In vivo/in vitro studies on the effects of cyclophosphamide on growth and differentiation of hamster palate.

A study was undertaken to examine the effects of cyclophosphamide (CP) on growth and differentiation of palatal tissues. An in vivo/in vitro approach was designed to analyze (1) whether the damage caused by in vivo administration of CP in the developing palate can be altered in vitro, and (2) to determine the effects of CP on the synthesis of collagen and glycosaminoglycan (GAG), which are essential for proper palate development. In addition, effects of vitamin B1 and/or B6 on in vivo modulation of CP teratogenicity was evaluated. Pregnant hamsters were given 30 mg/kg CP or 1 ml saline on day 10 of gestation. Control and CP-treated embryonic palates were dissected on day 11 of gestation and incubated in vitro in the presence or absence of CP. In order to allow metabolic activation of CP in vitro, either a slice of hamster liver or microsomal S9 fraction of liver was added to the culture medium. To study collagen and GAG synthesis, palates were obtained between days 10 and 13 of gestation, and incubated in growth medium supplemented with [14C]proline or [3H]glucosamine, as appropriate. The rates of collagen and GAG synthesis were determined. The results showed that, in the controls, the presence of a liver slice or S9 fraction in the culture medium had no effects on in vitro closure of palate. In vivo CP exposed palates did not fuse in vitro. When drug was given in vitro, or both in vivo and in vitro, palatal closure did not occur. CP reduced synthesis of both collagen and GAG in the vertically developing palate. The drug-treated shelves reoriented only after the rates of collagen and GAG synthesis were restored to the levels comparable to the control counterparts. Co-administration of vitamin B1 and B6 did not interfere with the teratogenicity of CP. It was suggested that CP treatment affected DNA synthesis and injured growing cells, which in turn reduced the synthesis of GAG and collagen and affected the expansion of shelf volume to delay the reorientation of the palatal shelves. Furthermore, it appears that in vivo treatment with CP changes the programming of palatal tissues to prevent the fusion process in vivo, which could not be altered in vitro.