Casimer T. Grabowski

Physiological changes in the bloodstream of chick embryos exposed to teratogenic doses of hypoxia

Abstract Exposure of 5-day chick embryos to moderate hypoxia (10.5% for 5 hours) precipitates malformations. Most defects are caused by the “edema syndrome,” marked initially by a significant increase in all body fluids. A comprehensive analysis of the blood serum of normal and treated chicks was made, using a variety of microchemical techniques. In the serum from hypoxia-treated embryos, lactic acid, free amino acids, potassium, and carbon dioxide increase; sodium, calcium, chloride, and glucose decrease; protein and inorganic phosphate remain constant. Exposure to hypoxia causes radical changes in the composition of the serum. Nevertheless, the pH and osmotic pressure remain unchanged. The existence of strong differences in sodium, potassium, and water concentration between the blood stream and the fluids which surround the extraembryonic capillaries is interpreted as an indication of significant regulatory activity by some embryonic or extraembryonic organ, perhaps the yolk sac. Changes in the concentration of these factors in the blood stream after exposure to hypoxia are best explained as the result of interference with osmoregulatory activity. Moderate hypoxia produces fluid and electrolyte disturbances in the chick embryo which eventually lead to the development of malformations. A consideration of aspects of the fluid physiology of the embryo is helpful in understanding the mechanism of teratogenic action of hypoxia.

Studies on the developmental toxicity of ozone. I. Prenatal effects.

Ozone, the predominant oxidant in photochemical smog, produces pulmonary and systemic toxicities, and yet possible effects on the fetus have received little attention. Long-Evans rats were exposed during early (Day 6–9), mid (Day 9–12) or late (Day 17–20) gestation, or throughout the period of organogenesis (Day 6–15) to concentrations of ozone varying from 0 to 1.97 ppm. No terata were produced by any regimen, but the resorption rate was increased following exposure to concentrations of ozone above 1.26 ppm in midgestation. Exposure to ambient levels (0.44 ppm) for 8 hr/day during the period of organogenesis produced no significant effects. Fetal heart rates, electrocardiograms, hematocrits, and plasma electrolytes were unaltered by exposure to 1 ppm during mid- or late gestation when examined on Day 20. Finally, sodium salicylate (150 mg/kg on Day 10) was shown to be synergistic with ozone (1 ppm in midgestation), in that the combined treatment decreased fetal weight, and to a lesser extent increased the resorption rate. These effects were minimal from either treatment administered alone.

Altered Electrolyte and Fluid Balance

Every embryologist knows that there are many important fluid-filled spaces in and around the intrauterine individual, such as the ventricles of the brain, the canal of the spinal cord, the pelvis of the ureter, the lumens of various portions of the gut and glands, the entire cardiovascular system, the blastocoele, amnion, allantois, and yolk sac. Every teratologist also knows that many developmental abnormalities involve deficiencies or overenlargements of these spaces, e.g., hydronephrosis, hydroureter, hydrocephalus and hydrocephalus-like conditions, exencephaly, hydramnois, and the occlusion of various hollow organs. Less well known are details of the composition of the fluids that fill these spaces, how their volume is controlled, and their precise role in the normal and abnormal morphogenesis of the structures with which they are associated. Although the extracellular proteins (e.g., plasma proteins and some enzymes) of some developing fluids are reasonably well known, less is known of small organic molecules, inorganic constituents, and the means by which their concentrations are controlled. It is often assumed that, apart from that performed by the placenta, little or no regulation is possible on the part of the embryo and young fetus, due to physiological immaturity of organs (Howard, 1957) or cells (Widdowson, 1968). The primary aim of this chapter is to review reports which demonstrate that a study of the embryonic and fetal fluids, their composition, and the physiology of their regulation is a valuable approach to an analysis of abnormal development caused by known teratogenic agents, as well as to provide insight into the role of these fluids in normal development.

Studies on the developmental toxicity of ozone: Postnatal effects

Pregnant rats were exposed to either 0, 1.0, or 1.5 ppm ozone during either mid gestation (Days 9-12) or late gestation (Days 17-20). The dams were allowed to deliver and the early morphological and behavioral development of their pups was monitored. Both exposure regimens transiently reduced neonatal growth rates. The late gestation exposure regimen produced retardations in early reflex development and in open field behavior. Finally, several males from this exposure regimen remained permanently stunted in growth.

Teratogenic Significance of Ionic and Fluid Imbalances

Some agents teratogenic to the chick embryo cause serum electrolyte and fluid inmbalances which initiate an edema syndrome leading to malformations. Differences in ionic composition of serum and yolk of normal chicks help explain how these imbalances can be produced.

