Anomalies of Cartilaginous and Ossified Axial Skeleton in Rat Fetuses Treated with Cyclophosphamide: Type, Frequency, and Stage Specificity

Abstract

The purpose of the present study was to assess the type, frequency and stage specificity of axial skeletal anomalies induced by cyclophosphamide (CP). Female Crj: Wistar rats were mated with male rats (vaginal sperm = gestation day 0, GDO). They were then injected intravenously with 5 mg/kg of CP in a saline solution on GD7, 8, 9, 10, 11, 12, 13, or 14, or with saline on GD8 for controls. Dams were sacrificed on GD20. Fetuses were stained with alcian blue and alizarin red S. Eighteen types of axial skeletal anomaly were detected, e. g., shifted ventral lamina, cervical rib, cleft arch of the atlas, axis fused to atlas, sternebral misalignment, bipartite sternum, fused ribs, change in pre‐sacral vertebral number, supernumerary rib, misdirected transverse process, anteriorly‐shifted spinous process, short spinous process, fused vertebral bodies, cartilaginous short rib at Th.7, short or absent rib at Th.12 or 13, dumbbell‐shaped vertebral body, wavy rib, and rib cartilage not attached to the sternal cartilage. The present experimental study indicates that: 1) observations of cartilaginous skeletons can provide more information than observations of ossified skeletons; 2) the pattern of CP‐induced anomalies along the anterior–posterior axis may depend on the sequence of development progression among structures, spinous process, vertebral arch, vertebral body, rib/transverse process; and 3) in more cranial vertebrae, anteriorly‐shifted anomalies, such as anteriorly‐shifted ventral lamina and anteriorly‐shifted spinous processes, rather than posteriorly‐shifted anomalies, may be the primary indicators for detecting developmental toxicity, since CP injection increased the frequencies of these anomalies.

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs

“…For example, cyclophosphamide produced a high incidence of wavy ribs (Padmanabhan and Hameed, 1985;Hessel et al, 1994). Data from several studies to determine the sensitive period of exposure for cyclophosphamide indicate that GD 14 or 15 were the most critical times (Padmanabhan and Hameed, 1985;Hessel et al, 1994;Igarashi, 1998).…”

Increases in discontinuous rib cartilage and fused carpal bone in rat fetuses exposed to the teratogens, busulfan, acetazolamide, vitamin A, and ketoconazole

“…Skeletal examinations were conducted with reference to an atlas of term fetuses and related literature. 5,6,[19][20][21] The examinations were conducted using stereomicroscope at a GLP facility by trained personnel.…”

“…Although cartilaginous changes are induced by several drugs such as cyclophosphamide and sodium valproate, in some cases Alizarin Red S staining alone could not detect the changes; and the importance of cartilaginous changes was not considered due to the unclear biological significance. [4][5][6][7] Among cartilaginous changes, discontinuous rib cartilage (DRC) and fused carpal bone (FCB) are easily identified in rat fetuses using Alcian Blue staining. In a previous study to evaluate the utility of Alcian Blue staining, we demonstrated that acetylsalicylic acid given at 125-500 mg/kg to pregnant rats on gestation day (GD) 10 induced irreversible cartilaginous changes consisting of DRC and FCB in the fetus.…”