Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1987

Abstract

This study demonstrates the use of submerged limb culture in teratologic testing. Pregnant mice were treated on day 11 of gestation (E11, plug date = E0) with 10, 20 or 40 mg of 5-fluorouracil (FU) per kg body weight. On E17, treated and untreated fetuses were examined for gross malformations and were fixed in 95% ethanol. Reduction of limb size and digital defects, including ectrodactyly (ED), syndactyly (SD), microdactyly and polydactyly were dose-dependent. In parallel studies, pregnant mice were treated on the morning of E11 and embryos were removed either 7h (E11) or 24h (E12) later for submerged limb culture. Changes in limb area showed a dose-response relationship while treatment had little effect on the shape of individual bones. This indicates the relatively unspecific nature of FU-induced embryotoxicity. E11 studies revealed a dose dependent response of ED, SD and fusion of the metacarpals/metatarsals (MC/MT) to the proximal phalanges. Unlike E11 cultures, middle phalanges were present but decreased in number as dosage increased. Limbs from embryos of untreated females were cultured (E11) in the presence of 0.002, 0.02, 0.2 or 2.0 mg FU/ml culture medium. The percentage of limbs void of paw cartilage or with decreased numbers of MC/MT was dose-dependent. A dose-dependent decrease in the deleterious effects of 0.02 mg FU/ml was observed when 0.2 or 0.02 mg thymine/ml was added to the cultures. In both culture and non-culture studies, hindlimbs (HL) were more affected than forelimbs (FL) and distal regions were more affected than proximal ones. In addition to the morphometric analyses, biochemical parameters of growth and differentiation were examined at 0, 36 and 72h of culture in untreated and treated limbs. Both DNA and protein of FU treated limbs were decreased compared to untreated controls. FL demonstrated greater capacity for regulation of losses in protein content, HL for DNA content. Submerged limb culture provides a useful model for the examination of xenobiotic effects on limb development and allows some comparative evaluation among in vivo, in vivo/in vitro and in vitro studies. (Abstract shortened with permission of author.)

Document Type

Dissertation - Open Access

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