Quantitative immunohistochemistry of metallothionein in rat placenta.

The presence of metallothionein (MT) was demonstrated in placentae from cadmium-exposed and control rats by an immunohistochemical technique, using peroxidase as label and the diaminobenzidine procedure for the staining reaction. The protein was found in different regions of the placenta, i.e. in trophoblastic labyrinth, in spongiotrophoblast and in visceral yolk sac. Cytophotometric analysis of the final reaction product revealed that the amount of MT was increased in the placental labyrinth of cadmium-exposed rats. Increases were found in both nuclei and cytoplasm of trophoblast cells in the labyrinth region. Possible roles of MT in the transport of zinc and in the carbohydrate metabolism are discussed.

Effects of cadmium exposure during pregnancy on cadmium and zinc concentrations in neonatal liver and consequences for the offspring.

The effects of cadmium exposure during pregnancy (by means of daily subcutaneous injections of 4.4 mumol/kg to the mother) on the neonates were investigated. No effect was observed on fetal or neonatal body weights, nor on neonatal liver weights. These parameters were examined up to 5 weeks after birth. The weight of neonatal thymuses was decreased 7 and 14 days after birth due to cadmium exposure of the mothers as compared with controls. This may be caused by zinc deficiency, because zinc concentrations in fetal and neonatal livers after cadmium exposure were found to be very low 20 days after conception and 5 h after birth. Cadmium concentration in neonatal liver decreased; however, cadmium in malignant liver increased as age increased. In the mother, cadmium was transferred to the milk, as it was demonstrated in the stomach contents of the pups. Simultaneous administration of zinc in amounts equimolar to cadmium did not have any noticeable effect on the amount of cadmium transferred to the fetus or on cadmium concentrations in any of the organs investigated. It could not prevent zinc deficiency in fetal and neonatal liver. In addition, growth retardation of the thymus from exposed pups could not be prevented by zinc administration.

Effects of cadmium exposure on glycogen phosphorylase activity in rat placenta as demonstrated by histochemical means.

Glycogen phosphorylase (PHO) activity was demonstrated histochemically in unfixed cryostat sections of placentae from cadmium-treated and control rats with the use of the semipermeable membrane technique. Staining of the newly synthesized glycogen was performed by lugol. A high activity was present in glycogen cells, spongiotrophoblast and visceral yolk sac from cadmium-treated and control animals. A low but distinct activity could be demonstrated in placental labyrinth from control rats in late pregnancy. Cadmium-exposed rats showed a considerably higher activity in the labyrinth during this period of pregnancy. The elevated PHO activity and concomitant higher glycogen content indicate a disturbance by exposure to cadmium of placental carbohydrate metabolism from day 18 onwards.

Cadmium and zinc concentrations in fetal and maternal rat tissue after parenteral administration of cadmium during pregnancy.

Cadmium (Cd) and zinc (Zn) concentrations were determined by solid sampling atomic absorption spectrometry (AAS) in rat maternal and fetal tissues after exposure to cadmium. Cadmium was administered subcutaneously as CdCl2 in saline daily during pregnancy. Two experiments were performed. In expt. I we investigated the tissue concentration at day 19 (gestational age) after administration of several doses: 0, 1.1, 2.2, 4.4, and 8.8 mumol Cd/kg/day. In expt. II the course of the Cd and Zn concentrations during pregnancy was investigated by collecting samples at days 14, 16, 18 and 20, after daily injections of 4.4 mumol Cd/kg. Cadmium concentrations in blood, maternal liver, placenta and fetal liver increased with dose and duration of exposure. Cadmium was heavily accumulated in the liver and transferred to the fetus only in small amounts. The zinc concentration in the maternal liver was positively correlated with the cadmium concentration. In the placenta the zinc concentration was not affected. Zinc in fetal liver was decreased from day 18 onward. Despite relatively high cadmium levels and decreased zinc levels in the fetus, we observed no adverse effects on various reproduction parameters, such as birth weights and obvious malformations.

Glycogen content of placenta and of fetal and maternal liver of cadmium-exposed rats. II: A quantitative histochemical study.

Quantitative data are presented of the glycogen contents in the placental labyrinth, fetal liver and maternal liver of 14-, 16-, 18-, 19- and 20-day pregnant rats exposed to cadmium during pregnancy. The values are obtained from periodic acid-Schiff-stained sections by microdensitometry. No changes due to cadmium exposure were observed in the glycogen content of maternal and fetal livers. However, at 18, 19 and 20 days of pregnancy, significantly higher amounts of glycogen were observed in the trophoblastic labyrinth of cadmium-exposed rats compared with control animals.

Light and electron microscopic investigation of the rat placenta after cadmium administration during pregnancy.

