Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro.

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.

Postnatal mirex cataractogenesis in rats: lens cation balance, growth and histology.

The effects of lactational mirex exposure on postnatal lens cation balance, lens growth and histology were examined in this study. Pregnant Long Evans rats were allowed to give birth, and litters were culled to eight individuals. Dams were intubated with 10 mg kg-1 day-1 mirex on days 1-4 postpartum. Pups were killed at 6-14 days of age, and lenses were removed, examined for opacities, weighed, and assayed for K+ and Na+ concentrations. Cataracts occurred in 71.0% of all treated litters and 58.1% of all treated individuals. Histologically, lenses from treated pups showed anterior and posterior cortical vacuoles by 8 days of age, greatly swollen lens fibers by 10 days of age, and areas of cell degeneration by 12-14 days of age. Lens K+:Na+ ratios were lower in treated groups on days 8-14 after birth, in both clear and cataractous lenses, and cataractous lenses were significantly higher in water content. Lens:eye weight ratios were not affected. The results of this study indicate that mirex-induced postnatal cataracts are due to lens degeneration subsequent to fiber cells swelling.

Plasma glucose and protein concentrations in rat fetuses and neonates exposed to cataractogenic doses of mirex.

Mirex was administered to rats during gestation or the early postnatal period and the effects on blood chemistry were studied, especially with regard to changes which might play a role in the known cataractogenicity of mirex. In the prenatal study dams were intubated with 6 mg/kg/day mirex on Days 8 through 15 of gestation, and fetal blood samples were obtained on Days 18 and 20. For postnatal studies, litters were culled to eight pups at birth. Dams were intubated with 10 mg/kg/day mirex on Days 1 through 4 postpartum, and blood was drawn from pups at ages 6 through 14 days. Glucose determinations were done on a Beckman ASTRA 8 autoanalyzer. Protein determinations were done by the method of Lowry et al. (O.H. Lowry, N. J. Rosebrough , A. L. Farr, and R. J. Randall (1951). J. Biol. Chem. 193, 165-175.) Plasma glucose levels were decreased by over 40% in mirex-treated fetuses which developed cataracts. Postnatal exposure to mirex did not alter plasma glucose. Mean plasma protein concentrations were significantly lower in treated litters on Days 12 and 14 postpartum, and treated pups with cataracts on Day 14 were hypoproteinemic compared to treated pups without cataracts. Hypoproteinemia is a common factor related to cataractogenesis induced by either prenatal or postnatal mirex exposure, and may possibly be a causative factor. Although hypoglycemia may be a contributing factor in prenatal cataractogenesis, it does not seem to be implicated in postnatal cataractogenesis.

Mirex-induced fetal cataracts: lens growth, histology and cation balance, and relationship to edema.

The effects of mirex on growth, histology, and K+ and Na+ concentrations of the fetal lens are examined. Pregnant Long-Evans rats were intubated with 6 mg/kg/day mirex in peanut oil on days 8-15 of gestation. Control dams received pure peanut oil. Fetuses were removed on days 18-21 and rated for extent of lens opacification and degree of edema. Lenses were removed, weighed, and assayed for K+ and Na+. Selected eyes were examined histologically. Cataracts were found in 49.6% of live treated fetuses on day 20, and the incidence and severity of cataracts were both correlated with the degree of fetal edema. Lens weights were reduced in treated litters whether or not cataracts occurred, but the reduction in cataractous lenses was greater. Sodium tended to be elevated and K+ reduced in lenses from treated fetuses, and the K+/Na+ ratio was significantly reduced in all treated groups. Histologically, treated lenses had swollen fibers on day 18 and areas of apparent cell breakdown by day 20. The swollen lens fibers, reduced K+/Na+ ratios and the correlation with edema suggest that fetal cataracts induced by mirex may be due to lens fiber cell breakdown subsequent to osmotic swelling.

The causes of perinatal death induced by prenatal exposure of rats to the pesticide, mirex. Part I: Pre-parturition observations of the cardiovascular system.

