Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Rabbit corticosteroid-binding globulin: primary structure and biosynthesis during pregnancy

The cDNA-deduced primary structure of rabbit corticosteroid-binding globulin (CBG) contains 383 amino acids (mol wt, 42,326), including three cysteine residues and four sites for N-glycosylation. It is primarily the product of a 1.68-kilobase hepatic mRNA, but small amounts of CBG mRNA were also found in maternal lung, spleen, and ovary and fetal kidney. In the fetus, hepatic CBG mRNA concentrations increase markedly after day 11 and were 2- to 5-fold higher than those in maternal liver during days 17-23. They then declined to very low levels at term (31 days). By contrast, maternal hepatic CBG mRNA levels did not increase until day 23; reached a peak at about day 27, and then declined to prepregnancy values by 3 days after delivery. In general, fetal and maternal hepatic CBG mRNA concentrations reflect the corresponding serum CBG levels. Our data, therefore, indicate that the marked changes in fetal and maternal plasma CBG levels during pregnancy reflect changes in the biosynthesis of the protein rather than alterations in compartmentalization or clearance.     

Octanoate metabolism in isolated hepatocytes and mitochondria from fetal, newborn and adult rabbit. Evidence for a high capacity for octanoate esterification in term fetal liver

Ketogenesis from endogenous fatty acids or from exogenous octanoate has been studied in isolated hepatocytes from fetal. 24-h-old newborn and adult rabbit. In fed adult rabbits, endogenous ketogenesis is low and increases sixfold in the presence of 2 mM octanoate. At birth, endogenous ketogenesis is low and markedly increases 24 h after birth but, in both cases, the addition of 2 mM octanoate does not increase the rates of ketone body production. Hepatocytes isolated from 24-h-old newborn or fed adult rabbits and incubated with [1-14C]octanoate show a preferential channeling of fatty acid into oxidation (84-92% of octanoate metabolized). In contrast, esterification represents 43% of the amount of octanoate metabolized at birth. Chromatographic analysis of labelled triacylglycerols shows that 76 +/- 2% of labelled fatty acids are identified as octanoate and all of the radioactivity in the octanoate peak is due to the carboxyl carbon. In hepatocytes from term fetus, the low capacity for octanoate oxidation is associated with a high capacity for esterification, whatever the octanoate concentration in the medium. Octanoate activated to octanoyl-CoA in the cytosol of fetal hepatocyte is not oxidized in the mitochondria since carnitine acyltransferase I has a low activity at birth in the rabbit. This suggests that only a part of the octanoate pool is activated outside the mitochondria. Factors involved in the direct esterification of octanoate into triacylglycerols in term fetal hepatocytes are discussed.     

A possible mechanism for cadmium-induced hypertension in rats

The mechanism of cadmium-induced hypertension was explored by measuring noradrenaline metabolism. Cadmium $\text{in vitro}$ was shown to inhibit both monoamine oxidase and catechol-$\text{O}$-methyltransferase, the two enzymes which inactivate the neurotransmitters noradrenaline and adrenaline. However, rats which were injected or fed (via the drinking water) with cadmium showed that, among the tissues surveyed, these two enzymes were inhibited significantly only in the aorta. $\text{In vitro}$, cadmium was found to inhibit noradrenaline binding to membranes from the heart, lung, and kidney, while stimulating binding to aortic membranes, which suggests that the effects may be specific. These results suggest that, in the aorta, cadmium may inhibit the two catabolic enzymes of noradrenaline, while at the same time stimulating noradrenaline-binding. Thus the effects of noradrenaline on vascular smooth muscle would be increased as well as prolonged.     

Precocious development in utero of certain UDP-glucuronyltransferase activities in rat fetuses exposed to glucocorticoids.

