Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos

Reproductive Toxicology 26 (2008) 292–297

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Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos Mi-Ra Kim, Ki-Nam Lee, Jung-Min Yon, Se-Ra Lee, Yan Jin, In-Jeoung Baek, Beom Jun Lee, Young Won Yun, Sang-Yoon Nam ∗ College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea

a r t i c l e

i n f o

Article history: Received 12 February 2008 Received in revised form 5 September 2008 Accepted 30 September 2008 Available online 5 October 2008 Keywords: Capsaicin Ethanol Teratogenicity Antioxidant Superoxide dismutase Glutathione peroxidase

a b s t r a c t Prenatal exposure to alcohol promotes the level of reactive oxygen species within embryos and results in developmental disorders. In this study, we investigated the effect of capsaicin (trans-8-methyl-Nvanillyl-6-nonenamide), the major pungent ingredient in red peppers, on ethanol-induced teratogenicity in mouse embryos (embryonic days 8.5–10.5). In response to ethanol administration (1.0 ␮l/ml), developmental parameters such as yolk sac circulation, allantois, heart, hindbrain, midbrain, forebrain, otic and optic systems, branchial bar, olfactory system, forelimb, hindlimb, and somites decreased significantly in comparison with those of control group (p < 0.05). However, the concurrent administration of capsaicin (1 × 10−8 ␮g/ml or 1 × 10−7 ␮g/ml) and ethanol significantly ameliorated most of the morphological scores excepting yolk sac circulation and hindlimb scores (p < 0.05). Furthermore, the levels of superoxide dismutase activity and cytoplasmic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNAs in the ethanol-treated embryos recovered to the levels observed in control embryos by capsaicin co-administration. These results indicate that capsaicin has a protective effect against ethanol-induced teratogenicity via an antioxidative activity. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Maternal alcohol consumption is a major cause of birth defects. Fetal Alcohol Syndrome (FAS) is characterized by preand postnatal growth retardation, neuronal damage (which may include abnormal brain morphology, neurological abnormalities, and developmental and intellectual impairment), a specific pattern of craniofacial abnormalities, and limb and cardiac defects [1,2]. Numerous studies have documented the effects of alcohol abuse during pregnancy, including the effects of exposure during the first trimester, a critical period of development for the central nervous system in the embryo [3]. Studies confining exposure to early gestation have concluded that dysmorphic facial features, a characteristic of FAS result specifically from ethanol exposure during the embryonic period, which encompasses the first 3–8 weeks of gestation [4]. The incidence of FAS has been estimated as 0.97 cases per 1000 live babies in developed countries [5].

∗ Corresponding author at: Laboratory of Veterinary Anatomy/RIVM (Core Res. Institute), College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel.: +82 43 261 2596 fax: +82 43 271 3246. E-mail address: [email protected] (S.-Y. Nam). 0890-6238/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2008.09.006

The mechanisms linking ethanol exposure to cellular damage are not clearly understood; however, during the last decade, oxidative stress has been increasingly recognized as one of the predominant causes of ethanol toxicity [6]. In the presence of ethanol, cellular damage caused by an excess of free radicals and reactive oxygen species (ROS) is a consequence of the peroxidation of lipids, nucleic acids, and proteins [7–9] and alterations in enzyme activity [10]. Maternal alcohol consumption induces an excess of ROS in fetal tissues which is responsible for some toxic responses to alcohol. For example in postimplantation embryo culture, alcohol or acetaldehyde-treated embryos exhibited retardation in embryonic growth and development in a concentration-dependent manner as well as an increase in the number of injured cells in the developing midbrain due to the presence of ROS [11]. Previous studies have shown that antioxidants can significantly improve adverse developmental outcomes and diminish the incidence and severity of major malformations that result from ethanol exposure in utero. Vitamin E supplementation has been known to significantly normalize lipid peroxidation and free radical generation in ethanol-fed rats [12,13]. Chen et al. [14] reported that cell death in the forelimb buds of embryos concurrently exposed to ethanol and a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134, was notably reduced compared to embryos from ethanol-treated dams. During early postnatal

