Acute effects of cadmium on the pregnant rat and embryo-fetal development.

In rats, of the Wistar-Porton strain, a single intravenous injection of 1.25 mg Cd2+ between days 9 and 15 of gestation results in a high incidence (80% of hydrocephalus, together with other malformations in the fetuses, examined on day 20. This dose is critical, since 1.1 mg Cd2+/kg is not teratogenic, while 1.35 mg Cd2+/kg kills all the embryos. Intravenous injection of Cd2+ to the pregnant rat on day 12 causes a dose-dependent inhibition of placental Zn2+ transport. At the teratogenic dose, Zn2+ transport is inhibited by about 75% at 4 hr. Thereafter, inhibition decreases with time but is still significant at 48 hr. At 20 hr after administration of Cd2+ the embryonic concentration of Zn2+ is depressed by 33%. In the whole embryo the activity of the Zn2+-dependent thymidine kinase is inhibited by about 60% at 4 hr and at 20 hr the DNA concentration is reduced significantly. Placental transport of 14C-leucine and 14C-uridine, as well as the embryonic incorporation of these precursors into protein and RNA is unaffected at least at short times after the administration of Cd2+. It is possible therefore, that the teratogenic effects of Cd2+ may be related to the inhibition of DNA synthesis in the embryo. ImagesFIGURE 1.FIGURE 2.

Pregnancy represents a period of high susceptibility to parenterally administered cadmium (Cd2+). In rats, Parizek (1,2) has shown that a single subcutaneous injection of 40 ,umole CdCl2/kg body weight on days 17 to 21 of gestation, results in destruction of the fetal portion of the placenta with death of the embryos in a high proportion of pregnant animals. In female rats of the Wistar-Porton strain used in our laboratory which have a gestation period of 21 days, the intravenous LD50 drops significantly from the virgin female level of 1.77 mg to 1.05 mg Cd2+/kg body weight on day 20 of gestation (p < 0.001; df = 30). Immediately after the administration of the LD50 dose, both gravid and nongravid animals develop flushed extremities, due to vasodilatation, rapid shallow respiration, apathy and flaccidity of muscles. At this dose, most deaths occur between 16 and 24 hr. With higher doses most of the animals succumb within 6 hr, the predominant pathological lesion being subpleural hemorrhage of the lung. Just before death these animals develop violent seizures. Palpation, immediately * Molecular Toxicology Section, MRC Toxicology Unit, Medical Research Council Laboratories, Carshalton, Surrey SM5 4EF, U.K.
after the animal has stopped breathing reveals that the heart is still beating, and this finding suggests respiratory paralysis. Both this respiratory failure and the initial muscular flaccidity probably are due to neuromuscular block. Such effects of Cd2+ have been observed by Forshaw (3) in isolated preparation of the rat hemidiaphragm and seem to result from a competitive displacement of calcium.
Pregnant animals that die between 16 and 24 hr after the administration of the LD50 dose of Cd2+ show vaginal bleeding at 8 hr, indicative of placental damage. Histology at this stage shows degeneration of the maternal part of the placenta (Fig. 1), which is in contrast to Parizek's findings (1, 2) that subcutaneous injection of Cd2+ salts is followed in all cases by rapidly progressive placental changes, chiefly of pars fetalis. The liver and the kidneys are edematous and hyperemic. The fetuses are pale in color and in distress, as shown by the presence of meconium-stained liquor amnii. At 20 hr the maternal animal is extremely pale, with severe hemorrhage per vaginum. Histology reveals that the placenta has lost its architecture and, as observed by Parizek (1), has turned into an extensive blood clot (Fig. 2). The liver exhibits periacinar necrosis, and the kidneys show vacuolation of the cells of the proximal convoluted tubules. Many areas in the cortex contain dilated tubules with flattened epithelium, and sometimes homogeneous casts that are probably proteinaceous. There are areas with complete degeneration of the proximal convoluted tubules. Inflammatory cells are never in large numbers but are scattered in areas containing altered tubules. The glomeruli and the distal convoluted tubules are spared. Thus, death at this stage may be due to a combination of retroplacental hemorrhage and hepatorenal failure. Nongravid females do not show these hepatic and renal changes at this dose (1.05 mg Cd2+/kg), though the lungs show occasional subpleural hemorrhage.
In addition to its increased toxicity at or near term, Cd2+ is known to cause congenital deformities in rats, mice, and hamsters (4-7), when administered during a particular period of gestation. In the present work a single intravenous dose of 1.25 mg Cd2+ between days 8 and 15 of gestation was found to be extremely teratogenic and to induce one or more deformities in a high percentage of the fetuses ( Table 1). The most frequent of these deformities is hydrocephalus, which is seen in 80o of the fetuses when examined, after delivery by cesarean section on day 20.
