Elsevier

Reproductive Toxicology

Volume 99, January 2021, Pages 152-159
Reproductive Toxicology

Valproic acid alters nitric oxide status in neurulating mouse embryos

https://doi.org/10.1016/j.reprotox.2020.08.012Get rights and content

Highlights

  • VPA treatment arouses a decrease of constitutive NOS and increase inducible NOS in neurulating mouse embryos.

  • VPA treatment increases the levels of tyrosine nitration and S-nitrosylation.

  • VPA treatment decreases antioxidant defense systems.

Abstract

The molecular bases of the teratogenic effects elicited by valproic acid (VPA) are not fully defined. It was previously shown that inhibition of nitric oxide (NO) synthesis is associated with an enhancement of the teratogenic effects of VPA, while amplification of NO signal by sildenafil prompted a dose-dependent reduction of VPA-induced neural tube defects. In this study, for the first time, the effect of VPA on the NO synthesis was evaluated in mouse embryos during early organogenesis. On gestation day 8, ICR-CD1 mice received 600 mg/kg of VPA. Eight and 24 h later embryos were collected and analyzed for NO synthase (NOS) isoform expression, and for molecular mechanisms involved in their modulation. As main finding, in utero embryonic exposure to VPA determined a time-dependent shift of NOS isoforms expression, with a down regulated expression and activity of constitutive NOS (cNOS) and an increased expression and activity of inducible NOS (iNOS). The teratological relevance of this information remains to be established.

Introduction

Valproic acid is widely used in the treatment of several neurological condition, including epilepsy, bipolar disorders and migraine. Gestational exposure to VPA has been consistently associated with increased risk of both malformations and neurodevelopmental disorders in humans and experimental animals, in a time and dose-dependent manner [[1], [2], [3], [4]]. The offspring of women receiving VPA during pregnancy shows an increased risk of neural tube defects (NTDs), cardiovascular defects, urogenital anomalies, skeletal malformations and limb defects [1,4]. Although several mechanisms have been postulated to explain VPA teratogenicity, including interference with folate metabolism, increased oxidative stress, inhibition of HDAC, NO depletion, epigenetic changes and inhibition of NMDA receptor [[1], [2], [3], [4], [5], [6], [7], [8], [9]], the exact mechanism(s) of VPA-mediated teratogenicity are yet unknown, and many basic questions have yet to be answered. Previous studies carried out in our laboratory suggested that NO can provide protection from VPA-mediated teratogenesis. When pregnant mice were treated during early organogenesis (gestation day 8) with a sub-teratogenic dose of the non-selective NOS inhibitor L-NAME the teratogenic effects of VPA were enhanced [7], as evidenced by the increased occurrence of axial skeletal malformations and anterior NTDs (exencephaly). On the other hand, treatment with sildenafil citrate, a type 5 phosphodiesterase inhibitor that is able to amplify the NO signal by blocking degradation of cGMP, dose dependently prevented the VPA-induced exencephaly [8]. The mechanism(s) behind these results remained undefined. According to recent suggestions, sildenafil may act by reducing the availability of NO for oxidation to peroxynitrite, or by prolonging NO in embryonic tissue and thereby minimizing VPA-induced NO depletion [10]. Attenuation of peroxynitrite-mediated inhibition of the NMDA receptor has also be proposed as a mechanism involved in the rescue effect of mediated by sildenafil [10].

NO is generated starting from l-arginine by a family of three NOS, nNOS (NOS1), iNOS (NOS2), and eNOS (NOS3). Each NOS isoform is encoded by a distinct gene, and is expressed in a variety of tissues and cell types. All nucleated mammalian cells possess at least one of the three conserved NOS enzymes. Neuronal NOS and eNOS are constitutively expressed mainly in the nervous system and the vascular endothelium, respectively, synthesizing a small amount of NO in a calcium-dependent manner both under basal conditions and upon stimulation. In contrast, iNOS is induced when stimulated by microbial endotoxins or certain pro-inflammatory cytokines, producing a larger amount of NO in a calcium-independent manner. NO is able to perform its physiological effects by the generation of its second messenger cGMP derived by the binding of NO with guanylate cyclase, a heme protein. It is important to notice that NO appears to be implicated in several biological phenomena that are crucial for organogenesis, including gene expression, cell growth, matrix remolding, proliferation, differentiation and apoptosis [[11], [12], [13]].

In order to expand information about the mechanistic basis for an involvement of NO system in VPA teratogenesis, in the present study, we set out to delineate the impact of a teratogenic insult with VPA on the NOSs expression status, and the molecular mechanisms involved in their modulation in neurulating mouse embryos. In addition, in order to gain further insight into the role of ROS in VPA-induced teratogenesis, we also evaluated redox-related state in embryonic tissue.

Section snippets

Animals and housing

Sexually mature and pathogen free ICR (CD-1) mice (24–26 g) were obtained from Harlan Italy (Udine, Italy). Upon arrival, animals were maintained in clear polycarbonate cages and allowed free access to rodent laboratory chow (Altromin-MT®, Italy) and filtered tap water. Animal room was maintained at 22 ± 1 °C with a relative humidity of 55 ± 5 %. The animals were maintained on a 12 -h light/dark cycle. Timed mating was produced by placing an individual male of the same stock into cages

Effects of VPA on NOSs in embryonic tissue

The first aim of this study was to investigate the effect of in utero exposure to VPA during neurulation on embryonic NOSs gene and protein expression (Fig.1). As shown in Fig. 1A, the control embryos (sham treated group) express both cNOS (eNOS, nNOS) and iNOS mRNAs at both time (8 h and 24 h), but with higher levels for cNOS. VPA treatment determined a downregulation of eNOS-mRNA levels at 8 h and 24 h, and at 24 h for nNOS compared to sham treated group, respectively. Regarding iNOS mRNA

Discussion

This study shows for the first time that a teratogenic insult with VPA given during early organogenesis is associated with an altered NOS expression in mouse embryonic tissue. In particular, data are consistent with a significant decrement of cNOS expression and a concomitant increase of iNOS after both 8 and 24 h exposure to VPA.

It has become increasingly clear that proper endogenous levels of NO are required for normal morphogenesis [24], and several studies are consistent with the concept

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors report no declarations of interest.

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