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Prenatal effects of retinoic acid on lumbar spinal cord development and liver antioxidants in rats Janka Vaˇsková a , Peter Patleviˇc c,∗ , Ladislav Vaˇsko a , Darina Kluchová b Department of Medical and Clinical Biochemistry, Pavol Jozef Sˇ afárik University in Koˇsice, Faculty of Medicine, Trieda SNP 1, 040 66 Koˇsice, Slovak Republic Department of Anatomy, Pavol Jozef Sˇ afárik University in Koˇsice, Faculty of Medicine, Sˇ robárova 2, 041 80 Koˇsice, Slovak Republic c Department of Ecology, University of Presov in Presov, Faculty of Humanities and Natural Science, Ul. 17 novembra cˇ . 1, 081 16 Preˇsov, Slovak Republic a

b

a r t i c l e

i n f o

Article history: Received 16 December 2013 Received in revised form 6 February 2014 Accepted 10 February 2014 Available online xxx Keywords: Liver Lumbar spinal cord Neuronal nitric oxide Postnatal development Retinoic acid Rat

a b s t r a c t During embryonic and early postnatal development, retinoic acid (RA) regulates genes that control neuronal differentiation and neurite outgrowth from the neural tube. The effects of high levels of RA on the CNS can be detected via nitric oxide (NO), which plays a crucial role in neural transmission. The aim of the study was to investigate the prenatal influence of high levels of RA on postnatal development of nitrergic structures in lumbar spinal cord and antioxidant status. RA was administered orally at a dose of 10 mg/kg body weight to pregnant female Wistar rats during days 8–10 of gestation. Neuronal nitric oxide synthase (nNOS) of lumbar spinal cord sections was processed for visualization via nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry on postnatal day one, day twenty-one and in adults. The results suggest that prenatal administration of high levels of RA is not associated with postnatal morphological changes in nNOS-positive neurons in the rat lumbar spinal cord. An estimation of the activity of enzymes related to the storage of retinoid in the liver showed possible side effects. Suppression and deepening of superoxide dismutase activity persisted into adulthood, and a concurrent downregulation of glutathione reductase was noted. A decrease in reduced glutathione persisted until adulthood when other compensatory mechanisms were probably active to maintain an appropriate level. © 2014 Elsevier GmbH. All rights reserved.

Introduction Retinoic acid (RA), a member of the retinoid family of lipids and a mediator of vitamin A activity, is an essential morphogen found in different species from invertebrates to metazoans including humans (Maden, 2008). While normal embryonic development requires retinoids, they can elicit adverse effects on the developing embryo if present in the wrong amounts, at the wrong stage or the wrong time (Sporn and Roberts, 1991). Embryos of the killifish, Fundulus heteroclitus exposed to low concentrations of RA (0.0001–0.1 ␮mol/l) develop normally, whereas those exposed to higher concentrations (0.5–100 ␮mol/l) develop characteristic dose-dependent defects (Vandersea et al., 1998). In mice,

Abbreviations: GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; IML, intermediolateral nucleus; NADPH-d, nicotinamide adenine dinucleotide phosphate-diaphorase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; RA, retinoic acid; SOD, superoxide dismutase. ∗ Corresponding author. E-mail address: [email protected] (P. Patleviˇc).

administration of RA on day 7 or day 8 of gestation causes retardation of general development, abnormal differentiation of the cranial neural plate and abnormal development of the hind-brain (MorrisKay et al., 1991). Administration of RA on day 9 of gestation induces dysmorphogenesis of the inner ear in mice (Frenz et al., 1996). Abnormalities in limb and neural plate development have been induced through RA administration between days 10 and 16 of mice gestation (Stafford et al., 1995). A study by Holson et al. (1997) found, that influence of teratogenic factors on gestation days 8, 9 and 10 of rats are critical for development of the CNS and can lead to various malformations. Therefore, in our study, we administered high doses of RA to pregnant female rats on gestation days 8, 9 and 10. Retinoids are involved in the mechanisms underlying the inflammatory response, and have been related to the generation and expression of nitric oxide (NO; Seguin-Devaux et al., 2002), prostaglandins (Devaux et al., 2001) and cyclooxygenases (Li et al., 2002). In addition to the widely known beneficial processes where NO plays a role (including neurotransmission, blood pressure regulation, immunomodulation), under certain conditions production of NO (as well as other NOS-derived oxidants) can be