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ethanol treatment in rats, ␣-tocopherol (vitamin E) and ␤-carotene supplementation attenuates or ameliorates ethanol-induced cell loss and neurobehavioral-associated deficits in vitro and in vivo [15]. Moreover, folic acid has been shown to be effective against ethanol-induced toxicity in vitro [16]. Capsaicin (8-methyl-N-vanillyl-6-nonemide) is the major pungent ingredient found in hot peppers of the plant genus Capsicum. The members of this family are used extensively in foods for both their coloring properties and their pungency, and are also utilized as a traditional medicine for the treatment of a wide range of disorders in different regions of the world. In contemporary medicine, capsaicin formulations are used topically to treat a variety of diseases associated with neurogenic pain, inflammation, and cancers that are caused by the stimulation of capsaicin receptors or the vanilloid receptor 1, an ion channel protein expressed by nociceptive primary afferent neurons [17,18]. Dietary capsaicin is effective in reducing the oxidant stress, which is indicated by countering the depleted antioxidant molecules and antioxidant enzymes in erythrocytes and liver, and decreasing the elevated lipid peroxide content [19,20]. Capsaicin potentially inhibits the peroxidation of various lipids [21] and reduces the generation of ROS in rat peritoneal macrophages [22]. One of the most effective intracellular enzymatic antioxidants is superoxide dismutase (SOD) that catalyzes the dismutation of superoxide anion to oxygen and to the less-reactive species hydrogen peroxide [23]. Also, cytoplasmic glutathione peroxidase (cGPx) efficiently catalyzes the reduction of hydrogen peroxide and organic hydroperoxides by glutathione [24] and phospholipid hydroperoxide GPx (phGPx) is a membrane-associated enzyme that can reduce phospholipid, cholesterol, and thymine hydroperoxides in cells [25]. In order to investigate whether capsaicin has a protective effect on ethanol-induced teratogenicity, we examined the morphological parameters of ethanol-and/or capsaicin-treated mouse embryos during their critical organogenesis periods (embryonic days 8.5–10.5) and analyzed the level of SOD activity and the expression of cGPx and phGPx mRNAs in the embryos as representative antioxidative enzymes. 2. Materials and methods 2.1. Experimental animals Virgin female ICR mice were mated overnight in an environment maintained at 21 ± 2 ◦ C and a relative humidity of 55 ± 10% with a 12-h light/dark cycle. Pregnancy was confirmed the following morning by the presence of vaginal plugs or spermatozoa detected in a vaginal smear; this was considered to be embryonic day (ED) 0.5. All procedures were conducted in compliance with the “Guidelines for the Care and Use of Animals” (Chungbuk National University Animal Care Committee according to NIH # 86-23). 2.2. Culture and treatment 2.2.1. Rat serum preparation Sprague–Dawley rat serum was prepared as follows: after collection, the blood samples were immediately centrifuged for 10 min at 3000 rpm, 4 ◦ C to clear the plasma fractions. The supernatant was then centrifuged again for 10 min at 3000 rpm, 4 ◦ C in order to completely separate the blood cells. The clear serum supernatant was decanted and pooled. The pooled serum was heat-inactivated for 30 min at 56 ◦ C in a water bath and either used immediately or stored at −70 ◦ C. The serum was incubated at 37 ◦ C and filtered through a 0.2 ◦ C filter prior to use in culture. 2.2.2. Whole embryo culture The whole embryo culture system was based on a previously described model [26]. Pregnant mice (n = 20) were killed by cervical dislocation on ED 8.5 and the uteri were removed and placed in a petridish containing sterilized Tyrode’s solution (pH 7.2). After removal of the decidua and Reichert’s membrane, embryos with an intact visceral yolk sac and ectoplacental cone were placed randomly in sealed culture bottles (3 embryos/bottle) containing 3 ml of rat serum and experimental agents. Capsaicin powder (Sigma, U.S.A.) was dissolved in 0.1% dimethylsulfoxide (DMSO; Sigma, U.S.A.) and used to prepare 1 × 10−8 ␮g/ml or 1 × 10−7 ␮g/ml serum solutions. The explanted embryos were exposed to experimental agents as follows: a

