Punicalagin improves chorioallantoic and yolk sac vasculogenesis and teratogenesis of embryos induced by nicotine exposure

Journal of Functional Foods 18 (2015) 617–630

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Punicalagin improves chorioallantoic and yolk sac vasculogenesis and teratogenesis of embryos induced by nicotine exposure Chunmei Lin, Jung-Min Yon, Beom Jun Lee, Jong-Koo Kang, Young Won Yun, Sang-Yoon Nam * College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 28644, South Korea

A R T I C L E

I N F O

A B S T R A C T

Article history:

Punicalagin is a functional ingredient in pomegranate juice, which has various beneficial

Received 19 May 2015

effects for health and prevention of disease. In this study, the anti-teratogenic effect of

Received in revised form 20 August

punicalagin (1 × 10−5 or 1 × 10−4 µM) was investigated in cultured mouse embryos and yolk

2015

sac-placentas exposed to nicotine (1 mM), one of the main toxins in cigarette smoke. Nicotine-

Accepted 28 August 2015

treated embryos revealed severe anomalies as well as impaired yolk sac vascularization,

Available online 14 September 2015

reduced labyrinth formation, and developmental arrest of blood islands in the chorioallantoic border. Furthermore, nicotine significantly altered the regulations of hypoxia- and

Keywords:

vascularization-related genes in yolk sac-placenta and the levels of oxidative stress, apop-

Punicalagin

tosis, and inflammation in both embryos and yolk sac-placentas. However, these detrimental

Nicotine

changes were remarkably improved in response to co-treatment with punicalagins. These

Teratogenesis

findings indicate that punicalagin could be crucial to the development of clinical strate-

Placenta

gies to attenuate nicotine-induced teratogenesis in embryos. © 2015 Elsevier Ltd. All rights reserved.

Yolk sac Vascularization

1.

Introduction

Punicalagin, which is a novel pomegranate fruit extract rich in polyphenols and is abundant in fruit husk, is extracted from pomegranate juice in quantities reaching >2 g/L juice and is responsible for the high antioxidant activity of pomegranate juice. Fruits and vegetables, which contain a diverse range of punicalagins, are believed to have properties important to the

prevention of cancer including antioxidant, anti-inflammatory, and anti-proliferative activities, as well as modulatory effects on subcellular signalling pathways, induction of cell cycle arrest, and apoptosis (Afaq, Saleem, Krueger, Reed, & Mukhtar, 2005; Aviram et al., 2000; Cerdá, Llorach, Cerón, Espín, & Tomás-Barberán, 2003; de Nigris et al., 2007). With rising rates of maternal smoking, its adverse effects in foetal development and growth have become a major public health issue (Cnattingius, 2004). Although the mechanisms

* Corresponding author. Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, South Korea. Tel.: +82 43 261 2596; fax: +82 43 271 3246. E-mail address: [email protected] (S.-Y. Nam). Abbreviations: ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; SOD1, cytoplasmic superoxide dismutase; GPx1, cytoplasmic glutathione peroxidase; Bcl-xL, B-cell lymphoma-extra large; Bax, Bcl2-associated X protein; TGF-β1, transforming growth factor-beta 1; HIF-1α, hypoxia inducible factor-1α; TNF-α, tumour necrosis factor-alpha; IL-1β, interleukin 1 beta; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; IGF-1, insulin-like growth factor-1; VEGFα, vascular endothelial growth factor alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase http://dx.doi.org/10.1016/j.jff.2015.08.029 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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linking maternal smoking exposure to cellular damage are not clearly understood, previous studies have shown that nicotine, one of the main toxins in cigarette smoke, causes adverse pregnancy outcomes and developmental anomalies via production of excessive reactive oxygen species (ROS) (Dennery, 2007). In addition, nicotine can cross the placental barrier and act directly and indirectly on foetal systems (Lichtensteiger, Ribary, Schlumpf, Odermatt, & Widmer, 1988). In mammals, the yolk sac placenta and chorioallantoic placenta facilitate the exchange of oxygen, nutrients, and wastes between the maternal and developing embryo during the first trimester. The yolk sac actively absorbs nutrients from the chorion and chorionic cavity, transports them to the embryo through yolk sac circulation, and acts as a transient placenta during early post-implantation before allantoic circulation is established (Cross, Werb, & Fisher, 1994; Jollie, 1990). The chorioallantoic placenta is composed of an outer trophoblastderived epithelium and an underlying vascular network (Cross et al., 2003). By embryonic 9.5–10 day, the associated vessels of allantois are fused into the chorionic plate to form a highly folded labyrinthine layer, which provides a large surface area for gas and nutrient exchange. The labyrinthine layer is divided into two syncytiotrophoblast layers that separate the foetal blood vessels from maternal blood spaces (El-Hashash, Warburton, & Kimber, 2010). Therefore, analyses of histopathological changes and functional destruction of the yolk sac placenta and chorioallantoic placenta are needed to understand the mechanism of teratogenicity and developmental toxicity induced by teratogens such as nicotine. Although previous studies have shown that supplements of some polyphenols could significantly reduce nicotineinduced oxidative injuries (Bakker, Timmermans, Steegers, Hofman, & Jaddoe, 2011; Kalpana & Menon, 2004; Lin et al., 2012), any information about anti-teratogenic effects of punicalagin is not available. In the present study, we investigated whether punicalagin exerts curative effects on nicotine-induced embryo malformation and placenta-yolk sac malfunction, as well as its regulatory mechanisms using a whole embryo culture system.

2.

Materials and methods

2.1.

