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

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Abstract
Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death 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. To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.