Teratogenic effects of proline analogue L-azetidine-2-carboxylic acid in hamster fetuses.

The teratogenic effects of L-azetidine-2-carboxylic acid (LACA) were evaluated in hamsters. Single or multiple intraperitoneal injections of LACA were given on gestational days 7-12. Fetal weights were significantly reduced in several LACA-treated groups as compared to controls. The frequency of external anomalies was significantly higher than controls in only two LACA-treated groups: 7.2% in 600 mg/kg on day 12 and 9.6% in 200 mg/kg/day on days 10-12. Subcutaneous hemorrhage and cleft palate were the most frequently occurring externally visible defects. Skeletal anomalies such as retarded ossification, synchronous vertebral ossification and shortening of bones were found in a high percentage of LACA-treated fetuses. The major teratological effect of LACA appeared to be the retardation of skeletal development.

The teratogenic effects of beta-aminopropionitrile in hamsters.

The teratogenicity of beta-aminopropionitrile (beta APN) was evaluated in golden Syrian hamsters; single doses of 1250, 2500, and 3750 mg/kg given on days 7-13 produced fetal mortality, growth retardation and malformations. All time-dose combinations produced gross external abnormalities with the highest rate (92.5%) occurring following day-12 treatment. High frequencies of skeletal abnormalities including deformities of the ribs, fibula, and scapula occurred after days-10-12 treatment. Deformed ribs occurred in 100% of fetuses exposed on day 11 to any of the dosages. The types of abnormality generally resembled those produced by lathyrogens in other species except for exencephaly and encephalocele, which occurred after treatment on day 7. These results indicate that betaAPN is a typical teratogen, capable of producing abnormalities of the central nervous system early in gestation, and may reflect a broader mechanism for its activity than previously suggested.

Neural tube lesions in the offspring of hamsters given single oral doses of lathyrogens early in gestation.

The lathyrogens aminoacetonitrile, D-penicillamine and semicarbazide were tested for their teratogenicity in hamster on day 7 of gestation at three different dose levels. The results showed that aminoacetonitrile and penicillamine were typical teratogens in hamsters, capable of causing significant embryotoxic effects consisting of increased fetal mortality, growth retardation and malformation. Semicarbazide at a dose of 100 mg/kg had only minor teratogenic effects, while slightly higher doses were lethal to the dams. The most severe fetal malformations following maternal exposure to aminoacetonitrile or penicillamine were exencephaly and encephalocele. Since lathyrogens inhibit the process of collagen cross-linking, the results of this study indicate that there may be an important relationship between collagen fiber formation in the embryo and neural tube closure.

Cytogenetic effects of delta-9-tetrahydrocannabinol (delta9-THC) on hamster bone marrow.

Single s.c. injections of 10 or 1000 mg/kg of delta9-THC did not induce discernable chromosomal damage but caused significant mitotic inhibition in the bone marrow of Syrian hamsters.

The morphogenesis of beta-aminopropionitrile-induced rib malformation in fetal golden Syrian hamsters.

In order to provide information on the mechanism of beta-aminopropionitrile (betaapn) induced teratogenesis, the pathogenesis of a fetal rib abnormality was studied at relatively short time intervals following maternal treatment with 2,500 mg/kg aqueous betaapn on day 11 of gestation. Histochemical tests of ribs from betaapn-exposed fetuses indicated a slight decrease in the level of glycosaminoglycans but at a time when the defect was already morphologically established. Ultrastructural observations on the chondrocytes of ribs from betaapn-exposed fetuses revealed alterations in mitochondrial structure indicative of a slight cytotoxic effect for the teratogen. The mitochondrial changes were transient, occurring initially at three hours after treatment and lasting for nine hours. Alterations in the size of collagen fibres in the cartilage of the fetal rib were also observed in the offspring of betaapn treated females. The mean diameter of collagen fibres in the ribs of control fetuses increased throughout the course of the study. The mean diameter of fibres in the fetuses of betaapn-exposed females failed to show any increase and was found to be significantly less than controls as early as three hours following maternal administration. The results suggested that the principal factor in the production of the fetal rib deformity was fundamentally the same as that known to affect the adult; namely a defect in the extracellular maturation of collagen.

Inhibition of beta-aminopropionitrile (beta APN)-induced skeletal teratogenesis by the flavonoid beta-hydroxyethylrutosides (HR) in hamster fetuses.

Since biochemical studies have shown that flavonoids such as beta-hydroxyethylrutosides (HR) protect against the damage to collagen induced by lathyrogens in adult rats, this compound was given to pregnant hamsters in order to determine its effects on the teratogenicity induced by beta-aminopropionitrile (beta APN). A dose of 2,500 mg/kg of beta APN alone given by gavage on day 11 produced a high frequency (69.5%) of skeletal anomalies in the offspring of hamsters. Administration of HR immediately following beta APN to pregnant animals resulted in a significantly decreased teratogenic response (P less than 0.05). These data provide evidence to support the view that the primary mechanism for the beta APN-induced skeletal dysmorphogenesis is the inhibition of cross linking during the maturation of collagen fibers.

Epithelial cell differentiation in organotypic cultures of fetal rat lung.

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.

Scanning electron microscopy of rat neurointermediate lobe cells in culture.

Enzymatically dispersed cells of the rat pars nervosa -- pars intermedia (NI) were observed by scanning electron microscopy after 1, 2 and 4 days in culture. The cells attached to the plate slowly, requiring about 3--4 days for the majority to adhere. The epithelial cells became attached singly and in clumps and branched chains, often over a bed of fibroblast-like cells. After the first day in culture, few surface features were evident on the NI cells, but by day 2, the surfaces showed a small number of blebs. In 4-day cultures, the cells revealed extensive areas with blebs and microvilli, and a few structures resembling cilia were observed. The adrenocorticotrophic hormone content of the cells after 4 days in culture was similar to that found in fresh tissue.

