Tumor promoter actions on rat embryonic development in culture

The many embryonic and developmental features associated with tumor promotion have prompted us to investigate the effects of a series of phorbol esters and related diterpene tumor promoters on mammalian embryogenesis. A culture system which supports the normal development of 10.4 day organogenesis-phase rat conceptuses was utilized. In this system, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent Stage I and II promoter, disrupted the morphology and function of the embryonic visceral yolk sac (dose required to affect 50% of conceptuses, 18 nM). The effect was characterized by an abnormal, progressive separation of the two cellular layers of the yolk sac, but cellular differentiation appeared to be uninterrupted. Parallel log dose-response lines for this effect were produced by phorbol-12,13-dibenzoate (dose required to affect 50% of conceptuses, 200 nM) and phorbol-12,13-diacetate (dose required to affect 50% of conceptuses, 300 nM) which are consistent with structure-activity relationships for other promotional actions of these compounds. In addition, the weak Stage I promoter, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, produced identical effects but was 1400 times less potent than was TPA, while mezerein, a potent Stage II promoter, was as potent as was TPA. These observations support the hypothesis that embryonic cells may be differentially sensitive to early- and late-stage promoters. Ethylphenylpropiolate, a nonpromoting hyperplastic agent in mouse skin, had no effect on yolk sac morphology or function at its maximum solubility (1.85 mM). Yolk sac disruption by TPA was potentiated by heat inactivation (56 degrees, 30 min) or 0.45-micron filtration of the culture medium. A more advanced stage of yolk sac development was less sensitive to TPA disruption since 11.4 day conceptuses, which were cultured for 30 hr, developed identical lesions, but TPA was at least 5-fold less potent. Thus, the tumor promoter-induced lesions of the rat yolk sac have some features consistent with late-stage tumor promotion and do not appear to be associated with general toxicity, hyperplasia, or alterations in cellular differentiation. We postulate that rat conceptuses maintained in vitro may provide an important model system for the study of the proposed mechanisms involved in chemical tumor promotion.