The first precocious development of UDP glucuronyltransferase in the mammalian fetus $\text{in utero}$ by a known compound of endogenous origin is described. Intraperitoneal injection of cortisol (8 mg) into maternal rats on days 14 and 15 of gestation stimulated fetal-liver transferase activity from near zero to $\text{1}\text{2}$ maternal levels by day 17; 0.3 mg dexamethasone, possessing a longer biological half-life, raised activity to full maternal level by day 16. In controls, injected with solvent only, fetal-liver transferase remained low on day 16. With both glucocorticoids, transferase stimulation was dose-dependent. Transferase activities were assayed in a range of digitonin concentrations from zero to above optimal for enzyme activation. Activities stimulated were towards $\text{o}$-aminophenol, $\text{p}$-nitrophenol, 1-naphthol and serotonin. Activities towards bilirubin, morphine and testosterone were not stimulated. The former group of activities are stimulated by glucocorticoids in culture and normally reach approximate adult levels just before birth; the latter group are not so stimulated on culture and normally reach adult levels after birth. Implications of these findings are discussed.     

Hepatic and extrahepatic N-acetyltransferase. Perinatal development using a new radioassay.

We report a sensitive and rapid radioassay method for p-aminobenzoic acid N-acetyltransferase. The principle of this assay involves acetylation of p-aminobenzoic acid with [1-14C] labeled acetyl coenzyme A and direct extraction of enzymically formed radioactive p-acetamidobenzoic acid into nonaqueous scintillation fluid. Using this radiometric assay, hepatic and extrahepatic tissue distributions from rat and rabbit were studied. Rabbit blastocyst and endometrial N-acetyltransferase specific activities were equivalent to hepatic activities. Perinatal development studies in rats and rabbits revealed that fetal and neonatal animals are capable of N-acetylation. Rat liver developmental studies exhibited two peaks of activity with the first peak occurring in the late fetus followed by a second peak 3 days after birth. Rabbit fetal and neonatal enzyme activity increased to adult levels by the second week after birth in liver and gut, however, lung showed a different developmental pattern. These studies demonstrate that fetal extrahepatic tissues, like adult tissues, play an important role in N-acetylation.     

A possible mechanism for cadmium-induced hypertension in rats

The mechanism of cadmium-induced hypertension was explored by measuring noradrenaline metabolism. Cadmium $\text{in vitro}$ was shown to inhibit both monoamine oxidase and catechol-O-methyltransferase, the two enzymes which inactivate the neurotransmitters noradrenaline and adrenaline. However, rats which were injected or fed (via the drinking water) with cadmium showed that, among the tissues surveyed, these two enzymes were inhibited significantly only in the aorta. $\text{In vitro}$, cadmium was found to inhibit noradrenaline binding to membranes from the heart, lung, and kidney, while stimulating binding to aortic membranes, which suggests that the effects may be specific. These results suggest that, in the aorta, cadmium may inhibit the two catabolic enzymes of noradrenaline, while at the same time stimulating noradrenaline-binding. Thus the effects of noradrenaline on vascular smooth muscle would be increased as well as prolonged.     

Studies on closure of the ductus arteriosus. XII. In utero effect of indomethacin and sodium salicylate in rats and rabbits

Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by $\text{in utero}$ contraction of the fetal ductus arteriosus when studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response $\text{in utero}$. The rat was found to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure $\text{in utero}$ to prostaglandin synthetase inhibitors with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.     

Biological evidence that separate hypothalamic hormones release the follicle stimulating and lutneinizing hormones

The first $\text{in}\text{utero}$ diagnosis of Sandhoff's disease was made in an at-risk fetus by the demonstration of deficient β-N-acetyl-hexosaminidase A and B activities in amniotic fluid components the day of amniocentesis. These enzymatic deficiencies were determined by enzyme assay and electrophoresis using 4-methylumbelliferyl-β-N-acetyl-glucosaminide as substrate. The concentrations of the neutral glycosphingolipids were quantified in amniotic fluid; the level of the glycosphingolipid substrate, globoside, was markedly increased in amniotic fluid from the at-risk fetus compared to that of fetal controls. In addition, ultrastructural examination demonstrated pathologic glycosphingolipid accumulation in uncultured amniotic cells. These enzymatic, chemical and ultrastructural procedures provided the rapid and accurate $\text{in}\text{utero}$ diagnosis Sandhoff's disease within three days of amniocentesis. The $\text{in}\text{utero}$ diagnosis was confirmed by the marked deficiencies of β-N-acetyl-hexosaminidase A and B in plasma and various tissues from the aborted fetus. These findings indicated that maternal hexosaminidases do not cross the fetal-placental barrier.     