Experimental animals

One male and three female ICR mice (8–10 weeks old) purchased from a commercial breeder (BioGenomics Co., Seoul, South Korea) were basically housed in a cage for mating and finally a total of 15 cages were used for embryo culture in this study (15 males and 45 females). The environmental conditions were controlled throughout the experiment, with an ambient temperature of 21 ± 2 °C, relative humidity of 55 ± 10%, air ventilation rate of 10 cycles per hour, and a 12:12 h light:dark cycle. The animals were fed standard mouse chow (Samyang Ltd., Incheon, South Korea) and tap water ad libitum throughout the experimental period. Pregnancy was confirmed the following morning (08:00) by the presence of vaginal plugs or spermatozoa detected in a vaginal smear collected after mating during the previous evening (20:00). This was considered to be embryonic day (E) 0.5. Pregnant mice were sacrificed by cervical dislocation euthanasia on E 8.5, after which embryos were

obtained. All experiments were approved and carried out according to the Guide for Care and Use of Animals (Chungbuk National University Animal Care Committee, CBNUA-608-13-01).

2.2.

Rat serum preparation

Serum of Sprague–Dawley male rats (10–12 weeks old) was prepared as embryonic culture fluid as follows. After collection, blood samples were immediately centrifuged for 10 min at 3600 × g and 4 °C to clear the plasma fraction of blood. The supernatant was then transferred to new tubes, which were centrifuged for 10 min at 3600 × g and 4 °C to separate the blood cells. The clear serum supernatant was subsequently decanted and pooled, after which the pooled serum was heatinactivated for 30 min at 56 °C in a water bath. The samples were then used immediately or stored at −70 °C. Serum was incubated at 37 °C and passed through a 0.2 µm filter prior to use in the whole embryo culture.

2.3. Whole embryo culture and nicotine and/or punicalagin treatments The whole embryo culture system was based on a previously described model (New, 1978). Briefly, animals were sacrificed via cervical dislocation at E8.5 between 09:00 and 10:00, and only embryos with somite numbers of 4–8 were utilized. After removal of the decidua and Reichert’s membranes, embryos with intact visceral yolk sacs and placentas were randomly placed into sealed culture bottles (three embryos/bottle) containing 3 mL of culture medium and different concentrations (1 × 10−5 or 1 × 10−4 µM) of punicalagin (Sigma, St. Louis, MO, USA) dissolved in saline and/or 1 mM of nicotine (163.8 µg/mL serum; Sigma), which was determined by our previous study (Lin et al., 2012). Furthermore, in our pilot studies to select the beneficial concentrations of punicalagin against nicotine-induced embryotoxicity, mouse embryos were exposed to several concentrations (10−7, 10−6, 10−5, 10−4, 10−3, 10−2, 10−1, and 1 µM) of punicalagin for 48 h and were analysed using a morphological scoring system. Consequently, two effective and economic concentrations of punicalagin (1 × 10−5 or 1 × 10−4 µM) were selected to do the following experiments (data not shown). Embryos were randomized into (1) control group, (2) nicotine group, (3) nicotine plus punicalagin (1 × 10−5 µM) group, and (4) nicotine plus punicalagin (1 × 10−4 µM) group. The embryos were incubated at 37 ± 0.5 °C in sealed culture bottles (three embryos/bottle) and rotated at 25 rpm. The culture bottles were initially gassed with a mixture of 5% O2, 5% CO2, and 90% N2 over a 17 h period at a flow rate of 150 mL/min. Subsequent gassing was performed at the same rate over 7 h (20% O2, 5% CO2, and 75% N2) and 24 h (40% O2, 5% CO2, and 55% N2). All embryos were cultured for 48 h using a whole embryo culture system (Ikemoto Rika Kogyo, Tokyo, Japan).

2.4.

Morphological scoring

At the end of the 48 h culture period, embryos were evaluated according to a morphological scoring system (Van Maele-Fabry, Delhaise, & Picard, 1990). Only viable embryos with

Journal of Functional Foods 18 (2015) 617–630

yolk sac circulation and a heartbeat were utilized for morphological scoring. Measurements of each viable embryo were obtained with 17 standard scoring items, plus the yolk sac diameter, crown–rump length, and head length. The morphological features that were assessed included the embryonic flexion, heart, caudal neural tube, brain (forebrain, midbrain, and hindbrain), otic and optic systems, olfactory organs, branchial arch, maxilla, mandible, limb buds (forelimb and hindlimb buds), yolk sac circulation, allantois, and somites.

2.5.

Histological examination

Embryo and yolk sacs were dissected in cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde. The samples were then dehydrated, embedded in paraffin and sectioned at 5 µm. Finally, samples were stained with haematoxylin and eosin.

2.6.

Lipid peroxidation measurement

Lipid peroxidation was determined by thiobarbituric-acidreactive species (TBARS) levels as described by Ohkawa, Ohishi, and Yagi (1979), with minor modifications. The TBARS concentration in the embryos was measured spectrophotometrically and expressed as the malondialdehyde (MDA) equivalents. The TBARS results are expressed as nmol MDA equivalents/mg protein. Embryos in each group were homogenized in chilled 10 mM phosphate buffer and then mixed thoroughly with 8.1% sodium dodecyl sulphate, 20% acetic acid, and 0.75% 2-thiobarbituric-acid solution. Next, the solution was heated for 30 min in a 95 °C oven. After cooling, the flocculent was removed by centrifugation at 4200 × g for 15 min. The absorbance of the upper layer was measured at 532 nm with a spectrophotometer and compared to the value from the prepared 1,1,3,3,-tetramethoxypropane standard curve. The protein content of the embryos was determined according to a previous method using bovine serum albumin as the standard (Lowry, Rosebrough, Farr, & Randall, 1951).

