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 treatm ent on gestational day (gd) 17, and direct injection appeared to be as effe ctive as maternal intraperitoneal injection in inducing limb hemorrhage. Fu rther studies examined protein nitration and electron transport inhibition in tissues of exposed fetuses. L-NAME caused significant increases in nitro tyrosine (NT) formation in limb but not in heart or brain, and reduced elec tron 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 rel atively specific inhibitor of iNOS, significantly moderated limb hemorrhage and protein nitration in distal limb. These results suggest that L-NAME wo rks directly on the fetal limb vasculature and indicate a cytotoxic role fo r peroxynitrite, a potent oxidant and nitrating agent that is the reaction product of nitric oxide and superoxide anion radical. We propose that L-NAM E and other vasoactive toxicants disrupt the fetal limb in a sequential pro cess. Initially, nitric oxide (NO) is depleted, causing hemorrhage and edem a in the limb. Within hours, iNOS is induced, resulting in cytotoxic tissue concentrations of NO and reactive nitrogen species that induce apoptosis a nd/or necrosis in the limb. We suggest that L-NAME exposure may serve as a model of vascular disruptive limb malformations. Teratology 60:151-160, 199 9. (C) 1999 Wiley-Liss, Inc.