Hepatic glutathione, metallothionein and zinc in the rat on gestational day 19 during chronic ethanol administration.

Ethanol, under certain conditions, alters the metabolism of sulfur amino acids, metallothionein (MT) and zinc. If chronic ethanol administration during pregnancy decreases the availability of sulfur amino acids or Zn, this deficiency could contribute to growth retardation of the fetuses, one of the features of fetal alcohol syndrome. The purpose of this study was to discern whether chronic ethanol administration to pregnant rats alters glutathione (GSH), MT or Zn content of selected tissues of the dams and fetuses. Sprague-Dawley rats were fed from gestational days 5 to 19 either the control diet ad libitum (AF), the ethanol diet ad libitum (EF) or the control diet using the pair-feeding technique (PF). On the 19th day of gestation, total hepatic GSH was significantly lower for the EF and PF dams than for the AF dams. Hepatic MT contents were similar for the AF and EF dams, and hepatic MT content was significantly greater for the PF dams than the AF and EF dams. The three groups did not differ regarding hepatic Zn content of dams or fetuses. In summary, on the 19th day of gestation, chronic ethanol feeding of pregnant rats did not lower the maternal hepatic GSH level below that of PF dams, did not induce hepatic MT in the dams and did not prevent fetuses from achieving body weights and hepatic Zn concentrations equal to those of controls.

Polycyclic aromatic hydrocarbon-DNA adducts in spontaneously aborted fetal tissue.

Fetal tissue and placentas from 15 human spontaneous abortions were evaluated for DNA adducts of polycyclic aromatic hydrocarbons (PAHs), using a competitive enzyme-linked immunosorbent assay (ELISA) with fluorescent end-point detection. PAH-derived adducts were found in 43% of placentas, 27% of fetal liver samples and 42% of fetal lung specimens, thus confirming that the human fetus is a target for DNA damage. As there was only 60% concordance between placenta and fetal lung or liver on the presence or absence of detectable PAH adducts, the placenta was not a good surrogate for adduct formation in other fetal organs. PAH-derived adducts in fetal liver and lung presumably form as a result of transplacental exposure to environmental stimuli. Since none of the positive fetal samples were from women who reported smoking during pregnancy, cigarette smoke is, in this case, an unlikely candidate and the adducts detected must be due to some other common source(s) of hydrocarbon exposure. The high frequency of positive samples in our small series casts some doubt on whether fetal PAH-DNA adducts identify a population at increased risk for transplacental carcinogenesis.

Isolation and partial characterization of a human amelogenin from a single fetal dentition using HPLC techniques.

A strategy based on high pressure liquid chromatography (HPLC) techniques for the isolation or a principal amelogenin molecule from a single human dentition is described. A partial sequence (33 residues) for this 24 kDa amelogenin is presented and related to earlier studies of human 5 kDa tyrosine-rich amelogenin polypeptides (TRAPs). A failure to identify amino acid residue #25 (tryptophan in other amelogenins) suggests that this 24 kDa amelogenin is the progenitor of the human TRAP-2 molecule and provides further support for the possibility of several human amelogenin gene products, generated by splice-junction selection, from the single amelogenin gene in the same individual. Alternatively, multiple amelogenins may arise by expression of both the AMELX and AMELY loci.

Transforming growth factor alpha in developing rats.

Transforming growth factor-alpha (TGF-alpha) concentrations were measured in lung, brain, liver, and kidney of rats at three different ages (20 days gestation and 9 and 50 days postnatal). TGF-alpha concentrations were maximal in the lung and brain by 20 days of gestation and showed minimal changes during nursing (day 9) and young adulthood (day 50). The liver, which also showed maximal TGF-alpha concentration by 20 days of gestation, demonstrated a progressive reduction with age to nadir values in the young adult. In contrast to the pattern in other tissues, kidney had the lowest concentration of TGF-alpha in late gestation and showed an increase by 50 days of age. As TGF-alpha acts via the epidermal growth factor (EGF) receptor, its function in development may be analogous to that of EGF. Thus TGF-alpha may have a role in lung maturation and postinjury repair, liver repair and regeneration, and neuronal cell growth.