2.7.

Superoxide dismutase (SOD) activity assay

Total SOD activity was assayed with a SOD Assay kit-WST (Dojindo Laboratories, Kumamoto, Japan). Briefly, the mouse embryos were homogenized and the protein concentrations of the supernatants were analysed by the Bradford method (Bradford, 1976). The supernatants were incubated with an assay reagent containing xanthine, xanthine oxidase, and a watersoluble tetrazolium salt, WST-1. The superoxide free radicals generated from the xanthine substrate by xanthine oxidase reduced WST-1 to WST-1 diformazan, which shows maximum absorbance at 450 nm. SOD in the embryos inhibited WST-1 reduction as it catalysed the dismutation of superoxide ions to molecular oxygen and hydrogen peroxide. The reduction of WST-1 was measured spectrophotometrically at 450 nm and SOD activity was calculated as an inhibition rate in which 1 U was defined as a 50% decrease from the control value over a period of 30 min at 37 °C. The results were presented as specific

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activity, which was determined as the total activity per embryo divided by the total amount of protein per embryo.

2.8. Quantitative real-time polymerase chain reaction (PCR) analysis Total RNA was extracted from cultured mouse embryos and placentas including the yolk sac using a Trizol Reagent kit (Invitrogen, Carlsbad, CA, USA). RNA samples were purified with a column using an RNA Clean-up kit (Macherey-Nagel, Bethlehem, PA, USA), after which total RNA (2 µg) was used in a cDNA Synthesis kit (Invitrogen). Real-time PCR was carried out in a 20 µg reaction volume using a SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and mouse embryonic or placenta cDNA (1.6 µg) as the template. Reactions were performed using a 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Genespecific primers were designed by TIB Mol-Bio Synthesis (Berlin, Germany). Specifically, the following primer sets were used: mouse insulin-like growth factor-1 (IGF-1 forward: 5′TCGGCCTCATAGTACCCACT-3′; reverse: 5′-ACGACATGATGT GTATCTTTATTGC-3′), transforming growth factor-beta 1 (TGFβ1 forward: 5′-TGGAGCAACATGTGGAACTC-3′; reverse: 5′CAGCAGCCGGTTACCAAG-3′), vascular endothelial growth factor alpha (VEGFα forward: 5′-AACGATGAAGCCCTGGAGTG-3′; reverse: 5′-GCTGGCTTTGGTGAGGTTTG-3′), hypoxia-inducible factor 1-alpha (HIF-1α forward: 5′-CACCAGACAGAGCAGGAA3′; reverse: 5′-TCAGGAACAGTATTT CTTTGATTCA-3′), SOD1 (forward: 5′-TGCGTGCTGAAGGGCGAC-3′; reverse: 5′-GTC CTGACAACAC AACCTGGTTC-3′), cytoplasmic glutathione peroxidase (GPx1 forward: 5′-TGTTTGAGAAGTGCGAAG TG-3′; reverse: 5′-GTGTTGGCAAGGCATTCC-3′), B-cell lymphomaextra large (Bcl-xL forward: 5′-TGACCACCTAGAGCCTTGGA-3′; reverse: 5′-TGTTCCCGTAGAGATCCACAA-3′), caspase 3 (forward: 5′-AAAGCCGAAACTCTTCATCAT-3′; reverse: 5′-GTCCCACT GTCTGTCTCA-3′), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB forward: 5′-CACTGCTCAGGTC CACTGTC-3′; reverse: 5′-CTGTCACTATCCCGGAGTTCA-3′), and cytokines [tumour necrosis factor-alpha (TNF-α forward: 5′TACCTTGTTGCCTCCTCTT-3′; reverse: 5′-GTCACCAAATCA GCGTTATTAAG-3′) and interleukin 1 beta (IL-1β forward: 5′TCACAAGCAGAGCACAAG-3′; reverse: 5′-GAAACAGTCCAG CCCATAC-3′)]. In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH forward: 5′-CGTGCCGCCTGGAGAAACC-3′; reverse: 5′-TGGAAGAGTGGGAGTTGCTGTTG-3′) primers were used as an internal standard to normalize target transcript expression. Data were analysed from nine independent runs using a comparative Ct method, as previously described by Livak and Schmittgen (2001).

2.9.

Statistical evaluation

Group differences were assessed via one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All analyses were conducted using the SPSS for Windows software, version 10.0 (SPSS Inc., Chicago, IL, USA). Morphological score data were compared using the Kruskal–Wallis nonparametric ANOVA and Dunn’s multiple comparison post hoc test. A p < 0.05 was considered significant. All data are expressed as the mean ± SEM.

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3.

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Results

3.1. Protective effects of punicalagin against nicotineinduced developmental defects in mouse embryos To examine whether punicalagin could improve the developmental arrest of embryos exposed to nicotine, embryos were evaluated according to the morphological scoring system (Van Maele-Fabry et al., 1990). After 48 h of culture, embryos exposed to nicotine alone (1 mM) exhibited severe growth retardation and developmental abnormalities (Fig. 1 and Table 1). Specifically, embryos exposed to nicotine showed significantly lower morphological scores for yolk sac diameter and circulation, allantois, flexion, crown–rump length, head length, and heart, as well as the hind, mid, and forebrains, otic, optic, and olfactory systems, branchial bars, maxillary and mandibular processes, forelimb, hindlimb, and number of somites relative to normal control embryos (p < 0.05). A significant decrease in the total morphological score (48.1 ± 7.35) was observed for embryos exposed to nicotine alone relative to the normal control embryos (64.3 ± 2.89; p < 0.05). However, when the embryos were exposed to nicotine plus punicalagin (1 × 10−5 or 1 × 10−4 µM), most of the morphological parameters were recovered

compared to those treated with nicotine alone (p < 0.05). The total morphological scores (56.9 ± 5.88 and 58.8 ± 3.88, respectively) for embryos that were co-treated with punicalagin were significantly higher than those for embryos treated with nicotine alone (p < 0.05). The abnormal developmental parameters in nicotine-treated embryos were significantly improved by cotreatment with punicalagin (1 × 10−4 µM, p < 0.05; Table 1). On the other hand, there was no significance between nicotine plus 1 × 10−5 punicalagin group and other groups, except for heart score (p < 0.05, Table 1).