Prenatal exposure to mirex, a chlorinated hydrocarbon insecticide, induces a high rate of perinatal death, but only a low incidence of visible abnormalities which could help to account for these deaths. This study is an attempt to determine the cause of these deaths. Pregnant rats were intubated with a moderate dose of mirex, in oil, 6 mg/kg/day, on days 8 1/2-15 1/2 of gestation. Observations, on 78 control and 136 treated fetuses, were made on the morning before parturition was expected. Fetuses were sequentially exposed and electrocardiograms obtained with the fetus attached to the placenta and uterus. ECGs were evaluated for rate of beat, regularity of beat, PR intervals, and other features. Fetuses were examined for edema level and vitality. None of the controls were dead and none had abnormal ECGs. Of the treated group, 14% were dead, 16% had a first-degree heart block, and 2% had a second-degree heart block. Some (6%) had slow, feeble atrial beats only, possibly a third-degree block, and appeared to be dying. The severity of the symptoms was proportional to the degree of visible edema. Most of the fetuses with little or no visible edema had normal ECGs, but the majority of the moderate to severely edematous fetuses (i.e., with a layer of subcutaneous edema across the back of 0.5 mm or more) had abnormal ECGs and/or were either dead or dying. These data show that the effects of mirex on the fetal cardiovascular system are a major factor in inducing prenatal death.

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.

An electrocardiographic study of cardiovascular problems in mirex-fed rat fetuses.

Sperm-positive female rats were intubated with pesticide, Mirex, in oil (5 to 10 mg/kg) on days 8 1/2 to 15 1/2. Controls were untreated or oil-fed. Testing was done on day 18 1/2. Fetuses were sequentially exposed and ECGs obtained with the fetus attached to the placenta and uterus. Counterparts of standard leads, I, II and III were used. Fetuses were weighed and examined afterwards. Swollen fetuses were rated on a scale of 1 (slight edema under chin) to 5 (3 mm edema across back). ECGs from 81 controls and 205 Mirex fetuses were obtained. They were evaluated for rate of heart beat, regularity of beat, PR intervals, and other features. One control exhibited an abnormality, a transitory period of premature atrial contractions. Mirex-fed fetuses frequently exhibited tachycardia, closely correlated with degree of edema. The heart rate increased from 150/minute in controls to 180 in slightly swollen to 224 in swollen fetuses. Mean PR intervals increased with degree of swelling and with dose. The frequency of first degree heart-block was also dose-related, ranging from 20% in the 5 mg/kg group to 77% in the 10 mg/kg group. Second-degree heart-blocks were found in 8%, 3% showed arrhythmias, and one had atrial flutter/fibrillation. These cardiovascular problems seem primarily related to the Mirex-induced edema and demonstrate that fetal edema is not innocuous. These data demonstrate the usefulness of fetal electrocardiography to detect functional teratology.

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.

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.

Responses of the rat foetus to maternal injections of adrenaline and vasopressin.

1. Injection of 0.5-2.0 units of vasopressin or 25-100 mug of adrenaline into the peritoneal cavity of pregnant rats produced a transient slowing of the foetal heart. The bradycardia could be induced in foetuses after 15-21 days of gestation. Foetal heart rates dropped from normal values of 140-180 beats/min, often to less than 20 beats/minute. The period of bradycardia was dose dependent and ranged from 30 to 65 minutes.2. Maternal injection of the hormones produced a fall in foetal blood pressure from an average of 54, often to less than 20 mm of water, in 17-day foetuses. Direct injection of the hormones into the pericardial sac of the foetuses had the opposite effect and pressures rose an average of 15 mm of water 1 min after the injection.3. During the period of bradycardia, the potassium concentrations in foetal serum rose from an average value of 8.9 mequiv/1. to an average of 17.3 mequiv/litre. Concentrations of serum sodium fell from 126.2 to 121.4 mequiv/1. during the bradycardia. No changes were detected in the concentrations of either calcium or chloride. Foetal P(O2) levels fell from 25 to 15, P(CO2) rose from 61 to 89 or more, and pH fell from 7.19 to 6.86 during the bradycardia.4. Maternal death and uterine clamping caused foetal bradycardia and a rise in foetal serum potassium to an average of 20.2 mequiv/litre.5. It is concluded that interruption of normal uterine blood flow by vasoconstruction (adrenaline or vasopressin) or direct blockage (uterine clamping) results in a transient hypoxia, bradycardia, and serum ion changes in foetuses.