Hydroxylamine moiety of developmental toxicants is associated with early cell death: A structure-activity analysis

Background: Cellular debris, an indicator of cell death, appears in limb bu ds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous in jection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea i nto the exocoelomic cavities of the embryos. This episode of early cell dea th appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. Methods: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogen esis, five structurally related hydroxylamine-bearing chemicals (hydroxylam ine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetoh ydroxamic acid, and hydroxyurethane) were administered at equimolar doses t o rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 mu m ol/embryo) injection on gestational day 12. Five additional chemicals, stru cturally similar to the hydroxylamine-bearing compounds, but possessing a t erminal amino group (-NH2) (ammonium hydroxide, methylamine, urea, acetamid e, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 m mol/kg or 1.30 mu mol/embryo) was co-administered with the hydroxylamine-be aring compounds to determine its effect on early cell death. Embryos were h arvested 4 or 8 hr after treatment and analyzed by light microscopy. Results: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compou nds produced an early episode of embryonic cell death. In all cases, the an tioxidant propyl gallate prevented or delayed the early episode of cell dea th observed after treatment with the hydroxylamine-bearing compounds. Conclusions: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compound s involves a free radical mechanism that requires the presence of a termina l hydroxylamine group for initiation. (C) 2000 Wiley-Liss, Inc.