Postnatal appearance of a short DNA repeat length in neurons of the cerebral cortex

A short DNA repeat length of 162 base pairs was present in neurons of the adult rabbit cerebral cortex while a 198 base pair repeat was found in cortical glial and kidney cells. The short DNA repeat length in cortical neurons was not evident in fetal or new born brain but it appeared during early postnatal development between 2 $\text{1}\text{2}$ and 3 $\text{1}\text{2}$ days in rabbit and between 4 and 7 days in mouse.     

Developmental differences in activation of cholinephosphate cytidylyltransferase by lipids in rabbit lung cytosol

Lung cytosolic cholinephosphate cytidylyltransferase is activated by lipids. We examined the lipid activation pattern as a function of development in rabbit lung from 27 days gestation through term (31 days) and in the adult. The enzyme in both the fetal and adult cytosol was dependent on lipids for activity. Extraction of the cytosol with acetone/butanol virtually abolished cytidylyltransferase activity, but the activity could be restored on addition of lipids extracted with chloroform/methanol from additional cytosol. Cytosolic phospholipids from the fetal lung reactivated cytidylyltransferase but both neutral lipids and phospholipids from the adult were required. The lipids had the same effect on cytidylyltransferase activity in delipidated cytosol from either the fetus or adult so the difference in activation pattern was attributable to the lipids rather than the protein. There was a shift from the fetal to the adult lipid activation pattern as development progressed. Further, there was a significant correlation between cytidylyltransferase activities in intact cytosols from developing lung and activities in delipidated cytosol in the presence of lipids from the same animals. Although these data suggest that lipids regulate cytosolic cytidylyltransferase activity in developing lung their physiological significance remains to be established.     

Dietary regulation of maternal and fetal cholesterol metabolism in the guinea pig

Studies to determine the effects of pre-natal interventions on maternal and fetal cholesterol homeostasis were carried out in the guinea pig. Guinea pig dams were fed either non-purified guinea pig diet or diet supplemented with either 1.1% of the bile acid binding resin cholestyramine or 0.25% cholesterol. Whole body rates of endogenous cholesterol synthesis were determined by quantitation of [3H]water incorporation into digitonin precipitable sterols in non-pregnant animals and at 40 and 60 days of gestation in the dam and fetus. Maternal hepatic cholesterol synthesis was reduced 87% by dietary cholesterol and was increased 3.5-fold with cholestyramine feeding. Fetal hepatic and peripheral tissue cholesterol synthesis rates peaked at 40 days gestation when peripheral tissue cholesterol synthesis was 5.7-fold higher and hepatic synthesis 6.2-fold greater than the near adult levels observed at 60 days. Cholesterol synthesis in the fetus was relatively insensitive to dietary manipulations; however, maternal cholestyramine treatment did result in a 1.4-fold increase in fetal carcass cholesterol synthesis at 60 days gestation. These data demonstrate that maternal cholesterogenic systems maintain responsiveness to dietary regulation during pregnancy; whereas fetal cholesterol homeostasis is relatively insensitive to dietary cholesterol throughout gestation yet may respond to induction by maternal cholestyramine treatment during the late gestation period.     

Synthesis of rat hepatic zinc thionein in response to the stress of sham operation

Following sham operation for adrenalectomy, a dramatic 30-fold increase in rat hepatic zinc thionein occurs, peaking at 18 hours after surgery. Hepatic cytosolic and serum zinc levels rise concomitantly with zinc thionein. Copper in hepatic thionein and cytosol rises only slightly and serum copper not at all during the period of observation. In the period 18 to 48 hours after surgery the content of hepatic zinc thionein decreases with a $\text{t}\text{1}\text{2}$ of 16.4 hours.Pretreatment with cycloheximide (1 mg/kg b.w.) two hours before surgery inhibits the rise in zinc thionein by 52%, the rise in cytosolic zinc by 56%, and actually causes a decrease in serum zinc by 33%. Pretreatment with the α-adrenergic receptor blocker, phetolamine (10 mg/kg b.w.), or the β-adrenergic receptor blocker, propranolol (10 mg/kg b.w.), 30 minutes before surgery also inhibited the rise in zinc thionein (82% and 60%, respectively) and cytosolic zinc (75% and 47%, respectively), and decreased serum zinc (38% and 44%, respectively) 19 hours after surgery.Treatment with corticosterone (40 mg/kg b.w.) alone or epinephrine (1–20 μg/kg b.w.) alone did not alter hepatic zinc thionein levels 18 hours late, although they each caused hypozincemia and epinephrine raised cytosolic zinc levels. Treatment with corticosterone and epinephrine together did, however, raise zinc thionein levels 3.2-fold (P<0.02).These experiments are consistent with the hypothesis that adrenal hormones are involved in the regulation of zinc metabolism, and, hence, zinc thionein in levels in rat liver following the stress of sham operation.     

