Melissa S. Tassinari

Resorption of implanted bone prepared from normal and warfarin-treated rats.

Bone that was virtually depleted of the vitamin K-dependent protein, osteocalcin, and 93% reduced in the concentration of its characteristic amino acid, gamma-carboxyglutamic acid, was obtained from rats treated with warfarin for 6 wk. Osteocalcin-deficient bone particles were resistant to resorption when implanted subcutaneously in normal rats. The relative resorption was 60% of control bone, as measured by histomorphometry as percent of bone particles in the field. Additionally, the number of multinucleated cells around the bone particles was reduced by 54%. These data suggest that osteocalcin is an essential component for bone matrix to elicit progenitor-cell recruitment and differentiation necessary for bone resorption.

Current and future needs for developmental toxicity testing.

A review is presented of the use of developmental toxicity testing in the United States and international regulatory assessment of human health risks associated with exposures to pharmaceuticals (human and veterinary), chemicals (agricultural, industrial, and environmental), food additives, cosmetics, and consumer products. Developmental toxicology data are used for prioritization and screening of pharmaceuticals and chemicals, for evaluating and labeling of pharmaceuticals, and for characterizing hazards and risk of exposures to industrial and environmental chemicals. The in vivo study designs utilized in hazard characterization and dose-response assessment for developmental outcomes have not changed substantially over the past 30 years and have served the process well. Now there are opportunities to incorporate new technologies and approaches to testing into the existing assessment paradigm, or to apply innovative approaches to various aspects of risk assessment. Developmental toxicology testing can be enhanced by the refinement or replacement of traditional in vivo protocols, including through the use of in vitro assays, studies conducted in alternative nonmammalian species, the application of new technologies, and the use of in silico models. Potential benefits to the current regulatory process include the ability to screen large numbers of chemicals quickly, with the commitment of fewer resources than traditional toxicology studies, and to refine the risk assessment process through an enhanced understanding of the mechanisms of developmental toxicity and their relevance to potential human risk. As the testing paradigm evolves, the ability to use developmental toxicology data to meet diverse critical regulatory needs must be retained.

Timing of implantation and closure of the palatal shelf in New Zealand white and Japanese white rabbits.

Two specific developmental events, namely implantation and palatal shelf closure, are of specific interest because they define, respectively, the beginning and the end of the treatment period in embryo-fetal developmental toxicity studies for pharmaceutical products. Thus, a detailed evaluation of the timing of implantation and closure of the hard palate is necessary to assure use of the proper exposure window in developmental toxicity studies in rabbits, the nonrodent species most commonly evaluated in regulatory developmental toxicology studies. The purpose of this study was to determine the timeline for implantation and closure of the hard palate in the New Zealand White rabbit, and to determine if this timeline differed in the Japanese White rabbit. To describe the timing of implantation, the uteri from does of the New Zealand White rabbit and the Japanese White rabbit were examined on gestation days (GDs) 5 through 8 for macroscopic evidence of implantation. To assess palatal shelf closure, fetuses were removed on GDs 17, 18, and 19 and fixed in Bouins solution. The fetuses were then categorized into five stages of palatal shelf closure: open (Stage I); approach of the palatal shelves (Stage II); partial closure of the hard palate (Stage III); full closure of the hard palate (Stage IV); and full closure of the soft palate (Stage V). In both the New Zealand White and Japanese White rabbit strains, implantation was initiated on GD 6.5 and was completed on GD 7. Partial closure of the palate began on GD 17.5, and by GD 19, closure of the hard palate was completed in all fetuses, and closure of the soft palate was completed in 75–96% of the fetuses. The timing of implantation and palatal shelf closure were comparable between the New Zealand White rabbit and the Japanese White rabbit. Therefore, treatment beginning on GD 7 and continuing until GD 19 encompasses the period of major organogenesis and is considered appropriate for use in developmental toxicity studies using either of these two strains of rabbits.

Cell Structure and the Regulation of Genes Controlling Proliferation and Differentiation: The Nuclear Matrix and Cytoskeleton

In this chapter and in the one which follows we will present concepts and experimental approaches associated with the relationship of proliferation to differentiation with emphasis on the contribution of cell structure to the regulation of cell growth and tissue specific gene expression. While these relationships are of broad biological relevance, we will focus primarily on development of the osteoblast phenotype with the understanding that analogous principles apply to the regulation of phenotype expression in general.

Cell Structure and Gene Expression: Contributions of the Extracellular Matrix to Regulation of Osteoblast Growth and Differentiation

The role of the extracellular matrix of specialized tissues in promoting cellular differentiation has long been recognized (1–2). Our studies have utilized rat osteoblast cultures (3–5) as a model system to examine a well defined extracellular matrix (ECM) and the coordinate regulation of the changes in cell structure and gene expression as related to its formation. These cells produce a mineralized ECM having a bone tissue-like organization analogous to embryonic bone (4–6). In contrast to tumor-derived or transformed osteosarcoma cell lines (7), those normal diploid bone derived cells in culture exhibit normal cell cycle regulated expression of genes which is functionally coupled to expression of differentiation specific genes (4). As the ECM develops, osteoblasts differentiate, progressing through three stages of development, a proliferation period, a period of matrix maturation, and a mineralization period (4). In the previous chapter, temporal expression of genes characterizing the osteoblast phenotype associated with this differentiation in vitro has been described in detail and is summarized in Figure 1.