Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos

Toxicological Sciences, Aug 2013

Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress–induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson’s disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations.

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Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos

Ava E.Schlisser 0 Barbara F.Ha 0 les 0 0 Department of Pharmacology and Therapeutics, McGill University , Montreal , Quebec, Canada - with IMARIS Software (Bitplane AG, Zurich, Switzerland). 3D isosurfaces of Statistical analyses. Statistical analyses were done by chi-square and twovehicle- and 600mg/kg hydroxyurea-treated embryos were generated. Onlwyay ANOVA, as appropriate, followed by a post hoc Bonferronis correction the lumbosacral somites were analyzed in this study because they were idfeonrtmi-ultiple comparisons using the GraphPad Prism computer program. The fied previously as a predominant site of 4-HNE protein adducts and nuclperiaorri level of significance was p<0.05. GAPDH translocation (Schlisser etal., 20Y1a0;n and Hales, 2006). Western blot analysis of cleaved caspase-3 and 4-HNE-protein adducts. Protein concentrations were determined using the Bio-Rad r esul Ts Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). For cleaved caspase-3 analysis and 4-HNE protein adduct determination, protDeinesprenyl Inhibited the Hydroxyurea-Induced Nuclear (20 g) from embryos from each treatment group were resolved by 10% Translocation of GAPDH in the Lumbosacral Somites Lactate assay. GD 9 embryos were pooled from vehicle- and drug-treated dams; there were four samples for each group with each sample representing two litters. Samples were homogenized in PBS, flash frozen, and stored at 80C. Lactate concentration was determined with the use of a Lactate Assay Kit (Biovision Research Products, Mountain View, CA). Ingredients, including lactate assay buffer, lactate probe (in anhydrous dimethyl sulfoxide), lactate enzyme mix, and L(+)-lactate standard (100nmol/l), were added to the homogenized samples. Lactate concentrations were determined by measuring the change in absorbance at 5n7m0. Lactate contents (nmol) were determined from the standard curve and adjusted for protein content, assessed using the Bio-Rad Bradford protein assay. Glutathione determinations. On GD 9, embryos were explanted into Hanks balanced salt solution, flash frozen in liquid nitrogen, and stored at 80C. Samples were defrosted and homogenized with 5-sulfosalicylic acid (5%, wt/vol). Both total (glutathione [GSH] + glutathione disulfide [GSSG]) and oxidized (GSSG) glutathione were assayed using the Microplate Assay foFri G.1. Analysis of the confocal images of GAPDH immunofluorescence GSH and GSSG from Oxford Biomedical Research (Cedarlane Laboratoriiens the lumbosacral regions of GD 9 embryos using IMARIS. The immunofluoLtd, Burlington, ON, Canada, Product no. GT40). Briefly, the reaction of GreSsHcence intensity of isolated nuclear GAPDH is represented here. HU 400, with Ellmans reagent (5,5-dithiobis-2-nitrobenzoic acid) gives rise to a p4ro0d0-mg/kg hydroxyurea; HU600, 600mg/kg hydroxyurea; D, deprenyl. Twouct that is quantified spectrophotometricallynamt (4S1P2ECTRAmax PLUS way ANOVA and a post hoc Bonferroni correction were done. Asterisks (***) 384, Molecular Devices, Sunnyvale, CA). A pyridine derivative is used asdaenote a statistically significant difference (p<0.001). denotes a significan thiol-scavenging reagent to assess the relative amounts of GSH and GdSifSfGer.ence between the hydroxyurea-treated groups in the absence and presence Oxidative stress is represented as the ratio of GSSG to GSH. of deprenyl (p<0.05). TABle1 Cesarean s ection o bservations