Immunohistological distribution of the secretory endometrial protein, 'pregnancy-associated endometrial alpha 2-globulin', a glycosylated beta-lactoglobulin homologue, in the human fetus and adult employing monoclonal antibodies.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any nonreproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of renin substrate in fetoplacental tissue.

Specific polyclonal rabbit anti-human renin substrate-antibodies were used in order to study the distribution of renin substrate immunoreactivity in human fetal and placental tissue. Renin substrate was immunohistochemically detected in human decidua and placenta, as well as in 19 weeks old human fetal liver and kidney. The presence of renin substrate in fetoplacental tissue supports the concept of a locally functioning renin-angiotensin system.

Analysis of 3'-azido-3'-deoxythymidine levels in tissues and milk by isocratic high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic (HPLC) assay was established to analyze levels of the antiretroviral agent 3'-azido-3'-deoxythymidine (AZT, zidovudine) in serum, milk and tissue extracts. After methanol precipitation, serum samples could be injected directly into the HPLC apparatus, whereas tissue extracts required further clarification. Recovery of AZT was virtually complete. Isocratic elution with a mobile phase consisting of 6% acetonitrile and 0.1 M ammonium acetate, pH adjusted to 4.5 with glacial acetic acid, resulted in good resolution of AZT and its metabolites; retention times for AZT and the internal standard, p-nitrophenol, were 20 and 37 min, respectively. Using this method, we have demonstrated that AZT crosses both the blood-brain and placental barriers and is excreted into milk at high levels.

Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them.

17 beta-hydroxysteroid oxidoreductases of human fetal and adult tissues: immunological cross-reactivity with an anti-human placental cytosolic 17 beta-hydroxysteroid oxidoreductase antibody.

To determine whether the immunological determinants of human placental 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) were present in 17 beta-HSORs of tissues of the human fetus and adult and of various non-human cells maintained in culture, western immunoblot analysis was conducted by use of a polyclonal antibody directed against determinants of the placental cytosolic enzyme. Tissues and cells were evaluated for the presence of immunocross-reactive proteins with a relative molecular mass (Mr) similar to that of placental 17 beta-HSOR (approximately 34,000). By use of homogenates of human fetal tissues, immunostaining of 17 beta-HSORs of Mr approximately 34 kDa was detected in trophoblast, fetal adrenal neocortex, fetal zone of the adrenal gland, liver, intestine, kidney, brain, lung, skin, heart, spleen, pancreas, chorion laeve, and, occasionally, amnion. Immunostaining at Mr approximately 34 kDa also was demonstrated by use of cytosolic preparations of fetal tissues and, in some cases, by use of unwashed microsomal fractions; this protein was either absent or present in almost undetectable amounts in washed microsomes, except for placenta and fetal brain. Immunostaining at approximately 34 kDa was demonstrated occasionally in decidua of pregnant women by use of homogenates, but was not detected in endometrium or myometrium of non-pregnant women, testis of an adult man, mouse and rat Leydig tumour cells, mouse and rat adrenal tumour cells, and normal bovine adrenocortical cells.

Human 'pregnancy-associated endometrial alpha 1-globulin', a 32 kDa insulin-like growth factor-binding protein: immunohistological distribution and localization in the adult and fetus.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

Expression of novel basement membrane components in the developing human kidney and eye.

The distribution of two novel human, basement-membrane (BM) collagens has been characterized by immunohistochemical analysis of developing and mature tissue using monoclonal antibodies specific for the non-collagenous (NC1) domain of each molecule. A distribution more restricted than that of type IV collagen was observed. In the kidney, the 28K parent molecules appear relatively late, at the early capillary-loop stage of glomerular development, whereas type IV collagen is present in all BM, including those of the ureteric bud, S-form, primitive glomerulus, and vessels. Antibody to the Alport familial nephritis antigen (a 26K peptide), which is missing from epidermal BM and glomerular BM in Alport syndrome, reacted with the ureteral bud BM and all stages of glomerular BM development from the early capillary-loop stage onward, but not with BM of more primitive glomeruli (vesicles and S forms). In the human fetal eye, the collagen molecules from which the 28K NC1 peptides are derived appear later in development than type IV collagen. They are present in trace amounts in Bruch's membrane but are not detected until after birth in the retinal internal limiting membrane and cuticular and non-pigmented epithelial BM of the ciliary process. In contrast, the BM of the lens capsule and Descemet's membrane were reactive with anti-28K antibodies early in development. In all instances, the 28K peptides are detected in BM that also contain the Alport antigen, although the later is present in some BM not containing the 28K peptides. The distribution of Alport antigen and type IV collagen in developing eye is similar to that observed in the mature eye. The 28K parent molecules appear to be expressed in concert with the maturation of the BM, coincident with fusion of glomerular endothelial and epithelial BM, whereas the lens capsule BM and Descemet's membrane contain these restricted components much earlier in gestation.