Toxic effects of cadmium on the developing rat lung. I. Altered pulmonary surfactant and the induction of respiratory distress syndrome

The effects of Cd on the growth of the fetal rat lung and the maturation of the pulmonary surfactant system were studied. Pregnant rats received sc injections of cadmium chloride on d 12-15 of gestation. Animals were sacrificed throughout late gestation. Fetal lungs were assayed for pulmonary surfactant lecithin and spingomyelin. Some animals were allowed to give birth and the neonates were observed for symptoms of respiratory distress. The treatment resulted in high fetal mortality and growth retardation. Lung-body weight ratios were reduced by 20-30% in treated fetuses. Pulmonary spingomyelin content was not affected by the Cd absolute quantity but not in lecithin-lung weight ratio on the last days of gestation. Parturition was delayed almost a full day by the Dd treatment, and birth weights were reduced. Of the treated neonates, 11% developed respiratory distress syndrome. All but one of these individuals died and had lungs with hyaline membranes. Prenatal exposure to Cd can (1) cause lung hypoplasia, (2) affect pulmonary surfactant, and (3) induce respiratory distress syndrome in term pups.

Plasma proteins and colloid osmotic pressure of blood of rat fetuses prenatally exposed to mirex

A significant effect of prenatal exposure to the pesticide Mirex is a fetal edema, which is responsible for many of the toxic effects of this agent. Pregnant rats were given 6 mg/kg mirex on d 8 1/2-15 1/2, a moderate dose that does not cause maternal mortality. The blood of 18 1/2- and 20 1/2-d rat fetuses was tested to determine a possible cause of the edema. There were no significant differences between fetal and maternal plasma or between control and treated fetal plasma with respect to sodium and potassium concentrations or total osmolality. The protein concentration of plasma of treated fetuses was lower than that of controls. This effect was proportional to the degree of edema. The concentration in 20 1/2-d controls was 25.2 mg/ml, that in mildly swollen treated fetuses 18.1 mg/ml, and that in severely swollen ones 13.5 mg/ml. Polyacrylamide gel electrophoresis showed quantitative and qualitative changes in plasma of affected fetuses. The colloid osmotic pressure was also reduced fron 8.0 mm in controls and unaffected treated fetuses to 4.8 mm in swollen fetuses. Since lowered plasma protein and colloid osmotic pressure cause tissue edema in adults, they are the probably cause of mirex-induced fetal edema.

Lactic Acid Accumulation as a Cause of Hypoxia-Induced Malformations in the Chick Embryo

Most hypoxia-induced malformations are caused by a syndrome involving tremendous edema followed by formation of clear blisters and hematomas. These, in turn, mechanically interfere with development. Studies of blood pH and lactic acid indicate that lactate accumulation initiates this syndrome. The effects of lactic acid injections parallel those of subjection to hypoxia, confirming this conclusion.

Ontogenetic changes in the osmotic pressure and sodium and potassium concentrations of chick embryo serum.

Abstract 1. 1. The osmotic pressure and sodium and potassium concentrations were measured in serum samples obtained from 3- to 20-day chick embryos, newly hatched and adult chickens. 2. 2. There are two different levels of serum sodium concentration and osmotic pressure in the life cycle of the chicken. The embryonic levels are 20–30 mequiv./1 lower than that of the adult, and remain relatively constant during the first 2 weeks of development. The transition to adult levels occurs in the first few days after hatching. 3. 3. The serum potassium concentration of the embryo (5·1 mequiv./1) is slightly lower than that of the adult (6·1 mequiv./1) and increases markedly during the perinatal period (8·3 mequiv./1).

The causes of perinatal death induced by prenatal exposure of rats to the pesticide, mirex. part II: Postnatal observations

Prenatal exposure to mirex induces a high rate of perinatal death without obvious cause. Part I of this study (Grabowski and Payne, 1982), in which fetuses were exposed to a moderate dose of mirex and observed just prior to parturition, it was found that 14% had died recently and an additional 9% were apparently dying with serious cardiovascular symptoms. In the present study, electrocardiograms of normal (oil‐fed controls) and mirex‐exposed rats (6 mg/kg·d of insecticide on d 8 to 15 of gestation) were obtained at birth and for several days afterwards. An ECG was obtained from most individuals within 5 min of birth. Pups were then weighed and tattooed so that individuals could be followed to postnatal d 5. Data on 70 control newborns were compared with that on 131 mirex‐treated pups. In the treated group, 23% were stillborn. Of those born alive, 8% died within 6 h of birth, and another 13% within 48 h. At birth, 14% had first‐degree heart blocks, and 2% had second‐degree blocks. All of those with second‐...