Neural tube defects are the most common malformations associated with diabetic pregnancies. Although the teratogenic effects of excess glucose have been investigated in in vivo and in vivo studies, a cellular basis for neural tube defects has not been elucidated. We used rat embryo culture to study the organogenesis period of development, with excess d-glucose added to the serum medium to induce neural tube anomalies. Light and electron microscopic examination of control 12-day-old embryos grown 48 hours in culture revealed blastlike cells with few organelles or cellular processes. Twelve-day-old embryos cultured in excess d-glucose had advanced cellular maturation with differentiation, including the presence of free polysomes and copious cell processes, regardless of whether they had an open neural tube. Cytoarchitectural changes such as decreased numbers of mitotic figures with mitotic cells in the mantle layer were focally distributed throughout the neural epithelium but with predominance at the site of failed closure. In vivo studies failed to demonstrate neural processes in day 12 normal embryos. Fourteen-day-old embryos grown in utero also had foci of cell processes in the neural tube but to a much lesser degree than that observed in the in vitro day 12 glucose-exposed embryos. The cellular aberrations in the excess d-glucose-treated embryos are characteristic of a premature maturational change. Since they are present in excess d-glucose-exposed embryos with or without failure of neural tube closure, these maturational and cytoarchitectural changes may contribute to the cellular basis for neural tube defects.

A B S T R A C T The activity of heme synthetase, which catalyzes the chelation offerrous iron to protoporphyrin to form heme, is deficient in sonicates of skin fibroblasts cultured from patients with protoporphyria. During culture in Eagle's medium supplemented with fetal calf serum, these cells do not accumulate protoporphyrin, however. This may be due to a minimal re quirement for heme synthesis, since glycine is incorporated into heme at a low rate which is similar to that in normal fibroblasts. In addition, the activity of 8-aminolevulinic acid (ALA) synthetase, the first and rate-limiting enzyme of heme biosynthesis which catalyzes the formation of ALA from glycine, is normal in lysates of the fibroblasts.Cultured fibroblasts were therefore incubated with ALA in order to bypass the rate-limiting step of heme biosynthesis. In the presence of 25 ,M iron, protoporphyrin was detected in protoporphyria cell lines when the concentration of ALA in the medium reached 50 ,M, but not in normal lines. As the concentration of ALA was increased above 50 ,uM, all lines accumulated protoporphyrin. However, the amount was 2-3 times more in cultured fibroblasts from patients with protoporphyria, reflecting their deficiency ofheme synthetase activity. When iron was not added to the medium, protoporphyrin accumulated to a similar degree in normal and protoporphyria fibroblasts; this was significantly more than that in the presence of iron. These studies indicate that excessive protoporphyrin accumulation in protoporphyria, which is due principally to deficient heme synthetase activity, may be modified by the rate of ALA formation in heme-producing tissues, and by the availability of iron.

Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

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