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vehicle control group (0.1% DMSO), an ethanol group (1 ␮g/ml), two capsaicin groups (1 × 10−8 and 1 × 10−7 ␮g/ml), and two ethanol plus capsaicin groups. Cultures were incubated at 37 ± 0.5 ◦ C and rotated at 25 rpm. All of the embryos were cultured for 48 h using a whole embryo culture system (Ikemoto Rika Kogyo, Japan) for morphological and antioxidative analyses. 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 ) [27]. 2.3. Morphologic scoring At the end of the 48 h culture period, embryonic features were evaluated according to the morphologic scoring system of Van Maele-Fabry et al. [28]. Only viable embryos with evidence of yolk sac circulation and a heartbeat were utilized for morphological scoring. Measurements of each viable embryo were obtained for the 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, neural tube, cerebral vesicles (forebrain, midbrain, and hindbrain), otic, optic and olfactory organs, branchial arch, maxilla, mandible, limb buds (forelimb and hindlimb buds), yolk sac circulation, allantois, and somites. The morphological score is the total of the points attributed to each of the 17 features [28]. 2.4. SOD activity assay Total SOD activity was assayed with a SOD assay kit-WST [29]. Briefly, the mouse embryos were homogenized and the protein concentrations of the supernatants were analyzed by the Bradford method [30]. The supernatants were incubated with an assay reagent containing xanthine, xanthine oxidase, and a water-soluble tetrazolium salt, WST-1. The superoxide free radicals generated from the xanthine substrate by xanthine oxidase reduced WST-1 to WST-1 diformazan, which absorbed maximally at 450 nm. SOD in the embryos inhibited the WST-1 reduction as it catalyzed the dismutation of superoxide ions to molecular oxygen and hydrogen peroxide. The reduction of WST-1 was measured spectrophotometrically at 450 nm. SOD activity was calculated as an inhibition rate at 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 activity, which was determined as the total activity per embryo divided by the total amount of protein per embryo. 2.5. Quantitative RT-PCR analysis Total RNA was extracted from the cultured mouse embryos using a Trizol reagent kit (Invitrogen, U.S.A.). The total RNA concentration was determined by UV absorbance. Two micrograms of total RNA were used for reverse transcription (RT) to generate cDNA using a cDNA synthesis kit (Bio-Rad, U.S.A.). The cDNA was employed as a template for subsequent PCR reactions. For quantitative RT-PCR, the cDNA was added to a 25 ␮l reaction solution containing components of the TaqMan Universal PCR Master Mix Kit (Applied Biosystems, U.S.A.). The reaction was carried out in a 7500 Real-Time PCR System (Applied Biosystems, U.S.A.), as described in the manufacture’s instructions. The Taqman probe primers to mouse phGPx (Assayed on demand # Mm00515041, Applied Biosystems) were used. The Taqman probe primers to mouse cGPx (cGPx forward primer: 5 -CCC CAC TGC GCT CAT GA-3 ; cGPx reverse primer: 5 -GGC ACA CCG GAG ACC AAA-3 ; cGPx Taqman probe: 5 -CGA CCC CAA GTA CAT C-3 ) were designed using Primer Express software (Applied Biosystems), following the criteria indicated in the program. The Taqman probes were FAM-labeled. Beta-actin primers were used as an internal standard (Assay on demand # 4352933E, Applied Bioscience) to normalize the expression of the target transcripts. Data were analyzed from triplicates of three independent runs using a comparative Ct method, as previously described by Livak and Schmittgen [31]. 2.6. Statistical evaluation Group differences were assessed via one-way ANOVA followed by Tukey’s multiple comparison test. All analyses were conducted using the Statistical Package for Social Sciences for Windows software, version 10.0 (SPSS Inc., IL, U.S.A.). Statistical significance was assessed at p < 0.05. All data were expressed as the mean ± S.D.

3. Results 3.1. Effects of capsaicin on organogenesis Two concentrations (1 × 10−8 ␮g/ml and 1 × 10−7 ␮g/ml) of capsaicin were tested for their ability to prevent ethanol-induced growth retardation in mouse whole embryo cultures. These concentrations were selected because they produced maximal capsaicin effects in our preliminary pilot study. All of the cultures

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Table 1 Effects of ethanol (EtOH) and/or capsaicin (Cap) treatment on mouse embryonic growth in vitro. Group

CON

EtOH

Cap10−8

Cap10−7

EtOH + Cap10−8

EtOH + Cap10−7

No. of embryos Yolk sac diameter (mm) Crown-rump length (mm) Head length (mm) No. of somites

20 4.7 ± 0.3 3.6 ± 0.5 1.7 ± 0.4 26.6 ± 2.2

20 4.5 ± 0.4 3.5 ± 0.4a 1.7 ± 0.3 24.9 ± 2.0a

21 4.7 ± 0.5 3.6 ± 0.3 1.6 ± 0.3 27.7 ± 1.5b

20 4.9 ± 0.5 3.6 ± 0.3 1.6 ± 0.2 27.2 ± 1.4b

24 4.9 ± 0.4 3.6 ± 0.3 1.6 ± 0.2 27.2 ± 1.7b

22 4.9 ± 0.5b 3.6 ± 0.4 1.6 ± 0.2 27.1 ± 1.7b

Each value represents the mean ± S.D. a vs. control (CON) group at p < 0.05. b vs. ethanol group at p < 0.05.

