.

Neoplastic transformation induced by furylfuramide and nitromethylfuran of embryonic hamster cells in tissue culture.

Secondary cultures of Syrian hamster embryonic fibroblasts were tested for transformation and neoplastic properties after exposure in vitro to furylfuramine (AF-2) and other nitrofurans. Typical morphological transformation was seen in five of six cultures between 30 and 186 days following treatment with 5-10X10(6) M AF-2 for 24 h. Transformation was seen in only one of four cultures 145 days after treatment for 6 h with AF-2. Treatment with 5-10X10(6) M NMF (5-nitro-2-methylfuran) for 24 h also induced transformation after 50 and 118 days in two cultures. In contrast, untreated cultures and cultures treated with 5-10X10(-6) M NFT [4-(5-nitro-2-furyl)thiazole] for 24 h were not transformed within 200 days. Three of the six lines transformed by AF-2 and both lines transformed by NMF also became tumorigenic 7-24 days after morphologic transformation. The other three transformed lines produced nodules which regressed within a few weeks of transplantation. Untreated and treated non-transformed lines did not produce tumors during an observation period of 6 months. The tumors were classified as fibrosarcomas. The ability to form colonies in soft agar was acquired by only one tumorigenic line.

Chromosomal aberration, mutation and morphological transformation of Syrian hamster embryonic cells after exposure to methylnitrosocyanamide.

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 X 10(-6) M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC or MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.

Genetic variation of pancreatic esterase isozyme in Japanese quail.

A genetic variation was found in pancreatic esterases of Japanese quail which appeared to be arylesterase. It was found on the cathode side in the agar gel electrophoresis. Three phenotypes, A,B and AB, were observed. These phenotypes were shown to be controlled by one autosomal locus, designated as Es-4, with co-dominant alleles Es-4A and Es-4B. Es-4 esterase isozymes were detected in all the individuals from about 4 days of age, but the activity was very weak. However, it gradually increased to reach a level almost the same as that of a mature quail from about 15 days of age.