32 Poster abstracts / Reproductive Toxicology 41 (2013) 21–34

Results: Fetuses from the first group appeared with cerebralhernia and umbilical hernia, while their weight was approxi-mately 0.42–0.45 g. They were underweighted compared to the4.58–5.36 g fetuses of the control group. The same verificationapplies for their placentas, which weighted 0.35–0.46 g comparedto the 0.77–1.14 g control group fetuses. Light microscopical study,showed pathological changes of the space between their pla-centa villi with apoptotic cells accumulation and ruptures of thematernal–fetal placenta barrier at the embryogenic part of the pla-centa.

Conclusions: Based on our study results, MTX’s use during preg-nancy even at a single dose could lead to reduced fetal development,congenital defects and maternal–fetal placenta barrier ruptures.Furthermore, the use of MTX during pregnancy increases the riskof congenital abnormalities and should be avoided.

Reference

[1] Sachdeva P, Patel BG, Patel BK. Drug use in pregnancy; a point to ponder! Ind JPharm Sci 2009;71(1):1–7.

[2] Chambers DC, Tutuncu NZ, Johnson D, Jones KL. Human pregnancy safety foragents used to treat rheumatoid arthritis: adequacy of available information andstrategies for developing post-marketing data. Arthritis Res Ther 2006;8(4):215.

http://dx.doi.org/10.1016/j.reprotox.2013.06.075

Inhibition of nitric oxide synthesis enhances the teratogenicresponse to valproic acid

Gian Mario Tiboni ∗, Francesca Marotta, Alberto Verrotti

Dipartimento di Medicina e Scienze dell’Invecchiamento, Università“G. d’Annunzio” di Chieti-Pescara, Italy

Introduction: The mechanism of valproic acid (VPA) teratogenic-ity is poorly known. A molecular interplay between nitric oxide(NO) and VPA has been reported. This study was carried out toinvestigate the potential consequences of NO deprivation on VPAteratogenicity.

Methods: On gestation day 8 (plug day = gestation day 0) ICR(CD-1) mice were injected with 20 mg/kg of the NOS inhibitorN(G)-nitro-l-arginine methyl esther (l-NAME). The dose of l-NAMEwas selected because known to be below the teratogenic thresh-old [1]. Thirty minutes later animals received a teratogenic doseof VPA (400 or 500 mg/kg). Developmental endpoints, includingintrauterine mortality, fetal growth and incidence of structuralabnormalities, were evaluated near the end of gestation.

Results: No treatment-related effects on maternal weightparameters after VPA, alone or in combination with l-NAME, weredetected. As main teratological effect, VPA induced axial skeletaldefects, affecting 35.2% and 67.0% of fetuses exposed to 400 mg/kgor 500 mg/kg, respectively. The spectrum of vertebral anomaliesincluded fused, asymmetric, and cleaved vertebrae, and vertebraewith asymmetric, cleaved, and dumbbell-shaped centrum. Abnor-mal vertebral phenotypes were found in thoracic, lumbar, sacral,and caudal vertebrae. Rib malformations consisted mainly of ribfusion. Regarding neural tube defects, exencephaly was observedin 2.3% and in 4.1% after VPA at 400 mg/kg or 500 mg/kg, respec-tively. Pre-treatment with l-NAME enhanced VPA teratogenicity. Asignificant increase in the rate of fetuses displaying axial skeletaldefects was observed when l-NAME was co-administered to VPAat 400 mg/kg, yielding this treatment regimen a 53.7% of skele-tally affected fetuses. This 50% increase in comparison to rateobserved after VPA 400 mg/kg alone (35.2%) was statistically sig-nificant. There was a trend toward increase in the rate of skeletallyaffected fetuses when l-NAME was co-administered to VPA at500 mg/kg group, from 67% (VPA at 500 mg/kg) to 76% (l-NAMEplus VPA 500 mg/kg), but there were no statistically significant

differences between these values among the groups. Concerningexencephaly, the low incidence (2.3%) of affected fetuses seen afterVPA 400 mg/kg alone was unaffected by l-NAME. On the other hand,when l-NAME was administered with VPA at 500 mg/kg a signif-icant enhancement in the rate of exencephalic fetuses resulted.Indeed, if only 4.1% of fetuses exposed to VPA at 500 mg/kg wasexencephalic, the percentage of affected fetuses raised to 22.2%after pre-treatment with l-NAME.

Conclusions: The present study shows that inhibition of NOsynthesis can result in an enhancement of the teratogenic effectsinduced by VPA.

Reference

[1] Tiboni GM, Marotta F, Barbacane L. Production of axial skeletal malformationswith the nitric oxide synthesis inhibitor NG-nitro-l-arginine methyl ester (l-NAME) in the mouse. Birth Defects Res B Dev Reprod Toxicol 2007;80:28–33.

http://dx.doi.org/10.1016/j.reprotox.2013.06.076

Prenatal and early postnatal administration of cyclooxygenaseinhibitors does not significantly change cartilage morphologyand physiology

F. Burdan 1,2,∗, A. Wrona 2, J. Szumiło 3, R. Klepacz 3, W. Wrona 4, J.Dudka 5

1 Department of Human Anatomy, Medical University of Lublin, Poland2 St. John’s Cancer Center, Lublin, Poland3 Department of Clinical Pathomorphology, Medical University ofLublin, Poland4 3rd Department of Gynecology, Medical University of Lublin, Poland5 Medical Biology Unit, Medical University of Lublin, Poland

Introduction: Drug administration during pregnancy and lac-tation may disturb fetal and neonatal physiology, since most ofthe xenobiotics easily cross the placenta barrier and pass intothe milk. The aim of the study was to evaluate the impact ofnon-selective (ibuprofen, piroxicam, tolmetin) and selective (DFU)cyclooxygenase-2 inhibitors on morphology of the elbow andshoulder joint; as well as on the expression of genes coding consti-tutive (COX-1) and inducible isoform (COX-2) of cyclooxygenase ata 7-day-old newborns exposed on the examined substances duringpre- and postnatal period.

Materials: The examined substances were administered topregnant rats from the 8 gestational day until the 7 lactationalday. Tolmetin and ibuprofen (8.5, 42.5 and 85 mg/kg/dose) wasgiven three times a day, while piroxicam (0.3, 1.5 and 3 mg/kg/dose)and DFU (0.2, 2.0 and 20.0 mg/kg/dose) once daily. Females weregiving birth naturally and on 7th day of lactation newborns weresacrificed. The limbs of the male pups were separated. After theroutine histological procedure and staining using histological andhistochemical methods, the joints morphology was evaluated. Theimmunoexpression of COX-1, COX-2 and prostaglandin receptorswas also checked. ELISA and RPA methods were employed to eval-uate the prostaglandins (PGE2, PGF2�) level and the expressionof genes coding both principal cyclooxygenase isoforms (COX-1,COX-2), respectively.

Results: The morphology of epiphyseal cartilages, distinguishingand accessory structures of the shoulder and elbow joint were notaffected in groups exposed to the examined substances. Secondaryossification centers and vascular boundless were found on the levelof epiphysis. A strong COX-1 immunostaining was observed in mostof chondrocytes, endothelial and synovial cells. However, COX-2immunoexpression was weaker and limited mainly to endothe-lium. Insignificant differences in morphology as well as intensityand/or localization of immunoexpresion of both COX isoforms were