3.2. Protective effects of punicalagin against defective yolk sac vascularization in mouse embryos treated with nicotine Yolk sac plays a crucial role in embryo development and survival. Therefore, we examined the effects of punicalagin on defective yolk sac vascularization in mouse embryos treated with nicotine. Many embryos treated with nicotine were shown to have pericardial effusion at E10.5, indicating osmotic imbalance and a common manifestation of disrupted yolk sac blood flow (Copp, 1995). Embryos exposed to nicotine showed abnormal yolk sac morphology with smaller size, pale colour, immature organization and impaired blood vessel formation.

Fig. 1 – Effects of nicotine and/or punicalagin on mouse organogenesis. (A) Normal control group. (B1–3) Embryos treated with nicotine (1 mM) alone show typical anomalies such as open brain, reduced forebrain, deformed posterior trunk, and regressed forelimbs. (C1–2 & D1–2) Nicotine plus punicalagin (1 × 10−5 µM and 1 × 10−4 µM) groups appear similar to the normal control group.

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Table 1 – Morphological changes in the cultured mouse embryos and yolk sac exposed to nicotine (1 mM) and/or 1 × 10−5 or 1 × 10−4 µM of punicalagin (PC). Growth Chemical (dose)

No. of embryo Yolk sac diameter (mm) Crown–rump length (mm) Head length (mm) No. of somites

Con

N

N + PC10−5

N + PC10−4

29 3.2 ± 0.34 2.6 ± 0.36 1.3 ± 0.18 31.8 ± 1.30

30 2.7 ± 0.33a 2.1 ± 0.26a 1.0 ± 0.17a 28.2 ± 2.59a

32 3.1 ± 0.40b 2.3 ± 0.25b 1.1 ± 0.16 30.9 ± 1.68b

32 3.0 ± 0.35b 2.3 ± 0.25b 1.1 ± 0.14b 31.4 ± 1.43b

Con

N

N + PC10−5

N + PC10−4

29 4.0 ± 0.36 2.1 ± 0.22 4.9 ± 0.10 4.9 ± 0.24 4.4 ± 0.28 4.6 ± 0.30 5.4 ± 0.55 4.8 ± 0.26 4.8 ± 0.30 3.6 ± 0.32 2.7 ± 0.30 2.7 ± 0.30 2.6 ± 0.60 5.0 ± 0.00 2.6 ± 0.28 0.7 ± 0.50 4.0 ± 0.19 64.3 ± 2.89

30 3.5 ± 0.48a 1.6 ± 0.22a 4.3 ± 0.78a 3.8 ± 0.61a 3.5 ± 0.75a 3.5 ± 0.75a 3.7 ± 0.79a 3.5 ± 0.76a 3.5 ± 0.76a 2.7 ± 0.46a 1.6 ± 0.46a 1.6 ± 0.44a 0.8 ± 0.68a 4.6 ± 0.56 1.9 ± 0.39a 0.0 ± 0.11a 3.8 ± 0.35 48.1 ± 7.35a

32 3.7 ± 0.26 1.9 ± 0.21b 4.8 ± 0.32b 4.1 ± 0.63 4.0 ± 0.42b 4.2 ± 0.49b 4.6 ± 0.60b 4.3 ± 0.57b 4.3 ± 0.58b 3.1 ± 0.38 2.1 ± 0.53b 2.1 ± 0.55b 2.3 ± 0.85b 5.0 ± 0.00 2.4 ± 0.50b 0.2 ± 0.48 3.8 ± 0.45 56.9 ± 5.88b

32 3.8 ± 0.26b 2.0 ± 0.17b 4.9 ± 0.26b 4.6 ± 0.47b,c 4.1 ± 0.15b 4.3 ± 0.32b 4.7 ± 0.59b 4.4 ± 0.35b 4.4 ± 0.38b 3.3 ± 0.30b 2.1 ± 0.56b 2.1 ± 0.55b 2.2 ± 0.75b 5.0 ± 0.00 2.5 ± 0.45b 0.5 ± 0.59b 4.0 ± 0.00 58.8 ± 3.88b

Development Chemical (dose)

No. of embryo Yolk sac circulatory Allantois Flexion Heart Hind brain Mid brain Fore brain Otic system Optic system Branchial bars Maxillary process Mandibular process Olfactory system Caudal neural tube Fore limb Hind limb Somites Total score

Each value represents the mean ± SEM. a vs. normal control (Con) group at p < 0.05. b vs. nicotine alone (N) group at p < 0.05. c vs. nicotine plus 1 × 10−5 punicalagin group (N + PC10−5) at p < 0.05.