Isolation of alpha-amino adipic acid from mature dermal collagen and elastin. Evidence for an oxidative pathway in the maturation of collagen and elastin.

Human serum alpha-2-macroglobulin has been found to be a major cadmium-binding protein $\text{in vitro}$. Serum and alpha-2-macroglobulin equilibrated with cadmium at the 0.20 ppm level were chromatographed over Sephadex and agarose gels to separate and estimate the molecular weights of the proteins. Alpha-2-macroglobulin was found to fragment into reproducible fragments when chromatographed on agarose gels showing different metal-binding fractions for cadmium and endogenous zinc. The distribution of cadmium on serum protein chromatograms was correlated with alpha-2-macroglobulin chromatograms. Cadmium was bound to fractions with molecular weights as high as 800,000 daltons with an affinity greater than that observed for serum albumin.     

Rat liver metabollothionein interactions of silver,zinc, and cadmium

Weanling rats were fed diets either alone or with combinations of silver, elevated zinc, or elevated cadmium for 7 weeks. The rats were then killed, and the silver, zinc, and cadmium proteins isolated from the livers by gel filtration and ion exchange chromatography. When silver was fed alone in diet, low levels of this metal were eluted as two peaks from the ion exchange column. In contrast, when silver was fed with cadmium or elevated zinc, three metal-containing peaks were eluted from this ion exchange column. Amino acid analysis revealed that the major proteins binding these metals are metallothioneins, as judged by high cysteine content.     

Epidermal growth factor receptors in fetal and maternal rabbit lung

The pattern of morphologic and functional development of lung during intrauterine period is influenced by several endogenous compounds. Recently Epidermal Growth Factor (EGF), when administered in vivo, has been shown to accelerate pulmonary maturation in fetal rabbit and sheep. We sought evidence for EGF receptor occurrence in fetal and maternal rabbit lung plasma membranes. The percent specific binding (mean ± S.E.M.) (125-I) EGF to LPM in the mother (n=5) and the fetus at term (n=7) was 1.08 ± 0.08 and 2.25 ± 0.12 per 175 μg of LPM protein respectively. The number of receptor sites per mg of LPM protein in the mother were significantly less than that in the fetus (44 ± 11 and 250 ± 24 × 10^?10, p < 0.001) with no apparent differences in Kd (2.10 ± 0.39 and 2.47 ± 0.24 × 10⁹). Presence of high affinity receptors for EGF in fetal and maternal lung plasma membranes suggests a direct role of EGF in fetal lung maturation.     

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Nonproliferating cultures of adult rat hepatocytes were found to lose 60–70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (95%) in the activity of the zinc metalloenzyme, δ-aminolevulinic acid dehydratase, as well as a loss (>85%) in the cellular content of immunoreactive δ-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both δ-aminolevulinic acid dehydratase activity and immunoreactive protein. Since the spontaneous, selective loss of cellular zinc appears to have specific effects on a relevant hepatic function, this culture system constitutes a novel $\text{in}$$\text{vitro}$ model of zinc deficiency in mature liver.     

Pulmonary phosphatidic acid phosphohydrolase. Developmental patterns in rabbit lung

1. The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueously dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PAmb-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PAaq-dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the Paaq- or the Pamb-dependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaq-dependent activities in the void volume (Vo) (50% total), a peak with an apparent molecular mass (Mr) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PAaq-dependent peak with Mr = 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PAmb-dependent activity in the Vo (15% total), a major peak with an apparent Mr = 390 kdaltons (44% total) and minor peaks with Mr = 240 kdaltons (16% total) and Mr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on he PAmb-dependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PAmb-dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profiles of the PAaq-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profiles of the PAaq-dependent activities in adult cytosol were monophasic.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.