Ikuo Hayasaka

Teratogenicity of Azosemide, a Loop Diuretic, in Rats, Mice and Rabbits

Teratogenic effects of azosemide, a loop diuretic, were investigated in rats, mice and rabbits. Azosemide was given orally to pregnant rats, mice and rabbits during organogenesis. The pregnant animals were killed at term and their fetuses were examined for external, visceral and skeletal abnormalities. In rats, azosemide at 10–30 mg/kg/day did not affect intrauterine growth, resorptions and rates of external and visceral malformations. Treatment with 90 mg/kg/day resulted in a significant increase in skeletal abnormalities such as wavy ribs, bent scapula and bent humerus. However, the skeletal abnormalities observed in term fetuses could not be found in adult offspring, indicating that they were temporary. In mice, 1250 mg/kg/day of azosemide caused maternal death, abortion, and retarded maternal and fetal weight. Treatment with 200–500 mg/kg/day did not induce fetal mortalities, external and visceral malformations. Skeletal abnormalities increased in dose‐dependent fashion. The type of abnormalities was identical to that encountered in rat fetuses. Furosemide as a positive control also produced similar types of skeletal abnormalities in mouse fetuses. In rabbits, azosemide did not have embryolethal or teratogenic effects even at the highest dose (6 mg/kg/ day), which caused maternal death.

Azosemide-Induced Fetal Wavy Ribs and Their Disappearance after Birth in Rats

ABSTRACT Azosemide produced bent long bones such as wavy ribs in rat fetuses, but these abnormalities could not be found in the adult offspring. In the present study, the morphological sequence from appearance to disappearance of wavy ribs was examined in cartilage‐bone double stained specimens of fetuses and pups from mothers treated with azosemide on day 16 of gestation. The first detected change of the skeletal abnormalities was inhibition of bone deposition in the ossification centers of fetuses on day 17 of gestation. A bend first appeared on day 18 of gestation, and consisted of cartilage and portion stained neither alcian blue nor alizarin red S. Ossification began at this stage. From day 19 of gestation onward, ossification progressed toward the ends of the cartilage model including the bent region. The bend disappeared in most pups as bone in the bent region grew on days 10–14 postpartum. The present findings imply that the bend may be caused by difference in growth between cartilaginous and unstained portions, and a surface remodeling of bones may straighten the bend in the subsequent bone growth.

Development and Disappearance of Wavy Ribs Caused by Azose‐mide in the Mouse Fetus

ABSTRACT Treatment of gravid mice (day 13 of gestation) with azosemide induces wavy ribs in their fetuses. The present study examined the morphological sequence of azosemide‐induced wavy ribs, from their appearance through disappearance, by means of cartilage‐bone double stain, hematoxylin and eosin stain, von Kóssas stain for calcium salts and alkaline phosphatase stain. Both endochondral and intramembranous ossifications of the ribs were inhibited in day 14 azosemide‐treated fetuses. A curvature of the ribs occurred on day 15 of gestation, and excessive uncalcified osteoid was present on the outer surfaces of the bent region. In the region of curvature, endochondral and intramembranous ossification, which is remarkable in normal, was not observed. Calcification, which began on day 14 in untreated fetuses, started including the bent region on day 16 of gestation. However, ribs in azosemide‐treated fetuses exhibited alkaline phosphatase activity like in the control, during days 14–16 of gestation. These observations suggest that the defective ossification in azosemide‐treated fetuses is caused by inhibition of calcium salts deposition on the uncalcified osteoid. During the lactation period, the bend of ribs was gradually normalized, and the bend was disappeared on day 9 postpartum.