Toshio Nakatsuka

Embryotoxic effects of L-691,121, a class III antiarrhythmic agent, in rats

L-691, 121 is a class III antiarrhythmic agent which blocks potassium currents, leading to prolongation of cardiac potential and prevention of cardiac arrhythmia. In a developmental toxicity study in rats, there was a dose-dependent decrease in embryonic/fetal survival, and death of the entire litter was seen at an oral dose of 0.8 mg/kg per day. The critical period for embryolethality was determined as gestational days (GD) 10–13. In a study where females received 1 mg/kg on a critical day (GD 10 or 12) and were killed at 24-h intervals, a high embryonic mortality was seen at 72 h (GD 10 treatment) or 48 h (GD 12 treatment) after dosing. The surviving embryos had morphological abnormalities such as enlarged cardiac tube and pericardium, generalized edema, and hematoma. In order to investigate a possible mechanism for the embryolethality, GD 11 embryos were dissected from females at 4h after dosing of 1 mg/kg and incubated for 5 h in vitro. The embryonic heart rates were decreased for the first 2 h after incubation but tended to recover to control levels thereafter. When GD 11 embryos were incubated for 4 h with the drug, there were decreases in the heart rates during the entire observation period. In a washout study where the embryos were transferred to drug-free medium after 1-h exposure, decreased heart rates recovered to control levels. In GD 11 embryos cultured for 24 h with the drug, there were gross abnormalities that consisted of altered yolk sac and embryonic circulation, and enlargement of cardiac tube and pericardium similar to those seen in the preceding in vivo study. These results suggest that decreased preceding in vivo study. These results suggest that decreased heart rates, reduced yolk sac circulation and the associated morphological abnormalities induced by L-691,121 are related to the embryolethality in rats.

Impairment of male fertility induced by muscarinic receptor antagonists in rats

Male rats were treated with a muscarinic receptor antagonist at 3, 10, and 100mg/kg/day for 4 weeks prior to mating with untreated females and their reproductive status was determined on gestation days (GD) 15-17. Treatment-related decreases in the pregnancy rate were observed at 100mg/kg/day without any effects on mating performance. Impairment of male fertility by this compound was also observed after treatment for 1 week, but there were no effects after a 1-week withdrawal period suggesting reversibility of the effect. There were no treatment-related effects on sperm production or motility, or testicular histopathology in any group. In order to determine whether the reduced fertility was a class effect of muscarinic receptor antagonists, atropine was examined. Males received atropine for 1 week at 62.5 and 125 mg/kg/day and were mated with untreated females. A low pregnancy rate associated with a decrease in the number of implantations was observed at 125 mg/kg/day. The effect on implantation was also observed at 62.5mg/kg/day. These findings suggest that the impairment of fertility in male rats induced by muscarinic receptor antagonists is a class effect, and has a relatively short onset of effect and is quickly reversible.

Effects of Calcium Channel Blockers on Cultured Rat Embryos

Gestational day 11 rat embryos were cultured for 20 h in the presence of nifedipine (NIF), diltiazem (DIL) and verapamil (VER), and the effects of these calcium channel blockers on embryonic heart rate and gross morphology were examined. Reductions of embryonic heart rate were dose dependent and reversible. Effects on the heart rate were seen at doses as low as 0.3, 1 and 20 μg ml−1 for VER, DIL and NIF, respectively. Retardation of embryonic growth and morphological abnormalities, which were related to circulation defects, were seen at termination. The minimum doses to produce 100% effect on gross morphology were 2, 6 and 40 μg ml−1 for VER, DIL and NIF, respectively. These findings suggest that embryolethality seen in vivo studies is partly due to circulation defects and the associated morphological abnormalities in rats.

Species Specificity in Induction of Wavy Ribs: Failure of Furosemide to Induce the Anomaly in Rabbit Fetuses

Abstract Despite the strong activity to induce wavy ribs in rat fetuses, furosemide does not seem to have such activity in the rabbit. Since wavy ribs are known to be repairable in the rat during the early postnatal period, there is a possibility that the anomaly once produced in rabbit fetuses disappeared before the examination at term because of their longer duration of gestation. In order to test this possibility, the authors administered furosemide to pregnant rabbits and examined fetuses shortly after the treatment period for the presence of wavy ribs. Furosemide even at doses which were apparently toxic to maternal animals failed to produce the anomaly. Based on these results and in view of the absence of its occurrence in the literature, it may be concluded that there is species specificity in the induction of wavy ribs and this anomaly does not occur in rabbit fetuses. The temporal difference between the onset of ossification and that of increased myometrial contractions may probably be involved in this species specificity.

Suppressive effects of Bay K 8644 on toxicity of calcium channel blockers in cultured rat embryos.

Previous study revealed that calcium channel blockers (CCBs) reduced embryonic heart rates (HRs) and produced morphological abnormalities when Gestational Day (GD) 11.5 rat embryos were cultured for 20 hr. The present study was to investigate whether a calcium channel agonist, Bay K 8644 (BAY), prevented CCB-induced embryotoxicity in vitro. GD 11.5 embryos were exposed to nifedipine (NIF), diltiazem (DIL), and verapamil (VER) either alone or in combination with BAY at 0.1, 1.0, and 10 micrograms/ml. These doses of BAY alone had no effect on gross morphology. Embryonic HRs were increased at 10 micrograms/ml of BAY, but were within control levels at 0.1 and 1.0 microgram/ml. The doses of NIF, DIL, and VER were 40, 6.0, and 2.0 micrograms/ml, respectively, and were the minimum concentrations to produce a 100% effect on morphological abnormalities. Embryonic HRs were reduced to 22, 31, and 34% below control levels in the NIF, DIL, and VER groups, respectively. The negative chronotropic effects of CCBs were inhibited by coadministration with BAY, depending on its concentration. When embryos exposed to each CCB were supplemented with BAY at 1.0 or 10 micrograms/ml, embryonic HRs were comparable to those of controls. Combined exposures of each CCB and 10 micrograms/ml BAY did not cause any morphological abnormalities. These results suggested that mechanisms of CCB embryotoxicity were directly related to pharmacological consequences of calcium channel blockage in developing rats.

Survey of fluctuating fetal body weights in Sprague-Dawley rats

During the past 9 years of reproductive and developmental toxicity studies in Sprague-Dawley rat, we noticed that the average fetal body weight on day 20 of gestation in 81 control groups fluctuated significantly from study to study. The other parameters such as number of implants, resorption rats and number of live fetuses were relatively stable. With information cooperatively given by the animal supplier, we could pin down the possibility of the fluctuation. The animal supplier introduced Sprague-Dawley rats into Japan from the U. S. twice in the past. The documents we analyzed indicated that the fluctuation in fetal body weight was caused by different two colonies which were introduced into Japan at the different times.