were terminated after 48 h and all embryos completely survived. And then we evaluated the embryos according to the morphologic scoring system (Tables 1 and 2). There was no significant difference between the morphological scores of the mouse embryos that were exposed to capsaicin at concentrations of 1 × 10−8 ␮g/ml and 1 × 10−7 ␮g/ml, and the embryos in the control group. In contrast, embryos exposed to ethanol exhibited significantly lower morphological scores for their crown-rump length, number of somites, yolk sac circulation, allantois, heart, hindbrain, midbrain, forebrain, otic system, optic system, branchial bar, olfactory system, forelimb, hindlimb, and somites than the embryos in the control group. When the embryos were simultaneously exposed to capsaicin and ethanol, most of morphological parameters recovered significantly compared to those treated with ethanol alone excepting yolk sac circulation and hindlimb scores (p < 0.05). Embryos that were treated with capsaicin alone (Fig. 1C and D) were morphologically similar to those in the control group (Fig. 1A). However, ethanol administration induced a variety of embryonic anomalies including an opening of the brain (arrows) and abnormal heart development (Fig. 1B). The co-incubation of cultured embryos with capsaicin significantly improved the ethanol-induced growth retardation (Fig. 1E and F).

ethanol, the SOD activity significantly recovered to 85% of that of the control group (p < 0.05). 3.3. Effects of capsaicin on the expression of GPx genes 3.3.1. Effects of capsaicin on the expression of cGPx mRNA (Fig. 3) The cGPx mRNA level in the mouse embryos that were exposed to ethanol (1 ␮l/ml) was 0.58-fold of the level detected in the control group (1-fold); this difference was statistically significant (p < 0.05). However, when 1 × 10−8 ␮g/ml or 1 × 10−7 ␮g/ml of capsaicin was added to the ethanol-treated embryos, the cGPx mRNA level recovered to 1-fold and 0.88-fold of control group, respectively (p < 0.05). 3.3.2. Effects of capsaicin on the expression of phGPx mRNA (Fig. 4) Mouse embryos that were exposed to ethanol (1 ␮l/ml) exhibited a significantly reduced phGPx mRNA level (0.88-fold) compared to the control group (1-fold; p < 0.05). However, when the embryos were treated with 1 × 10−8 ␮g/ml or 1 × 10−7 ␮g/ml capsaicin in the presence of ethanol, the phGPx mRNA level significantly increased to 2-fold and 1.39-fold of control group, respectively (p < 0.05).

3.2. Effects of capsaicin on SOD activity (Fig. 2) 4. Discussion Mouse embryos that were exposed to ethanol (1 ␮l/ml) exhibited a significantly reduced SOD activity (49%; p < 0.05) compared to those in the control group (100%). However, when the embryos were treated with 1 × 10−7 ␮g/ml capsaicin in the presence of

It has been well established that ethanol consumption by the mother disrupts fetal development in humans [32] and in several species of experimental animals [33]; however, the precise

Table 2 Effects of ethanol (EtOH) and/or capsaicin (Cap) treatment on mouse embryonic development in vitro. Group

CON

EtOH

Cap10−8

Cap10−7

EtOH + Cap10−8

EtOH + Cap10−7

No. of embryos Yolk sac circulation Allantois Flexion Heart Hindbrain Midbrain Forebrain Otic system Optic system Branchial bars Maxillary process Mandibular process Olfactory system Caudal neural tube Forelimb Hindlimb Somites Total Score

20 4.2 ± 0.4 2.7 ± 0.4 4.6 ± 0.4 4.0 ± 0.1 4.6 ± 0.5 4.7 ± 0.4 4.8 ± 0.5 4.6 ± 0.5 4.8 ± 0.3 2.6 ± 0.6 2.1 ± 0.6 1.9 ± 0.5 2.2 ± 0.5 4.3 ± 0.7 2.9 ± 0.2 1.6 ± 0.5 4.8 ± 0.4 61.3 ± 7.6

20 3.7 ± 0.5a 2.3 ± 0.4a 4.6 ± 0.4 3.2 ± 0.5a 3.4 ± 0.7a 3.4 ± 0.7a 3.8 ± 0.7a 3.5 ± 0.6a 3.4 ± 0.7a 1.3 ± 0.5a 1.6 ± 0.4 1.5 ± 0.2 1.6 ± 0.4a 3.9 ± 0.5 2.0 ± 0.6a 0.9 ± 0.4a 4.4 ± 0.4a 48.4 ± 8.5a