The vessels of the yolk sac treated with nicotine displayed less, thin, and disorganized structures. A ring of blood islands that was only present in E8.5 mouse embryos was observed around the chorioallantoic border in the placentas of E10.5 embryos (Fig. 2Ab). Histological analysis of yolk sac treated with nicotine revealed a clear difference in morphology relative to the control group. The walls of yolk sacs exposed to nicotine exhibited paucity of vessels, absence of hematopoietic cells, thin and disordered arrangement of endodermal cells, and intracellular vacuoles (Fig. 2Bb). In contrast, co-treatment with punicalagin (1 × 10−5 or 1 × 10−4 µM) attenuated the nicotineinduced defects in yolk sac vascularization in mouse embryos (Fig. 2Bc, d).

3.3. Potential for punicalagin to recover chorioallantoic circulation of placentas impaired by nicotine The first step in placenta development involves attachment of the allantois to the chorionic plate on about E8.5, followed by vascular invasion of the labyrinth layer (Palis & Yoder, 2001). Histological analysis of placentas treated with nicotine showed severe defects in their development and morphogenesis

relative to the normal control group (Fig. 3Ab). Fewer embryonic vessels filled with embryonic nucleated erythrocytes and lined with endothelial cells entered into the chorion from the allantois, and these failed to invade the labyrinth in the nicotine group (Fig. 3Bb). In contrast, embryonic vessels entered into the labyrinth, as well as the chorion from the allantois, then branched out to form a dense vascular network (formation of the labyrinth) in punicalagin co-treated groups as well as the normal control group (Fig. 3Bc, d). Moreover, chorioallantoic circulation of the placentas such as maternal sinuses and embryonic blood vessels formed from allantoic blood vessels was evident and chorioallantoic attachment was observed in punicalagin co-treated groups (Fig. 3Ac, d), suggesting that punicalagin could accelerate a functional chorioallantoic circulation.

3.4. Punicalagin improves Hif-1α gene expression in placentas treated with nicotine The Hif-1α mRNA levels in placentas exposed to nicotine (1 mM) decreased significantly to 0.57-fold that of the control group (1-fold) (p < 0.05). However, when 1 × 10 −5 or 1 × 10 −4 µM

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Fig. 2 – Punicalagin attenuates nicotine-induced defective yolk sac vascularization in mouse embryos. (A) Nicotine-induced yolk sac defects showing smaller size, pale colour, rudimentary vasculature, lack of vitelline vessels (black arrows), and a ring of blood islands (asterisk). A-a; Normal control group. A-b; Nicotine (1 mM) alone group. A-c; Nicotine plus punicalagin (1 × 10−5 µM) group. A-d; Nicotine plus punicalagin (1 × 10−4 µM) group. (B) Histological examination of yolk sac wall stained with H & E (bar: 50 µm). Yolk sac defects induced by nicotine include lack of vessels, absence of hematopoietic cells (white arrows), disordered arrangement of endodermal cells, and intracellular vacuoles (arrowheads). B-a; Normal control group. B-b; Nicotine (1 mM) alone group. B-c; Nicotine plus punicalagin (1 × 10−5 µM) group. B-d; Nicotine plus punicalagin (1 × 10−4 µM) group. EC; endodermal cells. MC; mesodermal cells.

punicalagin was added to the nicotine-treated placentas, the Hif-1α mRNA levels were significantly recovered to 0.70-fold or 1.10-fold that of the control group, respectively (p < 0.05; Fig. 4A).

However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated placentas, the TGF-β1 mRNA levels decreased to 1.20-fold or 1.10-fold that of the control group, respectively (p < 0.05; Fig. 4C).

3.5. Punicalagin attenuates nicotine-induced vascular defects in placentas by upregulation of the VEGFα gene

3.6.2.

VEGFα mRNA levels in placentas exposed to nicotine (1 mM) decreased significantly to 0.35-fold that of the control group (1-fold) (p < 0.05). However, when 1 × 10 −5 or 1 × 10 −4 µM punicalagin was added to the nicotine-treated placentas, the VEGFα mRNA levels were significantly recovered to 0.74-fold or 0.81-fold that of the control group, respectively (p < 0.05; Fig. 4B).

3.6. Punicalagin regulates gene expression of growth factors in placentas exposed to nicotine 3.6.1.

TGF-β1 gene expression pattern

The TGF-β1 mRNA levels in placentas exposed to nicotine (1 mM) were 1.38-fold that of the control group (1-fold) (p < 0.05).

IGF-1 gene expression pattern

The IGF-1 mRNA levels in placentas exposed to nicotine (1 mM) were 0.69-fold that of the control group (1-fold) (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated placentas, the IGF-1 mRNA levels increased significantly to 0.85-fold or 1.09-fold that of the control group, respectively (p < 0.05; Fig. 4D).

3.7. Punicalagin decreases nicotine-induced oxidative stress, but improves antioxidant status in mouse embryos Oxidative stress is the major mechanism through which nicotine-induced apoptosis leads to malformation in the embryos; accordingly, the severity of any malformation depends on foetal antioxidative capacity (Dennery, 2007). Therefore, we examined the oxidative stress and antioxidant levels in whole