Distribution of free amino acids in streptozotocin-induced diabetic pregnant rats, their placentae and fetuses.

Amino acid levels in the non-pregnant streptozotocin (STZ)-induced diabetic rat have been shown to be abnormal. Our preliminary studies showed that placental transport, fetal serum levels and tissue uptake of the non-metabolizable amino acid alpha-amino isobutyric acid (AIB) were decreased in STZ-diabetic pregnant rats. In the present experiments, amino acid concentrations were measured in maternal (MS) and fetal (FS) sera and placentae (PL) by high performance liquid chromatography (HPLC) after triple extraction in 80% ethanol. Control (C), STZ-diabetic (D) and insulin-treated diabetic (DI) animals were studied at 22 days gestation. Pregnant diabetic rats had low serum levels of Gln, Lys, and Ser and insulin treatment corrected Gln and Ser but not Lys levels. Branched-chain amino acids did not show the large elevation characteristic of the non-pregnant diabetic rat. Placental levels of Tau, Gln, HPr, Thr and Lys were depressed in the diabetic animals and insulin treatment only partially improved these amino acid profiles. Placental amino acid levels did not always reflect maternal serum levels. Serum levels of most amino acids were lower in the fetus of the diabetic rat than in the fetus of the control rat. The notable exception was Ala which was higher in the fetuses of the diabetic animals. Insulin treatment of the mother did not correct many of the fetal amino acid levels even though maternal and fetal serum glucose levels at the time of autopsy were normal. The ability to maintain normal serum levels of many amino acids is impaired in the fetus of the diabetic rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of epidermal growth factor receptor and lipocortin-1 in fetal and neonatal human lungs.

The ontogeny and distribution of the epidermal growth factor (EGF) receptor and lipocortin-1, a major cellular substrate of the EGF receptor, were evaluated in a developmental series of fetal and neonatal human lungs (8 to 41 weeks' gestation and stillborn to 16 days' postnatal age). The peroxidase anti-peroxidase technique with two polyclonal antibodies recognizing the EGF receptor and one polyclonal antibody recognizing lipocortin-1 were used for immunohistochemical localization. Extensive or scattered bronchiolar EGF receptor immunoreactivity appeared in the entire series of frozen lung specimens from 15 to 32 weeks' gestation. Bronchial glands exhibited EGF receptor immunostaining from 19 weeks onward, and immunoreactivity in bronchial epithelium was detected from 23 weeks onward. Most tracheas showed extensive lipocortin-1 immunoreactivity in the epithelium beginning at 10 weeks' gestation. Immunostaining was also seen in cells lining the ducts of submucosal glands after 15 weeks' gestation and in nonmucous acinar cells of tracheal glands after their appearance at 18 weeks' gestation. Bronchial epithelium exhibited lipocortin-1 immunoreactivity from 12 weeks' gestation onward. Bronchial gland necks became immunostained from 16 weeks' gestation onward, followed by acinar immunostaining as they subsequently developed. Bronchiolar epithelium was immunostained as early as 12 weeks, beginning with the largest airways, and by 24 weeks extending distally to the bronchioloalveolar portals. Lipocortin-1 immunostaining of larger conducting airway epithelium was primarily confined to ciliated cells. Neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II cells, fibrocytes, chondrocytes, or smooth muscle cells at any gestational age. These developmental patterns suggest that the EGF receptor and lipocortin-1 may participate in normal growth factor-induced proliferation of the conducting airways and their glands in the human fetal lung and trachea.