for Dams Treated With Hydroxyurea and/or Deprenyl on GD9 Deprenyl HU (400mg/kg) HU (400mg/kg) + deprenyl HU (600mg/kg) HU (600mg/kg) + deprenyl Number of dams 9 Implantation sites 12.02.8 Late resorptions 0.30.5 Viable fetuses 11.72.9 Fetal weights (g) 1.360.05 Note. The data are presented as mean/litter values SEM. Two-way ANOVA and a post hoc Bonferroni correction were used to determine significance. Asterisks (*) and (***) denote a statistically significant difference (p<0.05) and (p<0.0001), respectively, from controls; denotes a significant differen between the HU-treated groups in the absence or presence of deprenyl (p<0.05). of the hindlimbs (tibia and femur hypoplasia), hindlimb digit hypoplasia, shortening of the forelimbs (hypoplasia of the radius and ulna), partial ossification of the supraoccipital bon and partial ossification of ribs and sternum (Fig.3). A quantitative analysis of the predominant lumbosac-ral ver tebral, hindlimb, and tail defects observed after treatment wi hydroxyurea in the absence or presence of deprenyl is presented in Table2. Exposure to saline or deprenyl had either no effect or a minimal effect (mild shortening of the tail with depreny alone) on skeletal development (Table2). Fetuses from litte exposed to hydroxyurea alone had lumbosacral vertebral mal formations (400mg/kg: 10.0%; 600mg/kg: 53.3%), hindlimb malformations (40m0g/kg: 10.0%; 600mg/kg: 33.3%), and tail aplasia (400mg/kg: 40.0%; 600mg/kg: 86.7%). The incidence of lumbosacral vertebral and hindlimb malformations was significantly increased in the group exposed tom6g0/k0g hydroxyurea and deprenyl in comparison to those treated witmhg6/ 00 oD kg hydroxyurea alone (Table2). lnw o a d Effects of Deprenyl and Hydroxyurea on Glycolysis fed r o Deprenyl had no effect on glycolysis as assessed by lactmat h production (Fig.4). As anticipated, the exposure of organogenttpe : sis stage embryos to hydroxyurea decreased lactate producti/toon, / indicating that glycolysis was inhibited. Deprenyl coadmiscxnis i tration did not affect this hydroxyurea-induced decrease in l.oxactate production (Fig.4). frdo j o u r EffHecotmseoofsDtaespirsenyl and Hydroxyurea on Glutathione .lrsango / To assess the effects of deprenyl and hydroxyurea on thbye redox status of the embryos, we measured reduced (GSH) andu g e oxidized (GSSG) glutathione in embryos collecthedaf3ter tso hydroxyurea treatment (Fig.5). There were no significant dOif n ferences in GSH content in embryos treated with deprenyl altcobon or with 400mg/kg hydroxyurea in the absence or presence ofre 2 deprenyl compared with control (Fig.5A). Exposure tom6g0/0 ,12 kg hydroxyurea resulted in a significant depletion of emb10ry 4 onic GSH; the GSH content of embryos in the group exposed to deprenyl in combination with this dose of hydroxyurea was not different from control. The ratio of GSSG/GSH was computed as a measure of oxidative stress (Fig.5B). Deprenyl alone did not affect the GSSG/ GSH ratio. Treatment with either 400 or m60g0/kg hydroxy20(5) 1(1) 1(8) 2(2) 2(2) 1(1) 1(1) 2(2) 8(5)*** 16(8) 1(1) 1(1) 2(1) 2(1) 2(2) 2(2) 8(8)*** Deprenyl HU (400 mg/kg) HU (400 mg/kg) + deprenyl HU (600 mg/kg) HU (600 mg/kg) + deprenyl Fi G.3. Illustrations of some of the skeletal defects observed in GD 18 fetuses after exposure to saline (control) or depmregn/yklg ahnydr6o0x0yurea. Bones appear red (alizarin red S); cartilage appears blue (alcian blue). Malformations are indicated by arrows and described in the text. even in the absence of MAO-B (Leetal., 1997; Ta (...truncated)


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Ava E. Schlisser, Barbara F. Hales. Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos, Toxicological Sciences, 2013, pp. 391-399, 134/2, DOI: 10.1093/toxsci/kft115