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Fig. 3 – Punicalagin accelerates functional chorioallantoic circulation following the reduction induced by nicotine treatment. (A) Maternal and foetal border in placenta from the impaired chorioallantoic attachment. A-a; Normal control group. Formation of the labyrinth in the placentas. A-b; Nicotine (1 mM) alone group. Failure of chorioallantoic attachment. A-c; Nicotine plus punicalagin (1 × 10−5 µM) group. A-d; Nicotine plus punicalagin (1 × 10−4 µM) group. Formation of the labyrinth in the placenta. (B) The enlarged structures of the boxed area in A. B-a; Normal control group. Maternal sinuses (yellow arrowheads) and embryonic nucleated blood vessels (white arrows) formed from the allantoic blood vessels. B-b; Nicotine (1 mM) alone group with no detectable definitive labyrinth. B-c; Nicotine plus punicalagin (1 × 10−5 µM) group. B-d; Nicotine plus punicalagin (1 × 10−4 µM) group. Nucleated red blood cells entered into not only the chorion, but also the labyrinth layer following punicalagin cotreatments. gc, giant cells; sp, spongiotrophoblasts; la, labyrinth; ch, chorion; al, allantois. H&E. bar: 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

embryos by analysing the lipid peroxidation, SOD activity, and mRNA expression patterns of HIF-1α, SOD1, and GPx1. Mouse embryos exposed to nicotine alone exhibited a significantly increased lipid peroxidation level (10.28 ± 0.14 nM/mg) compared to that of the control group (6.43 ± 0.60 nM/mg, p < 0.05),

as well as decreases in HIF-1α, SOD1, and GPx1 mRNAs and SOD activity to 0.75-fold, 0.74-fold, 0.57-fold and 0.37 ± 0.02 U/mg of those of the control group, respectively. However, when embryos were treated with nicotine plus punicalagin (1 × 10−5 or 1 × 10−4 µM) concurrently, significant improvements such as

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Fig. 4 – Punicalagin (PC) attenuates nicotine-induced vascular defects in placentas via improvement of related gene expressions. (A) HIF-1α mRNA expression levels in the placenta. (B) VEGFα mRNA expression levels in the placenta. (C) TGF-β1 mRNA expression levels in the placenta. (D) IGF-1 mRNA expression levels in the placenta. The results are the means ± SEM. Significant differences between punicalagin-treated groups [1 × 10−5 µM (N + PC10−5) and 1 × 10−4 µM (N + PC10−4)] and normal control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize the target transcript expression.

decreased levels of lipid peroxidation (8.25 ± 0.17 and 6.73 ± 0.33 nM/mg), and increased levels of SOD activity (0.57 ± 0.01 and 0.62 ± 0.01 U/mg), HIF-1α mRNA levels (0.89-fold and 0.98-fold), SOD1 mRNA levels (0.89-fold and 0.91-fold), and GPx1 mRNA levels (0.85-fold and 0.99-fold) were observed in both groups relative to the control group, respectively (p < 0.05; Fig. 5).

3.8. Punicalagin regulates gene expression of proinflammatory cytokines in placentas and embryos exposed to nicotine 3.8.1.

TNF-α gene expression patterns

The TNF-α mRNA levels in placentas exposed to nicotine (1 mM) were 2.61-fold that of the control group value (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the

nicotine-treated placentas, the TNF-α mRNA levels were recovered to 2.31-fold or 1.37-fold that of the control group, respectively (p < 0.05; Fig. 6A). Also, in mouse embryos, the TNF-α mRNA level of mice exposed to nicotine was 2.03-fold that of the control group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated embryos, the TNF-α mRNA levels were recovered to 1.46-fold or 1.22-fold that of the control group, respectively (p < 0.05; Fig. 6D).

3.8.2.

IL-1β gene expression patterns

The IL-β mRNA levels in placentas exposed to nicotine (1 mM) was 1.86-fold that of the control group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotinetreated placentas, IL-β mRNA levels decreased significantly to 1.02-fold or 0.84-fold that of the control group, respectively (p < 0.05; Fig. 6B). Moreover, the IL-β mRNA level in embryos

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Fig. 5 – Punicalagin (PC) decreases oxidative stress and improves antioxidant enzyme status in mouse embryos exposed to nicotine. (A) Lipid peroxidation was determined by thiobarbituric-acid-reactive species levels and expressed as the malondialdehyde (MDA) level. (B) SOD activity levels. (C) HIF-1α mRNA expression levels. (D) SOD1 mRNA expression levels. (E) GPx1 mRNA expression levels. GAPDH was used as an internal standard to normalize the target transcript expression. The results are the means ± SEM. Significant difference between each treatment group vs. normal control (Con; *) or nicotine alone (N; #) group as evaluated by one-way ANOVA at p < 0.05. 1 × 10−5 µM PC; N + PC10−5. 1 × 10−4 µM PC; N + PC10−4. exposed to nicotine (1 mM) was 1.76-fold that of the control group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated embryos, IL-β mRNA levels decreased significantly to 0.48-fold or 0.46-fold that of the control group, respectively (p < 0.05; Fig. 6E).

3.9. Punicalagin regulates NF-κB gene expression in nicotine-treated embryos The NF-κB mRNA levels in mouse embryos exposed to nicotine (1 mM) increased significantly to 2.08-fold that of the control

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Fig. 6 – Gene expression patterns of proinflammatory cytokines in the placenta and embryos exposed to nicotine (1 mM) and/or punicalagin (PC; 1 × 10−5 and 1 × 10−4 µM) at embryonic day 8.5 for 2 days in vitro. (A) TNF-α mRNA expression in placentas. (B) IL-1β mRNA expression in placentas. (C) NF-κB mRNA expression in embryos. (D) TNF-α mRNA expression in embryos. (E) IL-1β mRNA expression in embryos. The results are the means ± SEM. Significant differences between punicalagin groups and the normal control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression.

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group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated embryos, the NF-κB mRNA levels significantly decreased to 1.46-fold or 1.02-fold that of the control group, respectively (p < 0.05; Fig. 6C).

to the nicotine-treated embryos, the Bcl-xL mRNA levels were significantly recovered to 0.96-fold or 1.03-fold that of the control group, respectively (p < 0.05; Fig. 7C).