Cell culture of embryonic chick duodenal cells: preparation of epithelial-fibroblast bilayers and homotypic cultures of fibroblasts and epithelial cells.

A method for the primary cell culture of trypsin-dissociated embryonic chick duodenum is described. Both heterotypic (epithelial cells and fibroblasts together) and homotypic (highly enriched cultures of epithelial cells or fibroblasts alone) cell cultures were established. Dispersed duodenal epithelial cells and fibroblasts grown in 10% fetal bovine serum (FBS) spontaneously aggregated and proliferated as a bilayer of cells with the epithelial cells growing on top of the fibroblasts. Changing the serum supplement to 6% chicken serum (CS) and 4% FBS when the fibroblast monolayer reached confluence resulted in epithelial cell proliferation. Homotypic cultures of epithelial cells and fibroblasts were prepared and analyzed by scanning electron microscopy and transmission electron microscopy. Fibroblasts, isolated by differential adhesion and grown in 10% FBS, did not demonstrate measurable alkaline phosphatase activity. Homotypic epithelial cell cultures, isolated by floating them off the fibroblasts with collagenase, and maintained on collagen in 6% CS/4% FBS, demonstrated higher alkaline phosphatase-specific activity (16.1 +/- 2.3 U/mg protein) compared with epithelial-fibroblast bilayer cell cultures (12.1 +/- 1.3 U/mg protein).

Differentiation-arrested rat fetal lung in primary monolayer cell culture. II. Dexamethasone, triiodothyronine, and insulin effects on different gestational age cultures.

Differentiation-arrested and hormone-depleted monolayer cultures were developed from rat fetal lungs of 18-, 19-, 20-, and 22-days gestation. Incorporation of [3H]-choline into saturated phosphatidylcholine increased, whereas the rate of cell division decreased, with advancing gestational age. Both functions were modified by physiological concentrations of glucocorticoids, thyroid hormones, and insulin. Dexamethasone (0.055-5.5 nM) increased [3H]-choline incorporation into total saturated phosphatidylcholine in immature cultures only, but caused secretion in mature (day-22) cultures. Triiodothyronine (0.055-5.5 nM) increased [3H]-choline incorporation into total and secreted saturated phosphatidylcholine at all gestational ages. Insulin (5-50 microU/ml) inhibition of [3H]-choline incorporation into saturated phosphatidylcholine was evident only in mature cultures. Dexamethasone (0.55 nM), triiodothyronine (5.5 nM), and insulin (50 microU/ml) also had gestation-dependent effects on cell division.

Differentiation-arrested rat fetal lung in primary monolayer cell culture. I. Development of a differentiation-arrested and growth-supporting culture system using carbon-stripped bovine fetal calf serum.

In vitro observations of fetal lung maturation are complicated by continued epithelial cell maturation in culture. Removal of trace quantities of cortisol from the culture system has been reported elsewhere by Torday to prevent epithelial cell differentiation, but also prevent cell growth. In this report we have developed monolayer cell cultures from 18-, 19-, 20-, and 22-day-gestation fetal rat lung in a culture system stripped of steroid and thyroid hormones. These cultures demonstrate lamellar body development comparable to that seen in vivo at the gestational age at which cultures were developed; the gestation-dependent increase in precursor incorporation into saturated lecithin observed in these cultures is similar to that reported in in vivo studies, yet cell growth in culture has been preserved.

Differentiation-arrested rat fetal lung in primary monolayer cell culture. IV. Paradoxical effect of a "fetal" pO2 on precursor incorporation into phospholipids and hormone responsiveness.

Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO2 of approximately 142 mm Hg, cultures developed in a pO2 of approximately 30 mm Hg, close to the normal fetal arterial pO2, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO2 approximately 30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO2. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung.

Topography of short portal vessels in the rat pituitary gland: a scanning electron-microscopic and morphometric study of corrosion cast replicas.

We applied scanning electron microscopy combined with imaging and morphometric techniques to analyze the dorsal topography and morphology of short portal vessels linking the capillary beds of the pituitary neural and anterior lobes in adult male albino rats. The pituitary microvasculature was replicated by intracarotid injection of Batson's No. 17 compound producing plastic casts that were advantageous for comprehensive morphometric analyses using an imaging device. The analysis revealed the existence of two types of portal vessels having quantitatively different morphological properties. The bilateral venular plexus of 3-4 vessels located at the base of the infundibular stalk (each venule measuring 300 microns in length and 32 microns in diameter) appears to be the major part of the short portal system in the dorsum of the rat pituitary gland. Narrower capillary-like shunt vessels (6.8 microns in diameter), of about the same length as the venules, were situated throughout other subregions of the intermediate lobe cleft. The short portal vessels of both types made direct anastomoses with the capillary networks in the neural and anterior lobes. The neural lobe capillaries were twice as numerous (1324 per mm2), and only half as wide (6.2 microns), as the sinusoidal capillaries in the anterior lobe (density of 637 per mm2; diameter of 13.7 microns). The topographical position of the portal venular system suggests that the caudolateral subregions of the pituitary neural and anterior lobes have a functional relationship dependent on rapid interlobe transfer of neurohumoral factors such as hormones via the portal blood. This process appears to be supplemented throughout the rest of the cleft between the two lobes by a small number of capillary shunts that supply the epithelial cell lobules of the intermediate lobe in situ. The findings collectively indicate that this portal system provides a constant stream of neurohumoral information that is shared moment-by-moment between the pituitary neural and anterior lobes.