The morphology of the rat placenta was studied after exposure to cadmium chloride during pregnancy, using optimal fixation conditions. In contrast to previous observations, no differences were observed after cadmium administration in relative volume densities of trophoblastic tissue, maternal lacunae, fetal capillaries and connective tissue, nor in trophoblastic thickness or other morphometric features. At the ultrastructural level, the amount of glycogen in trophoblast layer II was elevated in cadmium exposed rats, but other electron microscopic features (amount and localization of lipid, degenerative vesicles, thickness and general appearance of the trophoblastic and endothelial layers and thickening or multiplication of the basal lamina) were not changed. Results obtained from the present experiments do not support the suggestion that cadmium is responsible for structural changes in the placentae of human smokers.

Effects of chronic cadmium administration on placental and fetal development.

Cadmium was administered subcutaneously to pregnant Wistar rats: 0.49 mg/kg as CdCl2 in saline daily, starting at the day of conception. Placentas and fetal livers were collected on day 14, 16, 18, 19 and 20 of gestation. Livers and thymuses from the newborns were collected 5 hours after delivery (day 22) and 1, 2 and 5 weeks after delivery. In these tissues concentrations of cadmium and zinc were determined by solid sampling ETA-AAS. Furthermore, the effect of cadmium administration on the glycogen content of the trophoblastic labyrinth and the fetal liver was studied. The concentration of cadmium in the placenta increased with time of exposure, indicating accumulation of cadmium in this organ. In the fetal liver, cadmium was present in a very low concentration, which slightly increased with longer exposure. The concentration of zinc in the placenta tends to decrease between day 14 and day 20. This decrease was observed both in control and in cadmium-exposed animals. Zinc levels increased in fetal livers from control dams, whereas this rise was markedly reduced in fetuses from cadmium-exposed animals. Placentas from cadmium-exposed animals had a changed glycogen pattern as compared to the controls, namely higher glycogen contents of the labyrinth at the end of pregnancy. However, notwithstanding lower zinc levels in the fetus and changed glycogen deposition in the placenta, it is not quite clear whether cadmium affects fetal development. No changes were observed in fetal weights or birthweights, nor in glycogen deposition of the fetal liver. Indications were obtained for reduced neonatal thymic weights.

Glycogen content of placenta and of fetal and maternal liver in cadmium-exposed rats. I: A descriptive light microscopic study.

Female Wistar rats were injected subcutaneously with CdCl2 (0.49 mg Cd/kg/day) daily during pregnancy. Placentae, fetal livers and maternal livers were prepared for histological investigation of the glycogen content 14, 16, 18, 19 and 20 days after mating. The glycogen content was estimated semiquantitatively in periodic acid-Schiff-(PAS-)stained sections. The development of the glycogen content of the placenta and the fetal liver from days 14 to 20 of pregnancy in control animals is described and compared with the development of the glycogen content of these tissues from cadmium-exposed animals. No clear changes due to cadmium exposure were observed in the glycogen content of maternal and fetal livers, nor in the spongiotrophoblast and the islets of glycogen in the placenta. The glycogen content in the placental labyrinth increased from day 14 up to day 18 in both control and cadmium-exposed animals. In control animals, the glycogen content decreased after day 18, whereas it remained high in the placental labyrinth of cadmium-exposed animals; this might be considered as a feature of retarded maturation. Regulating mechanisms and similarities between the effects of cadmium exposure and the diabetic state are discussed.

Increase of cadmium-thiolate clusters as a measure of morphological non-toxic cadmium accumulation in the rat liver.

When cadmium is chronically administered to rats, an increase by more than 10% of protein bound disulphides and cadmium-thiolate clusters appears to be an indicator for non-toxic accumulation of cadmium in liver and kidney and probably in other organs as well. Using enzyme histochemistry, no damage could be observed in these livers, on the contrary, even signs of increased cellular activity could be demonstrated with specific staining for single stranded RNA. It is clearly demonstrated that in the case of 2 livers with the same quantity of accumulated cadmium morphological damage is completely dependent on dose and schedule of administration. However, despite the fact that cadmium is retained very well in rat livers showing an increase in protein-bound disulphides and cadmium-thiolate clusters, there are still small morphological changes, especially in cells and tissues that appear to have a relatively small potency for producing cadmium-binding proteins.

Light microscopic aspects of the rat placenta after chronic cadmium administration.

Female Wistar rats were injected daily with different doses of CdCl2 during the first 19 days of pregnancy. The placentas of highly exposed animals showed reduced weights as well as some light microscopic changes with respect to the amount of collagen in the basal membranes around fetal blood vessels and to the relative volume densities of the fetal blood vessels. These results are in accordance with changes, found in the placentas of smoking mothers, probably due to cadmium in cigarette smoke.