21 4.1 ± 0.2 2.8 ± 0.4 4.6 ± 0.3 4.1 ± 0.3 4.9 ± 0.3 4.8 ± 0.3 4.9 ± 0.3 4.7 ± 0.5 4.9 ± 0.4 2.5 ± 0.5 1.7 ± 0.5 1.7 ± 0.4 1.9 ± 0.4 4.0 ± 0.5 3.0 ± 0.2 1.5 ± 0.6 4.9 ± 0.4 60.6 ± 6.4

20 3.8 ± 0.8 2.6 ± 0.5 4.5 ± 0.4 4.0 ± 0.3 4.4 ± 0.6 4.4 ± 0.7 4.5 ± 0.7 4.4 ± 0.5 4.7 ± 0.5 2.5 ± 0.5 1.7 ± 0.6 1.7 ± 0.4 2.0 ± 0.3 4.1 ± 0.7 2.7 ± 0.5 1.3 ± 0.7 5.0 ± 0.2 58.0 ± 8.9

24 4.0 ± 0.3 2.8 ± 0.4b 4.4 ± 0.4 4.0 ± 0.2b 4.5 ± 0.6b 4.6 ± 0.7b 4.6 ± 0.7b 4.7 ± 0.5b 4.9 ± 0.4b 2.1 ± 0.2b 1.8 ± 0.7 1.6 ± 0.4 1.7 ± 0.5 3.9 ± 0.3 3.0 ± 0.2b 1.3 ± 0.6 4.9 ± 0.4b 58.5 ± 7.4b

22 4.0 ± 0.7 2.7 ± 0.5b 4.6 ± 0.3 3.9 ± 0.3b 4.7 ± 0.3b 4.6 ± 0.6b 4.6 ± 0.6b 4.5 ± 0.5b 4.5 ± 0.8b 2.2 ± 0.4b 1.9 ± 0.4 1.6 ± 0.4 2.0 ± 0.3b 4.0 ± 0.4 2.9 ± 0.3b 1.1 ± 0.5 4.9 ± 0.4b 58.6 ± 3.4b

Each value represents the mean ± S.D. a vs. control (CON) group at p < 0.05. b vs. ethanol group at p < 0.05.

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Fig. 1. Effects of ethanol and/or capsaicin on mouse embryonic development. (A) control group, (B) ethanol group (1 ␮l/ml): forebrain and hindbrain opening (arrows), (C) capsaicin group (1 × 10−8 ␮g/ml), (D) capsaicin group (1 × 10−7 ␮g/ml), (E) ethanol (1 ␮l/ml) plus capsaicin (1 × 10−8 ␮g/ml) group, (F) ethanol (1 ␮l/ml) plus capsaicin (1 × 10−7 ␮g/ml) group.

reasons for this disturbed embryonic development have not been clarified. One proposed mechanism suggests that the metabolism of ethanol to acetaldehyde produces oxidative stress and leads to physical dysmorphogenesis and central nervous dysfunction [34]. The teratogenicity due to ethanol-induced ROS generation and lipid peroxidation has been well demonstrated in rodent whole embryos grown in culture during the period of neurulation corresponding to weeks 3–4 of human gestation [35,36]. The representative malformations induced by exogenous exposure during this period of

development are neural tube defects and craniofacial anomalies, both of which have been reported as a consequence of excessive ethanol consumption during human gestation [37,38]. In this study, ethanol administration induced a variety of embryonic anomalies including an opening of the brain, abnormal heart development, and growth retardation. Hot chili peppers that belong to the plant genus Capsicum (family Solanaceae) are among the most heavily consumed spices throughout the world [39]. The primary pungent prin-

Fig. 2. Superoxide dismutase (SOD) activity in mouse embryos (E8.5) exposed to ethanol and/or capsaicin for 2 days in vitro. Each value (n = 9) represents the mean ± S.D. *vs. control group at p < 0.05, # vs. ethanol group at p < 0.05. CON; control group, EtOH; ethanol group (1 ␮/ml), EtOH + Cap10−8 ; ethanol (1 ␮g/ml) plus capsaicin (1 × 10−8 ␮g/ml) group, EtOH + Cap10−7 ; ethanol (1 ␮l/ml) plus capsaicin (1 × 10−7 ␮g/ml) group.