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detrimental owing to induced nitrosation and oxidative stress (Wink and Mitchell, 1998) or altered enzyme activities; for example P450-mediated drug metabolism (Miller, 2004). A family of three nitric oxide synthase 1 (NOS) isoforms produces nitric oxide (NO) and l-citrulline via a stepwise oxidation of the guanidinium nitrogen of l-arginine. Although all three NOS isoforms (two constitutively expressed isoforms; neuronal (nNOS) and endothelial (eNOS) and one inducible isoform (iNOS)) produce identical products, the function of NO varies widely in terms of physiological functions due to the varying localization of the isoforms within different cell populations of the body (Miller, 2004). Through NO production, the enzyme is involved in the promotion of axonal elongation and regulation of growth cone advancement (Van Wagenen and Rehder, 1999), and also causes cell death in the nervous system during development due to nitrosative stress (Freire et al., 2009). The histochemical reaction for NADPH-diaphorase (NADPH-d) colocalizes with neuronal NOS (nNOS), therefore the localization of NADPH-d is generally considered to be reflective of the presence of nNOS and is used as a specific histochemical marker for NO containing neurons (Kaur et al., 1999). In the spinal cord, NADPH-d activity has been detected in the dorsal horn, around the central canal, and at the intermediolateral cell column of the spinal cord (Xu et al., 2006). The liver is the principal organ responsible for the storage of retinol (Inder et al., 1999). The embryo is unable to synthesize retinol and is strongly dependent on the maternal delivery of retinol itself or precursors such as retinyl esters (retinyl palmitate) or carotenoids. Furthermore, the teratogenic effects of RA seem to appear about day 8 of gestation, possibly extending until day 12, during the initiation of abdominal formation (Quemelo et al., 2007). Retinol carrier molecules are present during this period, not only in the maternal placental decidua basalis, but also in the embryo from day 7 in the embryonic yolk sac and from day 11.5 post-coitum in the liver (Wendler et al., 2003). Administration of vitamin A and its derivatives in excess can lead to fibrosis and hepatocellular dysfunction (Choudhary and Swami, 2012). The toxicity mechanisms are poorly understood, but modulation of the hepatic function by vitamin A supplementation does affect the liver mitochondria structure and function (Leo and Lieber, 1999). Cellular responses to RA in vitro ranged from cell death to cell differentiation (Huang et al., 2006). We found no research studies that examined morphological changes of nitrergic structures in the lumbar spinal cord after prenatal influence of teratogenic RA. Our study focused on the NADPH-d/nNOS, around the central canal, dorsal horn and at the intermediolateral (IML) nucleus of the rat lumbar spinal cord until adulthood to assess if normal development was affected during this critical 3-day period of pregnant female rat overdose. Increased availability and utilization of RA by the embryo itself leads to the question of how the excess supply of RA could be used by the embryo, and the possibility that deviations from normal metabolic conditions might lead to the induction of oxidative stress in the retinol storage organ. Therefore, we also measured the activities of selected enzymatic and non-enzymatic antioxidants in liver mitochondria in the offspring of treated females.

Materials and methods Animals The study was carried out on the lumbar spinal cords and livers of Wistar rats of both sexes at postnatal day one, day twentyone and in adults. The rats were kept under standard conditions, subject to inspection from the Ethical Commission of the Medical ˇ in Koˇsice, Slovak Republic. Faculty, University of Pavol Jozef Safárik

