In explanted humeri of late fetal rats, retinoic acid was found to induce the release of proteoglycan followed by cartilage resorption. Tissue breakdown, which was demonstrated by losses of DNA, RNA, and protein, coincided with the appearance of necrotic cells. In control humeri chondrocytes were the main cell type, but in humeri treated for 4 days with retinoic acid the surviving cells were chondroblastlike. Sensitivity of proteoglycan release and tissue breakdown to retinoic acid decreased with age.The proteinase inhibitors cysteine, Trasylol, and soya and lima bean trypsin inhibitors did not antagonize the effects of retinoic acid. Phenylmethanesulfonyl fluoride suppressed cartilage resorption more effectively than proteoglycan release, while pepstatin merely suppressed cartilage resorption. The inhibition by EDTA of both the release of proteoglycan and cartilage resorption induced by retinoic acid was dose dependent. Zn abolished these effects, whereas Mn only relieved the release of proteoglycan induced by retinoic acid; this indicates that these two effects of retinoic acid are not necessarily linked.In view of our recent demonstration that the release of proteoglycan induced by retinoic acid requires RNA and protein synthesis, we suggest that the degradation of proteoglycans in response to retinoic acid is dependent upon continued synthesis of metalloproteinases.

Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.

Three retinoids of the arotinoid series, namely the free carboxylic acid Ro 13-7410, its ethyl ester Ro 13-6298, and the new arotinoid ethyl sulfone Ro 15-1570, were tested for their embryotoxic and teratogenic activity in rats. The retinoids were administered orally on either day 9 or 13 of gestation. Treatment on day 9 of gestation resulted mainly in malformations of the head and the trunk; whereas, on day 13 limb malformations were prominent. Ro 13-7410 and Ro 13-6298 were about 1000 times more embryotoxic and teratogenic than retinoic acid but induced a similar malformation pattern to retinoic acid. In contrast, the sulfur-containing arotinoid Ro 15-1570 was active at similar dose levels to retinoic acid but caused a peculiar malformation pattern on day 13 of gestation. This finding supports the hypothesis that the arotinoid ethyl sulfone Ro 15-1570 has unique biological properties, inducing no bone toxicity in adult rats and distinctly affecting limb development.

Daily oral treatment with retinoic acid (100 mg/kg bodyweight) induced regression of a transplantable rat chondrosarcoma. In a previous biochemical investigation we have shown that the tissue breakdown is preceded by the loss of proteoglycan. The present study describes the histological changes induced by retinoic acid. A decrease in the intensity of metachromatic staining with toluidine blue was noted already after 1 day and the discoloration was almost complete after 4 days correlating with the loss of proteoglycan. Especially in the perichondrium there was a rapid proliferation of fibroblasts and monocytes. Osteoclast-like cells were missing, but tumor nodules were eroded and split up by penetrating perichondrium. After 4 days of treatment larger necrotic areas were found, initially in the center of tumor nodules only. In other areas the majority of tumorous chondroblasts survived. Tumor nodules appeared partly mesenchyma-like with some fibroblast-like cells suggesting a dedifferentiation of chondroblasts by retinoic acid. we believe that tumor regression induced by retinoic acid involved proteoglycan degradation by chondroblasts themselves and chondroclast-like activity of monocytes and fibroblasts.

The activity of 18 vitamin D analogs on soft tissue calcification and growth impairment in neonatal rats and their effect on bone calcium mobilization, intestinal calcium absorption and binding to intestinal 1,25-dihydroxyvitamin D3 receptors in adult rats were compared. Depending on the chemical modification of the vitamin D parent compounds, they could be separated into active and inactive analogs. Cholecalciferol and ergocalciferol were similarly active, but epimerization of ergocalciferol at carbon 23 caused loss of activity. Hexafluorination at carbon 26 and 27 and the introduction of a double bond at carbon 22 or 23 had no or little effect on the activity. The loss of activity was caused by the introduction of a triple bond at carbon 23 and by hydroxylation at carbon 23, 26 or 28. The differentiation of human promyelocytic leukemia cells (HL-60) induced by these derivatives was used as a parameter for antitumour activity. All six analogs, which markedly affected calcium metabolism, were highly active in HL-60 cells. However, at least three derivatives were highly active in the antitumour test but failed to induce hypercalcemia. Thus, these results indicate that it could be possible to develop medically useful vitamin D derivatives devoid of hypercalcemic side-effects.