Fig. 3. RT-PCR analysis of cytoplasmic glutathione peroxidase (cGPx) mRNA in mouse embryos (E8.5) exposed to ethanol and/or capsaicin for 2 days in vitro. Each value (n = 9) represents the mean ± S.D. *vs. control group at p < 0.05, # vs. ethanol group at p < 0.05. CON; control group, EtOH; ethanol group (1 ␮l/ml), EtOH + Cap10−8 ; ethanol (1 ␮g/ml) plus capsaicin (1 × 10−8 ␮g/ml) group, EtOH + Cap10−7 ; ethanol (1 ␮g/ml) plus capsaicin (1 × 10−7 ␮g/ml) group.

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mRNAs in the ethanol-treated embryos recovered to levels similar to those detected in the control embryos, in response to capsaicin co-administration. Taken together, these results indicate that capsaicin has a protective effect against ethanol-induced teratogenicity via its antioxidative activity. Acknowledgement This work was supported in part by the Korea Research Foundation Grant funded by the Korean Government [MOEHRD, Basic Research Promotion Fund (KRF-2005-005-J15002 and KRF-2006312-E00151)]. Fig. 4. RT-PCR analysis of phospholipid hydroperoxide glutathione peroxidase (phGPx) mRNA in mouse embryos (E8.5) exposed to ethanol and/or capsaicin for 2 days in vitro. Each value (n = 9) represents the mean ± S.D. *vs. control group at p < 0.05, # vs. ethanol group at p < 0.05. CON; control group, EtOH; ethanol group (1 ␮l/ml), EtOH + Cap10−8 ; ethanol (1 ␮l/ml) plus capsaicin (1 × 10−8 ␮g/ml) group, EtOH + Cap10−7 ; ethanol (1 ␮l/ml) plus capsaicin (1 × 10−7 ␮g/ml) group.

ciple in Capsicum fruits has been identified as capsaicin [40]. The capsaicin content in peppers ranges from 0.1 to 2.5 mg/g [41], and the average of human capsaicin consumption is 0.5 to 4 mg/kg/day (EC Scientific Committee on Food, 2002, http://ec.europa. eu/food/fs/sc/scf/out120 en.pdf). When it applies to mouse, it would be 5 × 10−4 to 4 × 10−3 ␮g/mg/day. In this study, we used lower dose than that of mouse in postimplantational embryo (less than 1 mg/embryo), suggesting the metabolic rate in intestine and transfer rate into placenta in maternal environment. Capsaicin and its analogues have an effective antioxidant activity [21,42]. Capsaicin has been also found to scavenge radicals at/near the membrane surface and in the interior of the phospholipid membrane [21]. Furthermore, Lee et al. [43] reported that rats treated with capsaicin (3 mg/kg body weight) for 3 days showed a reduction in oxidative stress which was measured as malondialdehyde in the liver, lung, kidney, and muscle. In this study, we examined the protective effect of capsaicin against ethanol-induced teratogenecity in mouse embryos during their critical organogenesis periods using a postimplantation whole embryo culture technique and assessed the embryos in terms of 17 morphological parameters. Ethanol administration (1.0 ␮l/ml) induced significant embryonic defects in comparison with the embryos in the control group (p < 0.05). However, concurrent administration of capsaicin (1 × 10−8 ␮g/ml or 1 × 10−7 ␮g/ml) and ethanol significantly ameliorated the toxicity of the ethanol to the embryos (p < 0.05). In cells, SOD catalyses the dismutation of superoxide radicals (O2 •− ) to hydrogen peroxide, and either catalase or GPx converts the hydrogen peroxide into water [44]. PhGPx is an intracellular antioxidant that interacts directly with peroxidized phospholipids, cholesterol and cholesteryl ester [25]. Enhanced production of ROS has also been suggested to be involved in the teratogenic process of ethanol-exposed pregnancy in experimental animals [42,45] and pregnant women [46]. The mechanisms by which ethanolinduced ROS production disrupts fetal cells may be related to membrane damage by altered fluidity and interrupted transport systems [42,47]. ROS-mediated developmental disturbances can be treated with antioxidative compounds which have been successful in preventing ethanol-induced damage to embryos both in vitro [48] and in vivo [14,16,49]. Moreover, embryonic and fetal cells exposed to ethanol in vitro can be saved by the supplementation of antioxidants [42,50]. In fact, the available experimental evidence in favor of antioxidative treatment has led to suggestions of the prophylactic supplementation of ethanol-consuming pregnant women with antioxidative compounds, such as large doses of vitamin E and vitamin C [51]. In current study, the SOD activity and cGPx and phGPx

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