Acid hematin pigmentation of the cornea in stillborn fetuses.

During the postmortem histopathologic evaluation of eyes from stillborn fetuses we noted the presence of a prominent undescribed corneal pigment in 18 of 55 stillborn fetuses. The corneal pigment was frequently associated with documented meconium-stained amniotic fluid, and in no instance was a stained cornea coupled with recorded clear amniotic fluid. Pigmented corneas came from stillborn fetuses with a longer duration of intrauterine death than nonstained corneas. The pigment stained black with the Fontana-Masson stain, was birefringent, and treatment of tissue sections with 5% potassium permanganate and 5% oxalic acid as well as with saturated alcoholic picric acid solution removed the pigment indicating that it is acid hematin. The most likely cause of the acid hematin-stained corneas was tissue acidity created in utero with prolonged intrauterine death.

Transplacental transfer of human antinuclear antibodies in mice by injection of lupus IgG in pregnant animals.

Pregnant female Balb/c mice were injected with IgG fractions from patients with systemic lupus erythematosus, in order to study the in vivo passage of antinuclear antibodies (ANA) across the placenta. After injection of monospecific sera directed against nDNA, Sm, nRNP, Ro(SSA) and La(SSB), ANA were found in fetal circulation and trapped in the liver, spleen kidney and skin of fetus. Also, ANA were demonstrated in placental tissue and cord. The placental IgG-Fc receptors apparently played a major role in ANA entry into the fetus. Our study demonstrates that human ANA can be passively transferred into experimental animals to study their kinetics during pregnancy.

Sulphotransferase and its substrate: adenosine-3'-phosphate-5'-phosphosulphate in human fetal liver and placenta.

The activity of sulphotransferase (ST) towards 2-naphthol and the concentration of its endogenous substrate adenosine-3'-phosphate-5'-phosphosulphate (PAPS) were measured in human fetal and adult liver and in the placenta. The activity of ST (mean +/- SD; nmol/min/mg protein) was 0.28 +/- 0.06 (fetal liver); 1.82 +/- 0.44 (adult liver; p less than 0.001) and 0.021 +/- 0.014 (placenta; p less than 0.001). The concentration of PAPS (mean +/- SD; nmol/g wet tissue) was 10.1 +/- 0.9 (fetal liver); 23.4 +/- 2.4 (adult liver; p less than 0.001) and 3.6 +/- 1.1 (placenta; p less than 0.001). Both ST and PAPS were higher in fetal liver than in placenta. The difference between fetal liver and placenta was more marked for ST than for its substrate. Such a consideration was also drawn when fetal and adult liver were compared. Thus, the activity of the ST rather than the concentration of its substrate seems to be the limiting factor in sulphation.

Relative abundance and molecular size of immunoreactive insulin-like growth factors I and II in human fetal tissues.

Previous studies which have quantitated tissue mRNA transcripts have suggested that IGF II is expressed in substantially greater amounts than IGF I during human fetal development. However, this has not been tested directly by recovery and quantitation of IGF peptides from fetal tissues. Tissues from human fetuses were extracted in 1 M acetic acid, and insulin-like growth factor (IGF) I and II immunoreactive species separated from the supernatants by acidic gel filtration. When total immunoreactive IGF I or II eluting from Sephadex G50 between approximately 6 and 15 kDa molecular weight was estimated, allowing for likely blood contamination, tissues from five fetuses contained, on average, 230 fM/mg cell DNA IGF I and 690 fM/mg IGF II. The ratio of IGF II: IGF I peptide content varied from 1.6 in muscle to 7.8 in thymus. Separation of tissue supernatants, fetal plasma and fetal fibroblast-conditioned culture medium on Sephadex G75 revealed that immunoreactive IGF II eluted with molecular weights between 8 and 15 kDa, in addition to 7.5 kDa. While fetal tissues contain more IGF II than IGF I immunoreactivity, the likely presence of IGF II peptide does not agree with the great excess of mRNA for IGF II reported previously.