3.10. Punicalagin regulates the expression of apoptosisrelated genes in nicotine-treated placentas and embryos

3.10.2. Caspase 3 gene expression patterns

3.10.1. Bcl-xL gene expression patterns The Bcl-xL mRNA level in placentas exposed to nicotine (1 mM) was 0.56-fold that of the control group value (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated placentas, the Bcl-xL mRNA levels were significantly recovered to 0.91-fold or 0.96-fold that of the control group, respectively (p < 0.05; Fig. 7A). The Bcl-xL mRNA level in embryos exposed to nicotine (1 mM) was 0.79-fold that of the control group value (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added

The caspase 3 mRNA level in placentas exposed to nicotine (1 mM) was 1.25-fold that of the control group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated placentas, caspase 3 mRNA levels decreased significantly to 0.88-fold or 0.73-fold that of the control group, respectively (p < 0.05; Fig. 7B). The caspase 3 mRNA levels in embryos exposed to nicotine (1 mM) was 1.42-fold that of the control group (p < 0.05). However, when 1 × 10−5 or 1 × 10−4 µM punicalagin was added to the nicotine-treated embryos, caspase3 mRNA levels decreased significantly to 0.84-fold or 0.78-fold that of the control group, respectively (p < 0.05; Fig. 7D).

Fig. 7 – Expression levels of apoptotic genes Bcl-xL and caspase-3 between placentas (A&B) and embryos (C&D) exposed to nicotine (1 mM) and/or punicalagin (PC; 1 × 10−5 and 1 × 10−4 µM) at embryonic day 8.5 for 2 days in vitro. (A&C) Bcl-xL mRNA expression. (B&D) Caspase-3 mRNA expression. The results are the means ± SEM. Significant differences between punicalagin groups and the control (Con; *) or nicotine alone (N; #) group were examined at p < 0.05. GAPDH was used as an internal standard to normalize target transcript expression.

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4.

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Discussion

The first trimester is the most important and vulnerable stage and is most active for organogenesis (Dennery, 2007), and previous animal studies have demonstrated that antioxidants have a beneficial effect on nicotine-induced oxidative damage (Ashakumary & Vijayammal, 1996; Bakker et al., 2011; Kalpana & Menon, 2004). Therefore, we used a whole embryo culture system to investigate whether punicalagin could attenuate nicotine-induced embryo malformation. In the present study, we showed that the morphological scores of embryonic growth (yolk sac diameter, crown–rump length, head length, and somite number) and development (yolk sac circulation, allantois; heart; hindbrain, midbrain, and forebrain; otic, optic, and olfactory systems; branchial bars, maxillary and mandibular processes; forelimb and hindlimb) were significantly decreased by nicotine treatment. However, the selected concentrations of punicalagin (1 × 10−5 or 1 × 10−4 µM) were very effective at protecting embryos exposed to nicotine from most morphological anomalies, including abnormal heart development, deformed posterior trunk, regressed limbs, and an open and swollen brain. These findings indicate that punicalagin can effectively protect against nicotine-induced teratogenesis during embryonic organogenesis. Placenta, yolk sac and pre-fusion allantois are recognized as hematopoietic organs during development of the embryonic hematopoietic system in mammals (Alvarez-Silva, Belo-Diabangouaya, Salaün, & Dieterlen-Lièvre, 2003; Corbel, Salaün, Belo-Diabangouaya, & Dieterlen-Lièvre, 2007; Robin et al., 2009). Embryonic death is critically dependent on yolk sac abnormalities, including failed yolk sac hematopoiesis, yolk sac haemorrhage, deficient yolk sac angiogenesis and disrupted chorioallantoic fusion at E8 to E11.5 (Huang, Higuchi, Lasky, & Broze, 1997; Palis & Yoder, 2001; Watson & Cross, 2005). In the present study, yolk sacs treated with nicotine displayed severe defects in vascular development, including smaller size, pale colour, no organization and less, thin, and disorganized vessels. Furthermore, histological examination showed paucity of vessels, absence of hematopoietic cells, disordered arrangement of endodermal cells, and intracellular vacuoles. Hence, the yolk sac was impaired owing to decreased vascularization induced by nicotine. However, co-treatment with punicalagin (1 × 10−5 or 1 × 10−4 µM) improved the nicotineinduced defects of yolk sac vascularization in mouse embryos. On the other hand, yolk sac dysfunction induces an osmotic imbalance within the embryo resulting in swelling of the pericardia (Copp, 1995). In the present study, pallor and fewer vessels of yolk sac and pericardial effusion at E10.5 due to nicotine exposure were observed, suggesting that the embryonic malformation or growth retardation induced by nicotine treatment could be partially caused by osmotic imbalance as well as excessive oxidant stress. However, additional studies are needed to confirm this. Under normal embryogenesis in mice, the labyrinth layer begins to develop around E9.0 to 9.5 and subsequently functions as a nutrient transport unit. Allantois vessels are seen at E10, when embryonic vessels enter the labyrinth and chorion from the allantois (Kaufmann, 1992). In the present study, most labyrinth layers were abnormally thin in the nicotine group,

