The Nitric Oxide Synthesis Inhibitor Nω-Nitro-L-Arginine Methyl Ester (L-NAME) Causes Limb Defects in Mouse Fetuses: Protective Effect of Acute Hyperoxia

Abstract

In the present study the relationship between exposure to the nitric oxide synthesis inhibitor N -nitro-L-arginine methyl ester (L-NAME) and the induction of limb defects, with respect to stage specificity and dose dependency, was investigated in the mouse. ICR (CD-1) mice were dosed s.c with L-NAME at 50 or 90 mg/kg on gestation d 12, 13, 14, 15, or 16. A group of animals treated with vehicle on gestation d 14 served as control. Uterine contents were evaluated for teratogenesis on gestation d 18. A treatment-related disruption of limb development was noted. The effect was dose dependent and phase specific. L-NAME became teratogenically operational on gestation d 13 and elicited its maximum effect on gestation d 14, whereas no significant teratogenicity was observed when exposure occurred after gestation d 15. In utero exposure to L-NAME also reduced embryo viability relative to controls. When the higher dose was injected on gestation d 16, a significant number of dams delivered preterm. In a parallel study, the ability of hyperoxia to prevent limb teratogenesis was investigated. To this aim, a group of L-NAMEtreated animals (90 mg/kg s.c. on gestation d 14) were exposed to 98 to 100% O 2 for 12 h. L-NAME-treated mice breathing room air served as positive controls. In response to hyperoxia, a significant decrement of L-NAME-induced limb defects was found. This study characterizes for the first time the teratogenic capacity of L-NAME in the mouse. Results obtained with hyperoxia fit the hypothesis that hypoxic tissue damage may play a contributory role in L-NAME-induced limb defects. NO is a colorless gaseous molecule involved in the regulation of diverse important physiologic processes, including vascular tone, platelet reactivity, smooth muscle contractility, neurotransmission, and the cytotoxic action of immune cells (1). NO is generated endogenously during the conversion of L-arginine to citrulline by a family of enzymes known as NOS. At least three isoforms of NOS have been isolated, including the constitutive eNOS and neuronal NOS isoforms, and the inducible isoform, iNOS (1). Pharmacologic manipulation of NOS activity can be achieved by several classes of NOS inhibitors including L-arginine analogs, which act by denying NOS its substrate in a competitive manner (2).The L-arginine analog L-NAME, a nonselective inhibitor of NOS, has been shown to be teratogenic when administered to rats (3-8). Limb reduction defects with associated characteristic areas of macroscopic hemorrhage were the most common phenotypic alterations noted (3-8). L-NAME is not the only L-arginine analog known to cause dysmelia, as N -nitro-Larginine also induced limb defects (9). Notably, disruptions similar to those induced by L-arginine analogs have been noted in knockout mice lacking eNOS (10, 11). The current state of knowledge on L-NAME teratogenicity also includes the following facts: 1) the period of fetal vulnerability has an onset relatively late in gestation (3-5); 2) both oral (3-6) and parenteral (5) treatments are effective;...

Production of axial skeletal malformations with the nitric oxide synthesis inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) in the mouse

“…In agreement with previous observations (Tiboni and Giampietro, 2000;Tiboni et al, 2003), L-NAME failed to induce detectable signs of maternotoxicity, suggesting that alterations in maternal physiology were not involved in mediating the teratogenic action of L-NAME. Favoring the hypothesis that L-NAME acted by a direct effect on the developing conceptus is the notion that L-NAME can cross the placental barrier (Fantel et al, 1999).…”

“…Among them, the N G -nitro-L-arginine methyl ester (L-NAME) is the agent used most frequently. Insight gained with rat (Diket et al, 1994;Pierce et al, 1995;Fantel et al, 1997Fantel et al, , 1999Fantel and Person, 2002) and mouse (Tiboni et al, 2003) models have shown that in utero exposure to L-NAME is teratogenic, causing limb reduction defects. In contrast with the majority of teratogenic agents, which act by interfering with organogenetic processes, L-NAME-mediated limb teratogenesis seems to be dependent on disruption of already developed structures.…”

Impact of maternal endothelial nitric oxide synthase gene polymorphisms on blood pressure, protein excretion and fetal outcome in pregnancy

“…35,41 Similar defects have been described in mice deficient in endothelial NO synthase, 36,42 but not in other NO synthase isoforms. An association between the NOS3 A922G polymorphism and orofacial clefts has been reported.…”

Hypoxia and the Edema Syndrome: elucidation of a mechanism of teratogenesis

“…Nitric oxide has a demonstrated role in vasodilation, and genetic ablation (Gregg et al, 1998;Hefler et al, 2001;Tiboni et al, 2003) or chemical inhibition of nitric oxide synthase (Diket et al, 1994;Fantel et al, 1997Fantel et al, , 1999 produces limb defects preceded by hemorrhage, similar to what is observed following hypoxia. Almokalent and dofetilide are potassium channel-blocking drugs that have been shown in rats to produce defects associated with hemorrhage at different developmental stages including missing digits, limb defects, and tail defects (Abrahamsson et al, 1994;Danielsson et al, 1997;Webster et al, 1996).…”