Identification of lanosterol 14 alpha-methyl demethylase in human tissues.

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.

Developmental analysis of cephalic axial dysraphic disorders in arsenic-treated hamster embryos.

Parenteral injection of pregnant golden hamsters with inorganic arsenic salts early in gestation results, by term, in markedly elevated embryonic-fetal mortality (approximately equal to 50%) and, in surviving fetuses, a high (approximately equal to 90%) incidence of cephalic axial dysraphic disorders ("neural tube defects"), particularly exencephaly/anencephaly and encephaloceles. The present investigation traces the day by day development of these embryopathic effects of arsenic in the hamster with an emphasis on the pathogenesis of cephalic axial dysraphic disorders. Pregnant golden hamsters were given an intraperitoneal injection of sodium arsenate (20 mg/kg) on the 8th day (08.00) of their 16 day gestation period. Matched control dams were injected with an equivalent volume of distilled water by the same route and at the same stage of gestation. Experimental and control dams were sacrificed beginning 24 h after treatment and at regular daily intervals thereafter until term. Embryos and fetuses delivered from sacrificed dams were examined for abnormalities both grossly and histologically. In embryos delivered earliest after treatment (24-48 h) the principal deleterious effect of arsenic observed was retarded growth (elevation, approximation, and fusion) of the cephalic neural folds. This growth retardation ranged in severity among embryos. In the most severely afflicted there was a site wherein the opposing cephalic neural folds had completely failed to appose and fuse ("closure"). This failure of closure of all four tissue components of the neural folds (surface ectoderm, paraxial mesoderm, neural crest cells, neuroectoderm) resulted in a persistent dorsal opening in the head, i.e., cranioschisis aperta. The extent and appearance of this opening varied from a small, ovoid aperture in the dorsal midbrain (mesencephalic) region of the head to a widely open cleft involving the fore and hindbrain regions as well as the midbrain region. In less severely afflicted early embryos, the cephalic neural folds had elevated and met in the dorsal midline but had only incompletely fused, i.e., cranioschisis occulta. Microscopic study of these latter embryos revealed that in the affected region(s), complete closure of the surface ectoderm component of the neural folds had taken place, but only partial closure of the mesoderm, neural crest and neuroectoderm components. The different types of cephalic axial dysraphic disorders presenting in arsenic-treated fetuses delivered at later gestational stages (predominantly exencephaly and encephaloceles) could all be traced back and related to one or the other of these early forms of disturbed neurulation.

Trophoblast invasion and alteration of mesometrial arteries in the pregnant hamster: light and electron microscopic observations.

During gestation in the Syrian golden hamster, an extensive migration of trophoblast into the maternal uteroplacental arterial system takes place. The present light and electron microscopic study has focused on one facet of this phenomenon, namely the trophoblastic invasion and alteration of the mesometrial 'vascular knot' (a complex of anastamosing, small arterial channels carrying blood to the chorio-allantoic placenta). Beginning at about the 8th day of gestation, motile trophoblastic giant cells (TGC), originating from the primitive trophospongium (Träger), invade and migrate as a wave up the central maternal arteries of the decidua basalis. During the 12th and 13th days of gestation, they reach the mesometrium and successively settle in the lumina of the vascular knot arterial channels. Between this time and parturition (day 16) the arrested TGC sequentially remove and replace the arterial endothelium, breach the elastic lamina, and apparently destroy (by lytic activity) nearly all of the mural smooth muscle. Near term, the vascular knot arterial channels are composed almost entirely of TGC. The functional significance of these events, and, more generally, the phenomenon of trophoblastic invasion of maternal uterine arteries in mammalian species having haemochorial-type placentae, are still uncertain. These invasions may be requisite to adapting, structurally and physiologically, the maternal uterine arteries to support of the fetoplacental unit and/or to maintaining the immunologically privileged status of the latter.

Enhanced teratogenicity of orally administered lead in hamsters fed diets deficient in calcium or iron.

A preliminary investigation was carried out to determine if dietary deficiencies in calcium or iron enhance the embryotoxic and teratogenic effects of orally administered lead in hamsters. Females of experimental groups were given drinking water containing 0.05% or 0.1% lead acetate and a calcium or iron deficient diet for several weeks prior to and/or during pregnancy. The incidence of embryonic/fetal mortality and of abnormal fetuses was markedly increased in litters delivered from these animals near term (15th gestational day) compared with litters from control animals similarly exposed to leaded drinking water but fed complete diets.

Placental permeability during early gestation in the hamster. Electron microscopic observations using horseradish peroxidase as a macromolecular tracer.