The experiment was conducted in accordance with the “European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (86/609/EU) and with the approval of the State Veterinary and Food Administration of the Slovak Republic (No. 1696/07-221a). The rats had free access to food and drinking water. Retinoic acid administration Retinoic acid (R7632; Sigma, Schelldorf, Germany) was administered orally to 12 adult female rats at a dose of 10 mg/kg body weight (BW) on days 8, 9, and 10 of gestation (RA group) based on an earlier study (Holson et al., 1997). The control group consisted of 12 adult female rats given olive oil orally for the same period. Tissue processing At postnatal day one (P1), day twenty-one (P21) and in adults (P90), the experimental animals (8 animals per group, at P21 and P90 of both sexes in a 1:1 ratio, while individuals still came from the same litters) were deeply anesthetized with thiopental [50 mg/kg (i.v.)] and perfused transcardially with 500 ml of heparinized 0.9% saline solution, followed by 2000 ml of freshly prepared 4% paraformaldehyde (Sigma) + 0.1% glutaraldehyde buffered with 1 M sodium phosphate (pH 7.4). The lumbar spinal cord was removed, postfixed for 2 h in 4% paraformaldehyde, and then placed in 30% sucrose. Frozen horizontal segments of the lumbar spinal cord were sectioned at a thickness of 35 ␮m using a cryostat. Sections were collected in 0.1 M phosphate buffer (PBS) (pH 7.4) for histochemical processing. NADPH-d histochemistry NADPH-d was detected with the indirect method modified from Scherer-Singler et al. (1983) as follows: free floating sections were incubated for 1 h at 37 ◦ C in a solution containing 1 mg/ml of nitroblue tetrazolium (NBT; Sigma), 0.5 mg/ml ␤-NADPH (Sigma), and 1.25 mg/ml monosodium malate salt (Sigma) dissolved in 0.1 M PBS (pH 8.0), and 0.8% Triton X-100. Control sections were incubated in substrate-free media. Sections were monitored every 20 min to avoid overstaining. Following the reaction, the sections were rinsed in 0.1 M PBS (pH 7.4), mounted on slides, air-dried overnight, coverslipped with Entellan (Merck, Darmstadt, Germany) and examined by light microscopy. Micrographs of sections were taken by microscope Optika, model B600 Ti with digital camera system Moticam 2300. As a communication program between the camera and PC was used Motic Image Plus software, version 2.0 ML. Antioxidant enzyme activity The livers of anesthetized rats were removed and placed in an isolation medium containing 320 mM/l sucrose, 10 mM/l Tris and 1 mM/l EDTA (pH 7.4). Mitochondria were isolated from livers using the method of Fernández-Vizzara et al. (2010). Protein concentration was determined according to bicinchoninic acid (BCA) assay (Smith et al., 1985) and expressed as milligrams protein per milliliter of homogenate (mg prot/ml). Bovine serum albumin was used as a standard. Glutathione reductase activity (GR, E.C.1.6.4.2) was measured by the method described by Carlberg and Mannervik (1985) and the activity of glutathione peroxidase (GPx, E.C.1.11.1.9) by the method according to Zagrodski et al. (1998). Superoxide dismutase activity (SOD, E.C.1.15.1.1) was measured using an SOD-Assay Kit-WST (Fluka, Buchs, Switzerland) and that of reduced glutathione (GSH) was determined by the method of Floreani et al. (1997) using Ellman’s reagent. An M 501 Single beam

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UV/vis spectrophotometer was used for the enzyme assays (Spectronic Camspec Ltd., Leeds, UK). Data analysis Results are presented as the mean ± S.E.M. of at least three independent experiments. Statistical significance regarding NADPH-d histochemistry was determined by one-way ANOVA and analysis regarding antioxidant parameters by Student’s t-test. Differences were considered significant at P < 0.05. Results The distribution of NADPH-d-stained neurons and antioxidants in liver mitochondria at P1, P21 and P90 of both control and experimental groups was observed. nNOS-positive neurons at P1 In normal pups, the spatial distribution of nNOS-positive neurons in the medial and lateral parts of laminae I and II was not uniform at P1. nNOS-positive neurons were found in lamina III–IV of the dorsal horn as single neuronal bodies only (Fig. 1E). Multipolar neuronal cells stained weakly and their location was more frequently seen in the medial portion of the dorsal horn. nNOS-positive neurons were also located in the lateral part of the intermediate zone, in the IML nucleus (Fig. 1C). Many of these cells had obvious mediolateral processes. These neurons were less intensely stained for NADPH-d than the neurons surrounding the central canal, the middle of their neuronal body is luminous, and they are grouped in clusters. Few neurons were present approximately midway between the central canal and the IML nucleus; they were similar in shape and intensity of NADPH-d staining to the neurons in the IML nucleus. A band of small elongated, mostly bipolar nNOS-positive neurons with an anterior–posterior direction were located close to ependymal cells surrounding the central canal in the lumbar spinal cord at P1 (Fig. 1A). Clusters of nNOSpositive neurons were detected near the central canal. Neuronal outlines were continuous, more intensively NADPH-d stained with a light center of somata. Some neurons had thick, sparsely radiating dendrites projecting solely toward the intermediate zone. The central canal was closed, bordered by the slightly darker apical parts of ependymal cells. The ventral horn showed no nNOS positive neurons. Morphological images of the monitored areas of the spinal cord gray matter (Fig. 1B, D, and F) were similar to those in the control group. nNOS-positive neurons at P21 The superficial layer of the dorsal horn of the lumbar spinal cord showed a bilaminar configuration, in which lamina I–II was intensively immunostained for NADPH-d. A narrow strip between lamina I and II was only slightly NADPH-d positive (Fig. 2E). Neurons in both superficial and deep layers of the dorsal horn were mostly spindle-shaped, small, bipolar and the intensity of NADPHd staining rose from the media section toward lateral edge of the dorsal horn of the lumbar spinal cord. In the lamina III–IV, large multipolar neurons were located with an intense dark-stained neuronal body and uninterrupted NADPH-d positive processes. The IML nucleus showed colorful NADPH-d stained neurons ordered in clusters, but even here several neurons with a clear center were detected (Fig. 2C). Neurons in the central canal area were mostly NADPH-d positive; among them, a number of neurons with an illuminated center were also encountered. Neurons have fully enclosed bodies and rather long neuronal processes (Fig. 2A). There were no NADPH-d stained neurons detected in the ventral horn region of the

