Role of free radicals in the limb teratogenicity of L-NAME (N(G)-nitro-(L)-arginine methyl ester): a new mechanistic model of vascular disruption.

In continuing studies of limb effects resulting from fetal exposure to N(G)-nitro-(L)-arginine methyl ester (L-NAME), we examined the early time course of vascular changes and the effectiveness of fetal intraamniotic injection. Vascular engorgement and hemorrhage occurred within 4 hr of L-NAME treatment on gestational day (gd) 17, and direct injection appeared to be as effective as maternal intraperitoneal injection in inducing limb hemorrhage. Further studies examined protein nitration and electron transport inhibition in tissues of exposed fetuses. L-NAME caused significant increases in nitrotyrosine (NT) formation in limb but not in heart or brain, and reduced electron transport rates in limb. Three agents, alpha-phenyl-N-t-butylnitrone (PBN), a radical trap and inhibitor of inducible nitric oxide synthase (iNOS), allopurinol, an inhibitor of xanthine oxidase, and aminoguanidine, a relatively specific inhibitor of iNOS, significantly moderated limb hemorrhage and protein nitration in distal limb. These results suggest that L-NAME works directly on the fetal limb vasculature and indicate a cytotoxic role for peroxynitrite, a potent oxidant and nitrating agent that is the reaction product of nitric oxide and superoxide anion radical. We propose that L-NAME and other vasoactive toxicants disrupt the fetal limb in a sequential process. Initially, nitric oxide (NO) is depleted, causing hemorrhage and edema in the limb. Within hours, iNOS is induced, resulting in cytotoxic tissue concentrations of NO and reactive nitrogen species that induce apoptosis and/or necrosis in the limb. We suggest that L-NAME exposure may serve as a model of vascular disruptive limb malformations.