Distribution of hyaluronic acid and chondroitin sulfate proteoglycans in the presumptive aganglionic terminal bowel of ls/ls fetal mice: an ultrastructural analysis.

The terminal colon of the ls/ls mouse is aganglionic because an intrinsic defect prevents its colonization by cells migrating from the neural crest. Previous studies showed that laminin, type IV collagen, and glycosaminoglycans accumulate in the region of the presumptive aganglionic ls/ls bowel through which crest-derived cells would be expected to migrate. It was suggested that crest-derived cells might fail to enter the abnormal bowel because they receive inappropriate signals from a defective extracellular matrix. This hypothesis was evaluated by analyzing the ultrastructure of the extracellular matrix in mutant and control gut. Tissue was fixed in the presence of ruthenium red before or after selective enzymatic digestion. Heparan sulfate proteoglycan (diameter approximately equal to 15 nm) and chondroitin sulfate proteoglycan (diameter approximately equal to 20-50 nm) granules were found in both control and presumptive aganglionic gut. The heparan sulfate proteoglycan granules were primarily located within formed basal laminae, while chondroitin sulfate proteoglycan granules decorated plasma membranes and 5 nm hyaluronic acid microfibrils that formed a network in the extracellular matrix. At day E11.5, the mutant gut differed from the control in the following: 1) Hyaluronic acid microfibrils were longer and more numerous. 2) There were larger numbers of chondroitin sulfate proteoglycan granules associated with cell membranes and with hyaluronic acid microfibrils. By day E13 the spaces between mesenchymal cells of the outer wall of the control bowel contained a regular lattice of hyaluronic acid microfibrils studded with chondroitin sulfate proteoglycan granules. Instead of this lattice, tangles of excessively long hyaluronic acid microfibrils, coated more heavily than in the control with chondroitin sulfate proteoglycan granules, were found in the presumptive aganglionic gut. These results confirm that the extracellular matrix is abnormal in the presumptive aganglionic bowel of the ls/ls mouse; moreover, they also indicate that the defect involves not one, but several components of the extracellular matrix, as well as their distribution. The defective extracellular matrix is apparent at a time when crest-derived cells would be expected to be migrating in the terminal bowel and is located in their path. The observations thus support the idea that a localized abnormality of the extracellular matrix interferes with the colonization of the terminal bowel by crest-derived cells in the ls/ls mouse.

Ontogeny of ACTH(1-24) receptors in rat adrenal glands during the perinatal period.

Binding of ACTH to receptors was studied on crude adrenal membranes from fetal and newborn rats. 125I-Labelled ACTH(1-24) was used as the radioligand, the steroidogenic potency of which was 100-fold lower than that of unlabelled ACTH(1-24). Binding was specific, rapidly equilibrated and temperature dependent. Scatchard analysis of the binding data revealed a single class of binding sites with a dissociation constant of about 100 nmol/l at all stages of development studied. The concentration of ACTH receptors expressed per mg membrane proteins decreased in fetuses between days 17 and 21 of gestation and remained stable in newborn rats from weeks 1 to 4. The number of ACTH receptors expressed per adrenal increased regularly in fetal and newborn rats. The perinatal evolution of these concentrations of ACTH receptors is related to the increase in the size of the adrenals and the changes in cytoplasmic structures of the adrenocortical cells. When the number of ACTH-binding sites was expressed per microgram DNA, maximum values occurred in fetuses on day 19 of gestation, and minimum values in newborn rats, 1 week after birth. There was an excellent correlation between the plasma levels of immunoreactive ACTH and corticosterone and the number of ACTH receptors per microgram DNA during the perinatal period. Other results suggest that ACTH is able to up-regulate the number of its own receptors.

Carbofuran poisoning of pregnant woman and fetus per ingestion.

A case of carbamate pesticide poisoning of a pregnant woman by carbofuran ingestion is presented. The mother recovered from the poisoning in the hospital but necrosis of the fetus was found. Toxicological findings of the liver, brain, and kidney of the fetus revealed carbofuran in concentrations comparable with the mother's blood. Our findings in the case contribute to the research on permeation of the placental barrier by chemical substances.