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lumbar spinal cord. nNOS-positive neurons were observed in the experimental group, indicating that prenatal development of the spinal cord was affected by high levels of RA. There were no signs of morphological changes in nNOS-positive neurons indicating a prenatal neurodegenerative effect of RA (Fig. 2B, D, and F). nNOS-positive neurons at P90 The histochemical image of nNOS-positive neurons in the gray matter of the lumbar spinal cord in the RA group was fairly similar to that in the control group. In both groups, pericentral neurons were intensively stained for NADPH-d in both parts of the spinal cord, with clearly visible projections on a light background (Fig. 3A and B). Neurons were located in the dorsal part of the substantia intermedia centralis and lateral to the central canal. We have recorded multipolar cells with long, branched projections that continued into the lateral funiculus or media in the dorsal horn, among which we found several bipolar cells. In the IML nucleus of rat spinal cord, we observed the presence of numerous, closely grouped neurons with clearly stained dark bodies and projections (Fig. 3C and D). Neuronal projections were directed mostly to the central canal or lateral to the white matter. Parasympathetic preganglionic neurons were spread out between them in larger distances. Numerous nNOS-positive neurons were located in the superficial layers of the dorsal horn in a bilaminar arrangement (Fig. 3E and F). Neurons in lamina I were stained dark, followed by an non-colored band attributable to the outer part of lamina II and finally the stained bodies and projections of neurons in the inner part of lamina II. The bodies of neurons were circular, mostly bipolar with long, sparsely branching or numerous short projections. Among these cells, we found a few multipolar neurons. A small number of intensely dark stained neurons and their bodies were found in the deep layers of the dorsal horn, larger than those in lamina I–II. The projections of the neurons branched in the direction of lamina I–II, some directed laterally or ventrodorsally to the pericentral area. Antioxidant status in rat liver The activities of enzyme antioxidants as GR, GPx, and SOD as well as the content of the non-enzyme antioxidant, GSH in rat liver mitochondria at days P1, P21 and P90 were determined. As is shown in Fig. 4A, differences in SOD activities were significant at P1 and P21 when compared to controls (Fig. 4A). SOD activity levels at P1 were lower by 12.32% although at P21 they appeared to be increased by up to 36.72%. Upon reaching adulthood, the SOD activities of the experimental group oscillate, and the decrease in the activities of SOD is considered to be highly statistically significant. By conventional criteria, GPx and GR activities were not significantly changed (Fig. 4B and C), but showed an interesting trend. GPx activities decreased at P1 (a decrease of about 11.54% in comparison to controls) before increasing again in adulthood (more than 2.59%). GR, on the other hand, clearly demonstrated the opposite manner. GSH levels appear to be depleted at P1 (Fig. 4D). GSH levels were stable at P21 and showed no significant differences; even though the levels in adulthood were higher than in the control group (to 11.34%). Discussion NADPH-d histochemistry, as a valid method that permits localization of NOS activity, was used to characterize the nNOScontaining neurons in the rat lumbar spinal cord, and it was demonstrated that nNOS, responsible for the synthesis of NO, is discretely localized in the dorsal horn, around the central canal, and at the intermediolateral cell column of the spinal cord. NO as a gaseous neurotransmitter can influence synaptic transmission, plasticity,

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Fig. 1. Comparison of nNOS positive neurons in the lumbar spinal cord of control and RA group at P1. Figs. A, C and E indicate NADPH-d stained neurons in the control group. Figs. B, D and F indicate NADPH-d stained neurons in the RA group. Clusters of NADPH-d-positive neurons were detected near the CC and IML. Neuronal bodies of both groups were weekly colored with more intensively NADPH-d positive contours. Arrows indicate NADPH-d stained neurons in both groups. CC, central canal; IML, nucleus; DL, deep layers of the dorsal horn. Scale bar = 20 ␮m.