The permeabilities of the parietal yolk sac placenta and the preplacental region of the hamster conceptus during early postimplantation (day 8) were compared by means of electron microscopy and a macromolecular protein tracer, horseradish peroxidase (HRP). HRP was administered by injection into the maternal venous system; samples of the two placental tissues were obtained for examination at intervals between 4 minutes and 1 hour later. The three layers of the parietal yolk sac wall (from outer to inner: capsular trophoblast, Reichert's membrance, parietal endoderm) appeared to provide little impediment to the passage of HRP from perivitelline maternal blood spaces to the yolk sac cavity. HRP passed through the outer trophoblast layer, both by way of intracellular fenestrae (60-200 nm diameter) and narrower intercellular channels, and completely permeated the meshwork of Reichert's membrane within minutes after injection. The inner parietal endoderm cell layer was widely discontinuous and clearly presented no barrier to HRP movement. HRP reaching the yolk sac cavity was avidly endocytosed by the viceral yolk sac epithelium. In contrast to the parietal yolk sac, the preplacental region of the conceptus was impermeable to HRP. Zonular occluding junctions located between contiguous cells of the chorionic ectoderm layer of the preplacenta were the obvious barrier to the HRP molecules. These results suggest that in this rodent species, during the early postimplantation period of gestation, the pareital yolk sac placenta potentially plays a more important role in the maternal-embryonic transfer of macromolecular substances than does the preplacenta.

Iron and the liver: subcellular distribution of iron and decreased microsomal cytochrome P-450 in livers of iron-loaded rats.

To understand better the intracellular iron distribution and metabolic consequences of chronic hepatic iron overload, rats were given large doses of iron dextran or ferric citrate intraperitoneally. They accumulated large quantities of iron within Kupffer cells and hepatocytes. The relative subcellular iron distributions were similar in controls and iron-loaded rats, despite a ten- to 20-fold difference in hepatic iron concentration. Electron microscopy of whole liver and subcellular particulate fractions suggested that iron was present in highest concentration in lysosomes, which were rendered more labile by its presence. Nevertheless, quantitative iron determinations on all subcellular fractions, obtained by two preparative methods, showed that most of the iron was present in the "soluble" fraction. The amount of iron in the "microsomal" fraction varied, depending on the techniques used for preparation of this fraction. Cytochrome P-450 and total heme concentrations were decreased 40% to 50% in microsomes isolated from iron-loaded livers.

Freeze-fracture observations on the visceral yolk sac placenta of rats, mice and hamsters. With special reference to endodermal cell tight junctions.

Freeze-fracture replicas of visceral yolk sac from rats, mice and hamsters in late stages of gestation were studied by electron microscopy. Special attention was directed toward determining the types of junctional specializations that exist between the columnar endoderm cells of this placental membrane. In all three species, well-developed, zonular tight (occluding) junctions were found on the contiguous lateral surfaces of the endoderm cells. The tight junctional network, located in an immediate subluminal position, was from 0.2--0.5 micrometers in depth and consisted at any point of 2--5, interconnecting, approximately 9 eta wide, strands (P-face) or shallow furrows (E-face). Patch-like aggregations of irregular intramembrane particles, characteristic of desmosomes (maculae adherentes), also were observed at scattered sites below the tight junctions. However, no evidence of gap (communicating) junctions was encountered. The endoderm cells of the rodent visceral yolk sac have been shown to play a central role in the selective transport of macromolecular substances from the maternal to the fetal system. Tight junctions may be vital to this endodermal cell function by preventing random paracellular fluxes of macromolecules.

Ultrastructural observations on the maturation of the placental labyrinth of the golden hamster (days 10 to 16 of gestation).

Morphogenesis of the labyrinthine part of the chorioallantoic placenta of the golden hamster between day 10 of gestation and term (day 16) was studied by light and electron microscopy. During this period the labyrinth increases greatly in both size and complexity. Trabeculae of the labyrinth, thin partitions composed of trophoblastic tissue and fetal capillaries which delimit the maternal blood spaces, apparently proliferate both by appositional and interstitial growth. From the time of its formation (day 9 of gestation) until term the labyrinth is hemotrichorial in organization (i.e. three layers of trophoblast separate maternal blood from fetal capillaries). Both the inner and intermediate layers of trophoblast (layers III and II, respectively) are syncytial. The outer trophoblastic layer (III), which is in direct contact with maternal blood, remains cellular, although many of its component cells grow to giant cell dimensions ("labyrinthine giant cells"). Between the tenth and fourteenth days of gestation the anatomical barrier to diffusion between maternal and fetal blood is progressively reduced. This is accomplished both by gradual attenuation of the trophoblastic layers and fetal capillary endothelium and by the formation of discontinuities (gaps) in layer I, and diaphragmed fenestrae in fetal capillary endothelium. The labyrinthine placental barrier is fully developed and probably attains maximal functional efficiency by the fourteenth day of gestation. Late in the fifteenth day of gestation, a few hours before parturition, distinct degenerative changes are apparent in the trophoblastic layers and fetal capillaries of the trabeculae. The factors responsible for initiation these degenerative changes and the onset of parturition are still controversial.

Placental permeability of lead.

The detection of lead in fetal tissues by chemical analysis has long been accepted as prima facie evidence for the permeability of the placenta to this nonessential trace metal. However, only a few investigations, all on lower mammalian species, have contributed any direct experimental data bearing on this physiological process. Recent radioactive tracer and radioautographic studies on rodents have shown that lead crosses the placental membranes rapidly and in significant amounts even at relatively low maternal blood levels. While it is not possible to extrapolate directly the results of these experiments to humans because of differences in placental structure and other factors, the results do serve as a warning of the possible hazard to the human embryo and fetus of even low levels of lead in the maternal system.