embryonic blood vessels entered directly into the chorion from the allantois, and organized branching morphology of labyrinth layer was never established. Hence, nicotine led to failure of the labyrinth layer formation and subsequent impaired placenta development and function. In contrast, embryonic vessels in the nicotine plus punicalagin treatment groups entered into the labyrinth, as well as the chorion from the allantois and subsequently branched out to form a dense vascular network for formation of the labyrinth. Moreover, chorioallantoic circulation (maternal sinuses and embryonic blood vessels formed from the allantoic blood vessels) of the placentas was evident, and chorioallantoic attachment was completed in these punicalagin supplementary groups. These data suggest that punicalagin could accelerate functional chorioallantoic circulation by formation of the labyrinth. In the present study, nicotine caused defective yolk sac and chorioallantoic circulation due to substantially impaired vasculogenesis of the placenta. Although hypoxia is often thought of as being a pathological phenomenon, the mammalian embryo in fact develops in a low-oxygen environment. HIF activity is required for normal development of the placenta, and loss of Hif-1α in mouse causes a catastrophic failure in placenta formation, resulting in embryo lethality by E10.5 (Dunwoodie, 2009; Hustin & Schaaps, 1987). Our results showed that the levels of HIF-1α mRNA in both placentas and embryos decreased significantly following nicotine treatment, suggesting that embryos and placentas treated with nicotine can induce suppression of HIF-1 α transcription and placental defects. Hypoxia induces multiple responses to oxygen availability, including the expression of growth factors required for establishment of a functional circulatory system (Ramírez-Bergeron, Runge, Adelman, Gohil, & Simon, 2006). HIF protein can induce VEGFα, a growth factor important for angioblast specification and vasculogenesis in embryos. Furthermore, VEGFα is related to reduced yolk sac and placental defects (Breier, Clauss, & Risau, 1995; Damert, Miquerol, Gertsenstein, Risau, & Nagy, 2002). In the present study, we found that placentas exposed to nicotine can induce abnormal HIF-1 α transcription, which leads to TGF-1β activity and reduces VEGFα and IGF-1 expression, but that co-treatment with punicalagin improved vasculogenesis and HIF-1-mediated transcriptional activity in the placenta following nicotine exposure. Early organogenesis occurs in a relatively hypoxic environment, and the embryo is sensitive to oxidative stress (Dennery, 2007). We found that hypoxia induces oxidative stress and then abnormal organogenesis in mouse embryos by down-regulating HIF-1α and intracellular SOD genes (Yon, Baek, Lee, Yun, & Nam, 2011). Oxidative stress characterized by increased ROS is an important mechanism for nicotine-induced toxicity in mouse embryos (Dennery, 2007). During mouse embryogenesis, antioxidant enzymes (SOD1and GPx1) are highly expressed and play an important role in mouse embryos (Baek et al., 2005; Yon et al., 2008). In the present study, mouse embryos treated with nicotine showed increased lipid peroxidation, as well as reduced SOD1 and GPx1 mRNA levels and SOD activity. However, punicalagin co-treatment significantly decreased lipid peroxidation and increased antioxidant enzyme mRNA expression and SOD activity. These findings indicate that punicalagin exhibited antioxidant properties that could decrease oxidative stress by attenuating nicotine induced antioxidant enzyme depletion and lipid peroxidation.

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ROS not only have direct effects on cells, but also act as second messengers by regulating key transcription factors that alter gene expression in the embryo (Dennery, 2007). NF-κB is a transcription factor that promotes the transcription of genes involved in pro-inflammatory responses (Simmonds & Foxwell, 2008) and an important regulator of cytokine and anti-apoptotic gene expression (Flohé, Brigelius-Flohé, Saliou, Traber, & Packer, 1997; Haddad, Olver, & Land, 2000). NF-κB activation could be blocked by a variety of antioxidants including N-acetylcysteine, glutathione and β-carotene, or by overexpression of antioxidant enzymes such as SOD and GPx (Flohé et al., 1997; Kim, Seo, & Kim, 2011; Mercurio & Manning, 1999). In the present study, the embryos treated with nicotine (1 mM) showed significantly increased levels of the proinflammatory cytokines TNF-α and IL-1β, and NF-κB mRNA, as did placentas with yolk sac treated with nicotine. However, punicalagin co-treatment significantly decreased the proinflammatory cytokines and NFκB mRNA expression in the embryonic environment. In fact, oxidative stress is a potent inducer of apoptosis, which acts as a crucial physiological determinant in embryonic and neonatal development (Cheng, Cunningham, & Rubel, 2005; Wang & Han, 2009). In particular, apoptosis is typically accompanied by the activation of protein-splitting enzymes known as caspases (Lakhani et al., 2006). The activation of caspase 3, a reliable marker for the identification of apoptosis, induces chromosomal DNA fragmentation and the cellular morphological changes characteristic of apoptosis by cleaving proteins necessary for cell survival (Goldstein, Waterhouse, Juin, Evan, & Green, 2000; Yu et al., 2011). In the current study, Bcl-xL mRNA was significantly decreased, while caspase 3 was significantly increased following nicotine treatment in cultured embryos and placenta-yolk sac. However, these apoptotic changes induced by nicotine were attenuated by co-treatment with punicalagin. These findings indicate that punicalagin protects embryos against nicotine-induced teratogenesis via its anti-proinflammatory and anti-apoptotic effects.

5.

Conclusion

The results of these findings indicate that punicalagin might function by 1) improving defective yolk sac; 2) accelerating chorioallantoic circulation by chorioallantoic attachment; 3) improving vasculogenesis and HIF-1-mediated transcriptional activity; 4) decreasing oxidative stress, inflammation, and apoptosis in embryos and surrounding placenta and yolk sac exposed to nicotine during early organogenesis. Therefore, punicalagin could be crucial to the development of clinical strategies to attenuate nicotine-induced teratogenesis in mouse embryo. However, further studies are needed to determine proper food or punicalagin dosages to protect against nicotine exposure.

Acknowledgments This work was supported by the Priority Research Centers Program (2015R1A6A1A04020885) and Basic Science Research Program (2013R1A2A2A03016519) through the National Research

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Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.

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