neurotoxicity and development in the CNS (Li et al., 2006). It is well known that nNOS is in part closely bound to the N-methyl-daspartate receptor and is activated by the influx of calcium via the receptor-gated ion channel. This pathway to nNOS activation can contribute to excitotoxicity, i.e. glutamate-induced neuronal cell death. High levels of NO cause acute glutamate release from neurons and the high concentrations of extracellular glutamate can lead to cell death in neurons (Brown, 2010). In laminae I and II in mice, there was little or no expression of nNOS by P10, and nNOS activity in the superficial layer of the spinal cord increased gradually with age from P10 to P30 (Xu et al., 2006). It is well known that superficial dorsal horn neurons are the last of the spinal neurons to mature (Fitzgerald, 2005). In comparison with these findings, in our study there was no evidence of nNOS-positive neurons in laminae I and II of rat spinal cord at P1, while nNOS-expressing neurons were seen in laminae I

and II in both the control and experimental groups at P21 and P90. In the pericentral region and the intermediolateral cell column of rat spinal cord, nNOS-positive neurons were located at all ages in both control and experimental groups. Our results showed that nNOS activity was not altered in the pericentral region. In accordance with these localizations, it is well accepted that NO is involved in nociceptive processing and persistent pain as an intracellular and intercellular messenger in the spinal cord (Xu et al., 2006). In both the RA group and the control group, no nNOS activities were found in the ventral horn on P1, P21 and P90. In another case, oral retinoic acid at a dose of 10 mg/kg BW was associated with embryo-fetal alterations in Wistar rats (Seegmiller et al., 1997). The high systemic background of vitamin A increased by 50% the teratogenicity of a single dose (10 mg/kg) of RA given at day 9 (Nolen, 1989). Also, morphologically the general shape, shape of the lumen and spatial arrangement of the cell populations of floor

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Fig. 2. Comparison of nNOS-positive neurons in the lumbar spinal cord of control and RA group at P21. Figs. A, C and E indicate NADPH-d stained neurons in the control group. Figs. B, D and F indicate NADPH-d activity in the RA group. Neurons of both groups were intensively stained for NADPH-d although several neurons with a clear center were detected. Arrows indicate NADPH-d stained neurons in both groups. CC, central canal; IML, nucleus; SL, superficial layers of the dorsal horn. Scale bar = 20 ␮m.

plate and roof plate of the spinal cord appeared to be abnormal in group of rats treated with RA, when administered intraperitoneally as a single dose 7.5 mg/kg BW at day 11 of gestation (Namita et al., 2008). In our histochemical study, a 10 mg/kg dose of RA at gestational days 8–10 had no teratogenic effects on nNOS-positive neurons of rat lumbar spinal cord at P1, P21 or P90. Similarly, effect of RA on the teratogenicity was reduced after administration of 8 mg/kg on days 6–15 (Nolen, 1989). Various studies described above and our results in this current study complement in part claims of Rubin and LaMantia (1999) that the local availability of RA during mid-gestation establishes differences in RA-dependent gene expression in the embryonic thoracic/sacral versus cervical/lumbar spinal cord regions. However, there is little biochemical understanding of the factors and processes that facilitate and control transfer of retinoids from the maternal circulation to the embryo (Quadro et al., 2004).

The availability of retinol-binding proteins (RBPs) is considered critical, because they allow the storage of retinol in the liver, as well as the ability to transfer it from the liver to other tissues. The liver alone is able to turn over its retinoid stores without secreting these stores as retinol bound to RBP (Quadro et al., 2004). The binding capacity of RBP can be exceeded, leading to the storage of retinol as retinyl palmitate. In this case, retinyl ester is mobilized from the liver rather than retinol. Then, due to the detergent-like activity of retinyl ester, it may be responsible for the labialization of various organelle membranes, resulting in the release of organelle contents or alterations in membrane function (Choudhary and Swami, 2012). However, Quadro et al. (2004) indicated that retinol-RBP is not the only source of retinoids that reach the embryo. These authors established that retinyl esters in lipoprotein particles can be a significant source of retinoids for use by the fetus to support embryogenesis. This brief outline points out that the liver is the most likely organ to assess changes. As previously described, it is well-documented