In addition, anophthalmia, microphthalmia, gastroschiasis, and umbilical hernia are present in 45% of the fetuses. Though the incidence of hydrocephalus is highest when Cd2+ is given on day 10, this deformity also occurs in a great number of fetuses when the cation is administered on day 12 of gestation. As, at this stage of development, the embryos are of reasonable size, most of the following biochemical work has been done with animals treated with a dose of 1.25 mg Cd2+/kg body weight on day 12 of gestation. This dose is critical, since 1.10 mg does not produce malformations, while 1.35 mg Cd2+/kg kills all the embryos.
After the intravenous injection of isotopically labelled Cd2+ (1.25 mg/kg) to the pregnant rat on day 12 of gestation, extremely small amounts of Cd2+, i.e., about 16 ng/g wet weight tissue, are detectable in the embryos at 4 hr. At 24 hr, the amount is even smaller-about 5 ng Cd2+/g wet weight tissue. At these times the corresponding total placental concentrations of Cd2+ are 2 and 0.8 ,g/g wet weight,  a The maternal animals killed and examined on the 20th day of gestation. respectively; i.e., 100-150 times greater than the embryonic levels. This loss of Cd2+ from the 12-day embryo may be due at least in part to the absence of the high affinity Cd2+-binding protein, thionein, since the development of the liver and kidneys is just beginning at this time. Neither zinc-thionein nor cadmium-thionein is present in the 12-day embryos from normal and Cd2+-treated dams, respectively. In contrast, at 20 days the liver and kidneys of the normal fetus are well developed and the hepatic concentration of zinc-thionein is extremely high. When Cd2+ is administered to the mother at this stage of gestation, uptake of the cation by the fetus is greater at 24 hr than at 4 hr and, under these conditions, most of the cadmium that reaches the fetus is accumulated in the liver, specifically in the metallothionein fraction.
In agreement with these analytical measurements, 109Cd2+ can be detected by autoradiography in the livers of fetuses from pregnant animals that are dosed with the labeled cation on day 20 of gestation, but not in the whole embryos of females that are treated in the same way on day 12.
Although little Cd2+ reaches the embryo on day 12 of gestation, its presence in the placenta seems to interfere with the function of this organ, particularly with the transport of Zn2+; a cation for which the embryo has a high demand at this stage of development (8,9). Thus at 4 hr after a dose of 0.5 mg Cd2+/kg, transport of 65Zn+ across the placenta is inhibited by nearly 50%, and at 1.25 mg/kg it is reduced to about 25% of the control values (Table 2).
Even after a dose of Cd2+ as low as 0.25 mg/kg inhibition is appreciable, although not statistically significant. At 1.25 mg the inhibition persists and remains significant at 48 hr.
Quantitative interpretation of these results is difficult because of the changes caused by Cd2+ in the concentrations of Zn2+ in the plasma and other body compartments of the maternal animal. Nevertheless, as intravenous administration of Cd2+ results in an initial dose dependent depression of blood Zn2+, the effects of Cd2+ on total Zn2+ transport across the placenta are likely to be greater, not less, than those revealed by the measurements of 65Zn2+ uptake. As a result of the inhibition of Zn2+ transport, the Zn2+ concentration of the embryo is reduced by 33% at 20 hr after the intravenous injec-  (3 ,uCi) to the maternal animal. tion of 1.25 mg Cd2+/kg body weight to the maternal animal.
Maternal Zn2+ deficiency is known to lead to teratogenic effects in the rat (8,10) and to reduce thymidine incorporation (11), particularly in the developing brain of the 12-day embryo. Such effects have been attributed to the inhibition of the Zn2+dependent enzyme, thymidine kinase. When Cd2+ (1.25 mg/kg body weight) is administered to 12-day pregnant rats, followed at 4, 20, and 48 hr by a 15-min pulse of 14C thymidine (3 ,uCi), the transport of the latter at any time is found to be unaffected. Incorporation of thymidine into the embryonic DNA, however, is inhibited significantly at 4 and 20 hr although at 48 hr it is similar to that in the controls ( Table 3). The total DNA content of the embryo is little affected at 4 hr but at 20 and 48 hr it is reduced significantly.
Since Cd2+ blocks both thymidine incorporation into embryonic DNA and also the placental transfer of Zn2+, the former may be a direct result of the latter. Thymidine incorporation into embryonic DNA, however, is inhibited as early as 4 hr after the administration of Cd2+ to the maternal animal, that is before any measurable change has occurred in embryonic Zn2+ concentration. Furthermore, the effect of Cd2+ on placental Zn2+ transport is still apparent at 48 hr, whereas DNA synthesis has returned to the control level at this time. It is possible, therefore, that the small amounts of Cd2+ that are incorporated into the 12-day embryo have a direct inhibitory action on thymidine kinase. In vitro, this enzyme is known to be inhibited by Cd2+ (12). Inhibition of embryonic DNA synthesis, therefore, may be related to the teratogenic effects of Cd2+, particularly as the uptake and incorporation of 14C_ uridine and 14C-leucine by the embryos are not affected at 4 hr after the administration of Cd2+. Thus, at a time when DNA synthesis is inhibited appreciably, RNA and protein production are unaffected.