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Fig. 3. Comparison of nNOS-positive neurons in the lumbar spinal cord of control and RA group at P90. Figs. A, C and E indicate NADPH-d activity in the control group. Figs. B, D and F indicate NADPH-d stained neurons in the RA group. Neurons were intensively stained for NADPH-d in both parts of the spinal cord. Arrows indicate NADPH-d stained neurons in both groups. CC, central canal; IML, nucleus; SL, superficial layers of the dorsal horn. Scale bar = 20 ␮m.

that mitochondrial function is significantly compromised following spinal cord injury (McEwen et al., 2011). These originate from pregnant females administered with retinol during days 8–10 postcoitum, when overdose administration of RA during this time is conducive to malformations. We found that SOD activity was significantly lower (P < 0.001) compared to the control at the first postnatal day in the rat liver mitochondria, but the activity of GPx and GR showed no change when compared to control. Interestingly, the level of GSH was also significantly lower (P < 0.001). An explanation may lie in the alternative metabolic pathway of retinol. This may include P450s, which would actually contribute further to toxicity, as P450-mediated metabolism requires molecular oxygen and produces oxygen free radicals that can cause liver damage (Kono et al., 1999). It is well documented that mitochondria are a source of H2 O2 ; however, the release of O2 •− from mitochondria into the cytosol has yet to be definitively established (Han et al., 2003). Taking into account the

activity of GPx (which catalyzes the GSH-dependent reduction of substrate peroxides, e.g. peroxides free of fatty acids, phospholipids and cholesterol), followed by the activity of GR (which catalyzes the feedback reduction of oxidized glutathione), the increased formation of various peroxides may simply be caused by the insufficient dismutation of the superoxide radical (O2 •− ). Moreover, the mitochondrial intermembrane space contains several O2 •− scavenging pathways besides SOD, such as cytochrome c (Vander Heiden et al., 2000) as well as the voltage-dependent anion channel (Han et al., 2003). Besides SOD activities, the GSH levels also clearly showed an imbalance in redox homeostasis. It should be noted that GSH itself is directly involved in various detoxification reactions such as those against endogenously produced radicals and other toxic metabolites. The oxidative stress in the liver may be regarded as a serious finding. Firstly, because RA-metabolizing cytochrome P450s protects cells and tissues from exposure to RA during embryogenesis

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Fig. 4. Measured antioxidant parameters from rat liver mitochondria at postnatal days P1, P21 and P90. (A) Activities of superoxide dismutase, (B) activities of glutathione peroxidase, (C) activities of glutathione reductase, (D) levels of reduced glutathione (***P < 0.001).

by restricting RA access to transcriptional machinery by converting RA into rapidly excreted oxoderivatives such as 4-oxo-RA, 4-OH-RA, and 18-OH-RA (Tahayato et al., 2003; Taimi et al., 2004). Secondly, because of the close structural and functional similarities between NOS and cytochrome P450, many of the same redoxcycling and reductive reactions that occur with cytochrome P450 can also occur with NOS (Miller, 2004). The increase in oxidative stress conditions can be expected as a result of their real activities, while protecting other tissues from damage. Three weeks later, on day P21, SOD activities were significantly higher than in control animals (P < 0.001). Neither the activities of GPx and GR, nor the levels of GSH showed any change. The most recent concept is that most retinoid-induced toxicity results from nuclear receptor-mediated interaction and subsequent alteration in gene expression (Perrine et al., 2005). Therefore, low SOD activities at P1 could be the result of the down-regulation of the enzyme gene expression. Upon reaching adulthood, the SOD activities in the RA group were lower again, but levels of GSH were almost significantly higher compared to control rats. The observed state clearly does not relate to oxidative stress conditions. In conclusion, morphological analysis of NADPH-d stained neurons in the lumbar rat spinal cord showed that prenatal exposure to high levels of RA did not influence the biological nNOS activities, as no changes were observed at days P1, P21 or P90. The morphological presence of nNOS-positive neurons at P90 corresponds to the image at P21 in both parts of the spinal cord gray matter. It follows that such application does not cause any response in the spinal cord. It is an important observation that the administration of RA at a dose of 10 mg/kg body weight at days 8, 9, and 10 of gestation is a dose that affects the antioxidant status of the liver in the offspring and may provide the basis for subsequent changes in its functionality. Removing excess amounts of RA protects other organs from damage. At the same time it creates conditions of oxidative stress, which are temporary, though, this condition can greatly affect the response of the organism to other toxic substances in the period after birth.

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Please cite this article in press as: Vaˇsková J, et al. Prenatal effects of retinoic acid on lumbar spinal cord development and liver antioxidants in rats. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.02.003